FN3 domain-SIRNA conjugates and uses thereof

Abstract

The present disclosure relates to compositions such as siRNA molecules and FN3 domains conjugated thereto, as well as methods of making and using these molecules.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 380,112, filed October 19, 2022, and U.S. Provisional Application No. 63 / 505,898, filed June 2, 2023, each of which is incorporated by reference in its entirety.

[0002] The present embodiments relate to siRNA molecules that can be conjugated to fibronectin type III domain (FN3), and methods of making and using the molecules. [Background technology]

[0003] Therapeutic nucleic acids include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense, antagomir, antimira, microRNA mimics, supermira, U1 adaptors, and aptamers. In the case of siRNA or miRNA, these nucleic acids can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi). Because siRNA and miRNA constructs can be synthesized using any nucleotide sequence directed against the transcript of any target protein, the therapeutic applications of RNAi are extremely broad. To date, siRNA constructs have demonstrated the ability to specifically downregulate target proteins in both in vitro and in vivo models. Furthermore, siRNA constructs are currently being evaluated in clinical trials and approved for a variety of diseases.

[0004] However, siRNA constructs currently face two problems: first, their susceptibility to nuclease digestion in plasma; and second, their limited ability to access intracellular compartments where they can bind to RISC (RNA-induced silencing complex) when administered systemically as free siRNA or miRNA. To promote cellular uptake of oligonucleotides, certain delivery systems have been used, such as lipid nanoparticles formed from cationic lipids and other lipid components, such as cholesterol and PEG lipids, carbohydrates (such as GalNAc trimers), etc. However, these have not been shown to be successful in efficiently and effectively delivering siRNA to its intended target in tissues other than the liver.

[0005] CD40 and its ligand CD40L (or CD154) are transmembrane proteins expressed by immune cells, including B cells, T cells, and dendritic cells. CD40 functions to amplify immune responses by stimulating the activation and maturation of T cells and B cells. In autoimmune diseases, CD40's role in activating the immune system also includes the production of autoimmune antibodies. For example, studies have demonstrated a role for CD40 in neuroinflammatory diseases such as rheumatoid arthritis, thyroid autoimmune disease, type 1 diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, and lupus nephritis (see, e.g., Zheng et al., Arthritis Res. & Therapy, 2010, 12:R13; Peters et al., Semin Immunol., 2009, 21(5):293-300; and Ripoll et al., PLoS One, 2013, 8(6):e65068).

[0006] What is needed are compositions and methods for delivering therapeutic nucleic acids, such as small interfering RNA (siRNA), to intended cellular targets to downregulate CD40 production and expression in subjects suffering from autoimmune diseases. Additionally, what is needed are FN3 domains with properties optimized for clinical use that are capable of specifically binding to CD71, and methods of using such molecules for novel therapeutics that enable intracellular access via receptor-mediated internalization of CD71. The present embodiments meet these and other needs. Summary of the Invention

[0007] Provided herein are compositions comprising siRNA molecules comprising a sense strand and an antisense strand, such as those provided herein. In some embodiments, the siRNA molecule targets the CD40 gene. In some embodiments, the siRNA further comprises a linker covalently bound to the sense strand or antisense strand of the siRNA. In some embodiments, the linker is attached to the 5'-end or 3'-end of the sense strand or antisense strand. In some embodiments, the siRNA molecule further comprises a vinyl phosphonate modification on the sense strand or antisense strand. In some embodiments, the vinyl phosphonate modification is on the 5'-end or 3'-end of the sense strand or antisense strand. In some embodiments, the sense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1890, 1893, 1941, 1942, 1944, 46-178, 312-331, 1850, 1851, 1891, 1892, 1894-1928, 1932-1940, 1943, 1945-1959, 2298, 2302, 2304, 352-356, 673-805, 939-958, 2070, 2071, 2110-2148, 2152-2179, 2300, 2306, and 2308. In some embodiments, the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2290, 2293, 2051, 2052, 2054, 179-311, 332-351, 1960, 1961, 2000-2038, 2042-2050, 2053, 2055-2069, 2291, 2292, 2294-2297, 2299, 2303, 2305, 356-359, 806-938, 959-978, 2180, 2181, 2220-2258, 2262-2289, 2301, and 2309. In some embodiments, the siRNA molecule comprises an siRNA pair set forth in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, or Table 5B.

[0008] In some embodiments, the composition further comprises one or more FN3 domains conjugated to the siRNA molecule. In some embodiments, the one or more FN3 domains comprise a CD71-binding FN3 domain. In some embodiments, the FN3 domain comprises an amino acid sequence at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or identical to a sequence selected from any one of SEQ ID NOs: 570, 672, 1848, 1773, 1849, 1767, 360-569, 571-644, 663-67, 1395-1772, 1774-1766, and 1768-1847.

[0009] In some embodiments, the one or more FN3 domains comprise at least two FN3 domains linked by a peptide linker, in some embodiments, the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 645-661.

[0010] Also provided herein is a composition having the formula: (X1) n -(X2) q -(X3) y -L-X4; C-(X1) n -(X2) q -L-X4-(X3) y ; (X1) n -(X2) q -L-X4-(X3) y -C; C-(X1) n -(X2) q -L-X4-L-(X3) y ; or (X1) n -(X2) q -L-X4-L-(X3) y -C, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension, L is a linker, and X4 is a nucleic acid molecule such as an siRNA targeting CD40, such as those provided herein. C is a polymer, e.g., PEG, an albumin-binding protein, or an aliphatic chain that binds to a serum protein, and wherein n, q, and y are each independently 0 or 1. In some embodiments, X1, X2, and X3 bind to the same or different target proteins.

[0011] Also provided herein are compositions having the formula A1-B1, wherein A1 is (C) n -(L1) t -X s and B1 has the formula X AS -(L2) q -(F1) y or A1 has the formula (F1) n -(L1) t -X s and B1 has the formula X AS -(L2) q -(C) y having the formula: C is an aliphatic chain that binds to a polymer, e.g., PEG, an albumin-binding protein, or a serum protein; L1 and L2 are each independently a linker; X S is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule, X AS is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule, F1 is a polypeptide comprising at least one FN3 domain; wherein n, t, q, and y are each independently 0 or 1; X S and X AS form a double-stranded oligonucleotide molecule to form a composition / complex that targets CD40.

[0012] In some embodiments, provided herein is a method of treating an immune disease in a subject in need thereof, the method comprising administering to the subject a composition, such as any of the compositions provided herein. In some embodiments, provided herein is a method of reducing mRNA expression of a target gene in a cell, such as an immune cell, the method comprising contacting the immune cell with any of the compositions provided herein. In some embodiments, provided herein is a method of delivering an siRNA molecule to a cell, such as an immune cell, in a subject, the method comprising administering to the subject a pharmaceutical composition comprising any of the compositions provided herein. In some embodiments, provided herein is a method of delivering an siRNA molecule targeting CD40 to CD71-expressing immune cells in a subject, the method comprising administering to the subject a pharmaceutical composition comprising any of the compositions provided herein, wherein the siRNA molecule down-regulates mRNA expression of CD40 in the CD71-expressing immune cells.

[0013] Further provided herein are methods for reducing one or more serum cytokines, the methods comprising administering an siRNA molecule targeting CD40 to one or more CD71-expressing immune cells. In some embodiments, the one or more serum cytokines comprise IFN-γ, IL-6, TNF-α, IL-12, IP-10, RANTES, or any combination thereof. In some embodiments, the one or more CD71-expressing immune cells comprise B cells, T cells, or a combination thereof.

[0014] Further provided herein are methods for selectively reducing a population of CD71-expressing immune cells, the methods comprising administering an siRNA molecule targeting CD40 to the population of CD71-expressing immune cells. In some embodiments, the population of CD71-expressing immune cells comprises B cells, T cells, or a combination thereof. [Brief explanation of the drawings]

[0015] [Figure 1]FIG. 1 shows a flow chart of the steps and properties assessed for in silico screening of CD40 siRNAs. [Figure 2] FIG. 2 shows titration curves for exemplary CD40 siRNAs in Raji cells (FIG. 2, panel A) and A20 cells (FIG. 2, panel B). [Figure 3A] FIG. 1 shows relative in vitro CD40 mRNA expression in activated, CD40 ligand-exposed donated human dendritic cells treated or not with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates. [Figure 3B] FIG. 1 shows in vitro IL-12 production in provided human dendritic cells, activated or non-activated, exposed or not exposed to CD40 ligand, and treated or not treated with exemplary CD71-binding FN3 domain and CD40-targeted siRNA conjugates. [Figure 4] Figure 1 shows relative in vitro CD40 mRNA expression in donated human dendritic cells activated and treated with increasing concentrations of an exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugate. CD40 mRNA expression in treated cells is normalized to mRNA expression in activated, untreated dendritic cells. As the concentration (nM) of the conjugate increases, relative CD40 mRNA expression decreases in all donors. [Figure 5]

[0023] Figure 1 shows the relative in vitro CD40 protein expression over time in provided human dendritic cells that were activated and treated with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates, or activated and untreated ("Activation only"). CD40 protein expression in treated cells is normalized to protein expression in activated, untreated dendritic cells. [Figure 6]FIG. 1 shows in vitro cytokine production in provided dendritic cells activated and treated with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates, dendritic cells activated and treated with a negative control, and activated, untreated dendritic cells. [Figure 7] FIG. 1 shows in vivo serum cytokine levels in mice activated and treated with an exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugate, mice activated and treated with a negative control, mice activated and treated with a CD71-binding FN3 domain only, mice activated and treated with vehicle, and naive mice that were neither activated nor treated. [Figure 8] 1 shows in vivo serum cytokine levels in mice induced with the animal disease model EAE and activated and treated with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates. Also shown are EAE mice activated and treated with a negative control, EAE mice activated and treated with the CD71-binding FN3 domain alone, EAE mice activated and treated with vehicle, and healthy naive mice that were neither activated nor treated. [Figure 9] 1 shows the in vivo frequency of B cells in draining lymph node tissue and spinal cord tissue collected from mice induced with EAE, an animal disease model, and activated and treated with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates. Also shown are EAE mice activated and treated with a positive control, EAE mice activated and treated with vehicle, and healthy naive mice that were neither activated nor treated. [Figure 10] 1 shows the in vivo frequencies of dendritic cells, CD8 T cells, and CD4 T cells in spinal cord tissue collected from mice induced with EAE, an animal disease model, and activated and treated with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates. Also shown are EAE mice activated and treated with a positive control, and EAE mice activated and treated with vehicle. [Figure 11] 1 shows the in vivo frequencies of lymphocytes, monocytes, and macrophages in spinal cord tissue collected from mice induced with EAE, an animal disease model, and activated and treated with exemplary CD71-binding FN3 domain and CD40-targeting siRNA conjugates. Also shown are EAE mice activated and treated with a positive control, and EAE mice activated and treated with vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0016] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0017] "Fibronectin type III domain" or "FN3 domain" refers to a polypeptide sequence that frequently occurs in proteins including fibronectin, tenascin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 distinct FN3 domains present in human teniscin C, the 15 distinct FN3 domains present in human fibronectin (FN), and non-naturally occurring synthetic FN3 domains, such as those described in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the third FN3 domain of teniscin (TN3) or the tenth FN3 domain of fibronectin (FN10). As used throughout, "sentilin" also refers to an FN3 domain. Additionally, the FN3 domains described herein are also referred to as FN3 domains in the variable heavy chain (V H ) and / or light chain (VL ) structure and therefore is not an antibody.

[0018] "Autoimmune disease" refers to pathologies and conditions in which an individual's immune response is directed against the individual's own constituents, resulting in an undesirable and often debilitating condition. As used herein, "autoimmune disease" is intended to further include autoimmune conditions, syndromes, and the like. Autoimmune diseases include, but are not limited to, Addison's disease, allergies, allergic rhinitis, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune atrophic gastritis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune parotitis, autoimmune uveitis, celiac disease, primary biliary cirrhosis, benign lymphocytic vasculitis, COPD, colitis, coronary heart disease, Crohn's disease, diabetes mellitus (type 1), depression, diabetes including type 1 and / or type 2 diabetes, epididymitis, glomerulonephritis, Gut-Pasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammation Autoimmune diseases include, but are not limited to, conditions in which the affected tissue is the primary target, and in some cases, a secondary target. Such conditions include, but are not limited to, AIDS, atopic allergies, bronchial asthma, eczema, leprosy, schizophrenia, hereditary depression, tissue and organ transplantation, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus phenomenon, anaphylaxis, and alcohol and drug addiction.

[0019] "Capture agent" refers to a substance that binds to a specific type of cell and allows that cell to be isolated from other cells. Exemplary capture agents are magnetic beads, ferrous fluids, encapsulation reagents, molecules that bind to specific cell types, etc.

[0020] "Sample" refers to similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Exemplary samples include tissue biopsies, fine needle aspirates, surgically removed tissues, organ cultures, cell cultures, and biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, secretory tissues and mucosal secretions of organs, vaginal secretions, ascites, fluids of the pleural, pericardial, peritoneal, abdominal, and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media and lavage fluids.

[0021] "Substitute," "substituted," "mutate," or "mutated" refers to changing, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of that sequence.

[0022] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as a substitution, insertion, or deletion.

[0023] "Specifically binds" or "specific binding" refers to the binding of an FN3 domain to its target, e.g., CD71, at a concentration of approximately 1 x 10 -6 M or less, for example, about 1 × 10 -7 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, approximately 1×10 -12 M or less, or approximately 1 x 10 -13 The dissociation constant (K D) Alternatively, "specific binding" refers to the ability of an FN3 domain to bind to its target (e.g., CD71) at least 5-fold above a negative control in a standard solution ELISA assay. Specific binding can also be demonstrated using the proteome arrays described herein. In some embodiments, the negative control is an FN3 domain that does not bind to CD71. In some embodiments, an FN3 domain that specifically binds to CD71 may have cross-reactivity to other related antigens, e.g., the same given antigen from other species (homologues), such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troGlodytes (chimpanzee).

[0024] A "library" refers to a collection of variants. A library can be composed of polypeptide or polynucleotide variants.

[0025] "Stability" refers to the ability of a molecule to remain folded under physiological conditions such that the molecule retains at least one of its normal functional activities, such as binding to a given antigen, such as CD71.

[0026] "CD71" refers to the human CD71 protein having the amino acid sequence of SEQ ID NO: 3 or 4. In some embodiments, SEQ ID NO: 3 is the full-length human CD71 protein. In some embodiments, SEQ ID NO: 4 is the extracellular domain of human CD71.

[0027] "Tencon" is the consensus sequence: LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1), Refers to the synthetic fibronectin type III (FN3) domain described in US Patent Publication No. 2010 / 0216708.

[0028] "Immune cells" refer to cells of the immune system classified as lymphocytes (T cells, B cells, and NK cells), neutrophils, or monocytes / macrophages. Immune cells also include dendritic cells. "Dendritic cells" refer to a type of antigen-presenting cell (APC) that plays an important role in the adaptive immune system. The main function of dendritic cells is to present antigens to T lymphocytes and to secrete cytokines that can further regulate the immune response directly or indirectly. Dendritic cells have the ability to induce primary immune responses in inactive or resting naive T lymphocytes.

[0029] A "vector" refers to a polynucleotide that can replicate within a biological system or can be moved between such systems. A vector polynucleotide typically contains elements that function to facilitate replication or maintenance of the polynucleotide within the biological system, such as an origin of replication, a polyadenylation signal, or a selectable marker. Examples of such biological systems can include cells, viruses, animals, plants, and reconstituted biological systems that utilize biological components capable of replicating the vector. The polynucleotide comprising the vector can be a DNA molecule or an RNA molecule, or a hybrid of these.

[0030] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0031] "Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0032] "Polypeptide" or "protein" refers to a molecule containing at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides, less than about 50 amino acids, are sometimes referred to as "peptides."

[0033] "Valency" refers to the presence of a designated number of binding sites specific for an antigen in a molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of 1, 2, 4, and 6 binding sites specific for an antigen in a molecule, respectively.

[0034] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably.

[0035] "Isolated" refers to a homogenous population of molecules (e.g., synthetic polynucleotides or polypeptides, such as FN3 domains) that have been substantially separated and / or purified from other components of the system in which they are produced, such as recombinant cells, as well as proteins that have been subjected to at least one purification or isolation step. An "isolated FN3 domain" refers to an FN3 domain that is substantially free of other cellular material and / or chemicals, and encompasses FN3 domains isolated to greater degrees of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0036] When used in reference to cell movement, "migration" means that a cell moves from one location to another. For example, a cell (e.g., a white blood cell or immune cell) moving from a blood vessel to a tissue can be said to be migrating from the blood vessel to the tissue. Migration can also include the process of "margination," which refers to the movement of a cell from the inside of a blood vessel toward the blood vessel wall. Migration can also include the attachment of a cell to the blood vessel wall, as well as crossing the blood vessel wall to enter a tissue.

[0037] composition In some embodiments, compositions are provided that include a polypeptide, such as a polypeptide comprising an FN3 domain, linked to an oligonucleotide molecule. The oligonucleotide molecule can be, for example, an siRNA molecule. In some embodiments, the FN3 domain is a CD71-binding FN3 domain provided herein. In some embodiments, the oligonucleotide is a CD40 siRNA that binds to CD40 RNA, such as the mRNA provided herein. In some embodiments, the composition further includes a polymer provided herein.

[0038] In some embodiments, the siRNA molecule is a double-stranded RNAi (dsRNA) agent capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand (passenger strand) and an antisense strand (guide strand). In some embodiments, each strand of the dsRNA agent can range from 12 to 40 nucleotides in length. For example, each strand can be 14 to 40 nucleotides in length, 17 to 37 nucleotides in length, 25 to 37 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0039] In some embodiments, the sense strand and antisense strand typically form a double-stranded dsRNA. The duplex region of the dsRNA agent can be 12 to 40 nucleotide pairs in length. For example, the duplex region can be 14 to 40 nucleotide pairs, 17 to 30 nucleotide pairs, 25 to 35 nucleotide pairs, 27 to 35 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the duplexed region is selected from 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotide pairs in length.

[0040] In some embodiments, the dsRNA comprises one or more overhang regions and / or capping groups of the dsRNA agent at the 3'-end, 5'-end, or both ends of the strand. The overhang can be 1-10 nucleotides, 1-6 nucleotides, e.g., 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, or 1-2 nucleotides in length. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the gene sequence being targeted, or it can be another sequence. The first and second strands can also be linked, for example, by additional bases to form a hairpin, or can be linked by other non-basic linkers.

[0041] In some embodiments, each nucleotide in the overhang region of a dsRNA agent can be independently a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modification, such as 2-F, 2'-O-methyl, 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), and any combination thereof. For example, TT (UU) can be the overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the gene sequence being targeted, or it can be another sequence.

[0042] The 5' overhang or 3' overhang on the sense strand, antisense strand, or both strands of dsRNA agent can be phosphorylated.In some embodiments, the overhang region contains two nucleotides with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate between the two nucleotides, and these two nucleotides can be the same or different.In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand, or both strands.In one embodiment, this 3' overhang is present on the antisense strand.In one embodiment, this 3' overhang is present on the sense strand.

[0043] dsRNA agent can only contain a single overhang, which can enhance the interference activity of dsRNA without affecting overall stability.For example, the single-stranded overhang is located at the 3'-end of the sense strand, or alternatively at the 3'-end of the antisense strand.dsRNA can also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), or vice versa.Generally, the antisense strand of dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without being bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favor the loading of the guide strand into RNA-induced silencing complex (RISC).For example, the single overhang comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0044] In some embodiments, a dsRNA agent can also have two blunt ends at either end of the dsRNA duplex.

[0045] In some embodiments, all nucleotides in the sense strand and antisense strand of dsRNA agent can be modified.Each nucleotide can be modified with the same or different modifications, and modifications can include one or more changes of one or both of non-linked phosphate oxygen and / or one or more changes of linking phosphate oxygen;Modification of ribose sugar components, for example, two hydroxyls on ribose sugar;Large-scale replacement of phosphate moiety with " dephosphorylation " linker;Modification or replacement of naturally occurring base;And replacement or modification of ribose phosphate backbone.

[0046] In some embodiments, all or some of the bases in the 3' or 5' overhang can be modified, for example, using the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar, such as deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl (2'-OMe) modified instead of the ribosugar of the nucleobase, and modifications at the phosphate group, such as phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate modifications. The overhang does not need to be homologous to the target sequence.

[0047] In some embodiments, each residue in the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain multiple modifications. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0048] In some embodiments, at least two different modifications are typically present on the sense and antisense strands, and these two modifications may be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, or other modifications.

[0049] In one embodiment, the sense and antisense strands each comprise two different modified nucleotides selected from 2'-fluoro, 2'-O-methyl, or 2'-deoxy.

[0050] dsRNA agent can further comprise at least one phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide linkage.Phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide linkage modification can occur at any nucleotide of sense strand or antisense strand, or both, at any position of chain.For example, internucleotide linkage modification can occur at any nucleotide on sense strand and / or antisense strand, and each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can have a shift with respect to the alternating pattern of internucleotide linkage modification on antisense strand.

[0051] In some embodiments, dsRNA agent comprises phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide linkage between two nucleotides.Internucleotide linkage modification can also be carried out to connect overhang nucleotide with the terminal pairing nucleotide in double-stranded region. For example, at least 2, 3, 4 or all overhanging nucleotides can be linked via phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide linkages, and optionally there can be additional phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide linkages that connect overhanging nucleotides with the paired nucleotides adjacent to overhanging nucleotides.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides, and the third is the paired nucleotide adjacent to overhanging nucleotides.In some embodiments, these terminal three nucleotides can be at the 3'-end of antisense strand.

[0052] In some embodiments, dsRNA compositions are linked by modified base or nucleoside analogue as described in United States Patent (USP) 7,427,672, which is incorporated herein by reference.In some embodiments, modified base or nucleoside analogue is referred to as linker or L in the formula described herein.

[0053] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof: [ka] In the formula, the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group which may have a substituent; R1 and R2 are the same or different and each represents a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected with a protecting group for nucleic acid synthesis, or -P(R4)R5, where R4 and R5 are the same or different and each represents a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with 1 to 5 alkyl groups; and X represents OMe or F.

[0054] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, where R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl, lower alkoxy, halogen, or cyano group, or a silyl group.

[0055] In some embodiments, the modified base or nucleoside analog has the structure of Formula I and salts thereof, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a trityl group, a dimethoxytrityl group, a monomethoxytrityl group, or a tert-butyldiphenylsilyl group.

[0056] In some embodiments, the modified base or nucleoside analog has the structure of Formula I and salts thereof, where R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl, lower alkoxy, halogen, or cyano group, a silyl group, a phosphoramidite group, a phosphonyl group, a phosphate group, or a phosphate group protected with a protecting group for nucleic acid synthesis.

[0057] In some embodiments, the modified base or nucleoside analog has the structure of Formula I and salts thereof, where R2 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyldiphenylsilyl group, —P(OC2H4CN)(N(i-Pr)2), —P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphenyl group.

[0058] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein the base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following alpha groups: a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0059] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoro ... Amino-6-fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0060] In some embodiments, the modified base or nucleoside analog has the structure shown in formula IB and salts thereof: [ka] In the formula, the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group which may have a substituent; R1 and R2 are the same or different and each represents a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected with a protecting group for nucleic acid synthesis, or -P(R4)R5, where R4 and R5 are the same or different and each represents a hydroxyl group, a protecting group for nucleic acid synthesis, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a functional molecular unit substituent; m represents an integer of 0 to 2; and n represents an integer of 0 to 3. In some embodiments, m and n are 0.

[0061] In some embodiments, the modified base or nucleoside analog has the structure of formula IB and salts thereof, where R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl, a lower alkoxy, a halogen, or a cyano group, or a silyl group.

[0062] In some embodiments, the modified base or nucleoside analog has the structure of formula IB and salts thereof, where R1 is hydrogen, acetyl, benzoyl, methanesulfonyl, p-toluenesulfonyl, benzyl, p-methoxybenzyl, trityl, dimethoxytrityl, monomethoxytrityl, or tert-butyldiphenylsilyl.

[0063] In some embodiments, the modified base or nucleoside analog has the structure of formula IB and salts thereof, where R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl, lower alkoxy, halogen, or cyano group, a silyl group, a phosphoramidite group, a phosphonyl group, a phosphate group, or a phosphate group protected with a protecting group for nucleic acid synthesis.

[0064] In some embodiments, the modified base or nucleoside analog has the structure of formula IB and salts thereof, where R2 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyldiphenylsilyl group, —P(OC2H4CN)(N(i-Pr)2), —P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphenyl group.

[0065] In some embodiments, the modified base or nucleoside analog has the structure of Formula IB and salts thereof, where R3 is a hydrogen atom, a phenoxyacetyl group, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted with 1 to 3 aryl groups, a lower aliphatic or aromatic sulfonyl group, e.g., methanesulfonyl or p-toluenesulfonyl, an aliphatic acyl group having 1 to 5 carbon atoms, e.g., acetyl, or an aromatic acyl group such as benzoyl.

[0066] In some embodiments, the modified base or nucleoside analog has the structure shown in formula IB and salts thereof, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear import signal peptide.

[0067] In some embodiments, the modified base or nucleoside analog has the structure of formula IB and salts thereof, wherein the base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following alpha groups: a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0068] In some embodiments, the modified base or nucleoside analog has the structure shown in formula IB and salts thereof, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoro ...fluoropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected -amino-6-fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloro Purin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0069] In some embodiments, the modified base or nucleoside analog has the structure of formula IB and salts thereof, where m is 0 and n is 1.

[0070] In some embodiments, the modified base or nucleoside analog is a DNA or RNA oligonucleotide analog containing one or more unit structures of a nucleoside analog having the structure shown in Formula II, or a pharmacologically acceptable salt thereof, provided that the linkage between each nucleoside in the oligonucleotide analog is the same phosphodiester bond [--OP(O2)] as that in natural nucleic acids. - )O--], as well as one or more phosphorothioate bonds [--OP(O)( S- )O--], phosphorodithioate bond [--O2PS2--], phosphonate bond [--PO(OH)2-], phosphoramidate bond [--O=P(OH)2-], or mesylphosphoamidate bond [--OP(O)(N)(SO2)(CH3)O--], and the bases may be the same or different between these structures, provided that two or more of one or more types of these structures are included: [ka] In the formula, the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group which may have a substituent, and X represents OMe or F.

[0071] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula II, wherein the base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following alpha groups: a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0072] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula II, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, or an amino group protected with a protecting group for nucleic acid synthesis. 2-amino-6-fluoropurin-9-yl having an amino group, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9 -yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-methoxy-1 , 2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis,2-dihydropyrimidin-1-yl.

[0073] In some embodiments, the modified base or nucleoside analog is a DNA or RNA oligonucleotide analog containing one or more unit structures of a nucleoside analog having the structure represented by formula IIB, or a pharmacologically acceptable salt thereof, provided that the linkage between each nucleoside in the oligonucleotide analog is the same phosphodiester bond [--OP(O2) - )O--], as well as one or more phosphorothioate bonds [--OP(O)( S- )O--], phosphorodithioate bond [--O2PS2--], phosphonate bond [--PO(OH)2-], phosphoramidate bond [--O=P(OH)2-], or mesylphosphoamidate bond [--OP(O)(N)(SO2)(CH3)O--], and the bases may be the same or different between these structures, provided that two or more of one or more types of these structures are included: [ka] In the formula, the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group which may have a substituent, R3 includes a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, or a functional molecular unit substituent, m represents an integer of 0 to 2, and n represents an integer of 0 to 3. In some embodiments, m and n are 0.

[0074] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, where R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl, a lower alkoxy, a halogen, or a cyano group, or a silyl group.

[0075] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has a structure represented by formula IIB, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a trityl group, a dimethoxytrityl group, a monomethoxytrityl group, or a tert-butyldiphenylsilyl group.

[0076] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl, lower alkoxy, halogen, or cyano group, a silyl group, a phosphoramidite group, a phosphonyl group, a phosphate group, or a phosphate group protected with a protecting group for nucleic acid synthesis.

[0077] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, where R2 is a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a tert-butyldiphenylsilyl group, -P(OC2H4CN)(N(i-Pr)2), -P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphonyl group.

[0078] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, wherein R3 is a hydrogen atom, a phenoxyacetyl group, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted with 1 to 3 aryl groups, a lower aliphatic or aromatic sulfonyl group, e.g., a methanesulfonyl group or a p-toluenesulfonyl group, an aliphatic acyl group having 1 to 5 carbon atoms, e.g., an acetyl group, or an aromatic acyl group such as a benzoyl group.

[0079] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has a structure represented by formula IIB, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear import signal peptide.

[0080] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, wherein the base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following alpha groups: a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0081] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, or an amino group protected with a protecting group for nucleic acid synthesis. 2-amino-6-fluoropurin-9-yl having an amino group, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9 -yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-methoxy-1 , 2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis,2-dihydropyrimidin-1-yl.

[0082] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has the structure shown in Formula IIB, wherein m is 0 and n is 1.

[0083] In some embodiments, the dsRNA agent contains mismatch(es) with the target, or within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of a particular pairing, the simplest approach is to examine each pair individually, but next-neighbor or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0084] In some embodiments, a dsRNA agent may contain a phosphorus-containing group at the 5'-end of the sense strand or antisense strand. The 5'-end phosphorus-containing group may be 5'-end phosphate (5'-P), 5'-end phosphorothioate (5'-PS), 5'-end phosphorodithioate (5'-PS2), 5'-end vinylphosphonate (5'-VP), 5'-end methylphosphonate (MePhos), 5'-end mesylphosphoramidate (5'MsPA), or 5'-deoxy-5'-C-malonyl. When the 5'-end phosphorus-containing group is 5'-end vinylphosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer, such as trans-vinylphosphate or cis-vinylphosphate, or a mixture thereof. Representative structures of these modifications can be found, for example, in U.S. Patent No. 10,233,448, which is incorporated herein by reference in its entirety.

[0085] In some embodiments, the nucleotide analog or synthetic nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some cases, the modification includes H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, C-C 10 The alkyl moiety may further comprise a modification, such as an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazine, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some cases, the alkyl moiety further comprises an additional heteroatom such as O, S, N, or Se, each of which may be further substituted with an alkyl group as described above. In some cases, the carbon of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some cases, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0086] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. Exemplary chemical structures of a 2'-O-methyl modification of an adenosine molecule and a 2'O-methoxyethyl modification of a uridine are shown below. [ka]

[0087] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, where an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some cases, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving cellular uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is shown below. [ka]

[0088] In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which an oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of an LNA is shown below. The representation shown on the left highlights the chemical connections of the LNA monomer. The representation shown on the right highlights the locked 3'-endo (3E) conformation of the furanose ring of the LNA monomer. [ka]

[0089] In some cases, the modification at the 2' hydroxyl group includes ethylene nucleic acid (ENA), e.g., 2'-4'-ethylene bridged nucleic acid, which locks the sugar conformation into a C3'-endo sugar pucker conformation. ENA is part of the bridged nucleic acid class of modified nucleic acids, which also includes LNA. Exemplary chemical structures of ENA and bridged nucleic acid are shown below. [ka]

[0090] In some embodiments, additional modifications at the 2' hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0091] In some embodiments, nucleotide analogs include 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, such as 5-(2-amino)propyluridine, 5-halo Cytidine, 5-hauridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thymidine Modified bases include, but are not limited to, auridine, other thio bases such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, quasi-uridine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines, such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety and nucleotides having non-ribosyl sugars or analogs thereof. For example, the sugar moiety is, or is based on, mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles in some cases. The term nucleotide also includes what are known in the art as universal bases.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebulin.

[0092] In some embodiments, the nucleotide analogs further include morpholinos, peptide nucleic acids (PNAs), methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, 1',5'-anhydrohexitol nucleic acids (HNAs), or combinations thereof. Morpholinos or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics that of natural nucleic acids by deviating from the usual sugar and phosphate structures. In some cases, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In such cases, the backbone changes remove all positive and negative charges, forming morpholino neutral molecules that can cross cell membranes without the aid of cellular delivery agents, such as those used by charged oligonucleotides. [ka]

[0093] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar rings or phosphate linkages, and the bases are attached to oligoglycine-like molecules, which are appropriately spaced to eliminate backbone charge. [ka]

[0094] In some embodiments, one or more modifications optionally occur at the internucleotide linkage. In some cases, the modified internucleotide linkage is phosphorothioate, mesyl phosphoramidate, phosphorodithioate, methyl phosphonate, 5'-alkylene phosphonate, 5'-methyl phosphonate, 3'-alkylene phosphonate, boron trifluorodate, 3'-5'-linked or 2'-5'-linked boranophosphate and selenophosphate, phosphotriester, thionoalkylphosphotriester, hydrogen phosphonate linkage, alkyl phosphonate, alkyl phosphonothioate, aryl phosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkyl phosphoramidate, aminoalkyl phosphoramidate, thionophosphoramidate, phosphoro Phosphorothioate antisense oligonucleotides include, but are not limited to, piperazidate, phosphoroanilothioate, phosphoroanilidate, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazos, methylenedimethylhydrazos, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, thioamidate, riboacetyl linkage, aminoethylglycine, silyl or siloxane linkage, saturated or unsaturated and / or substituted and / or heteroatom-containing alkyl or cycloalkyl linkages containing 1 to 10 carbon atoms, morpholino linkages, amides, polyamides in which bases are directly or indirectly bound to the aza nitrogens of the backbone, and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate linkages. Mesyl phosphoramidate antisense oligonucleotides (MsPA ASOs) are antisense oligonucleotides containing mesyl phosphoramidate linkages.

[0095] In some cases, the modification is a methyl or thiol modification, such as a methyl phosphonate modification, a mesyl phosphoramidate, or a thiol phosphonate modification. In some cases, the modified nucleotide includes, but is not limited to, a 2'-fluoro N3-P5'-phosphoramidite.

[0096] In some cases, modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)).

[0097] In some embodiments, the one or more modifications further optionally include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications of the nucleotide analog at the 3' or 5' end. For example, the 3' end optionally includes a 3' cationic group or includes a 3'-3' inverted nucleoside at the 3' end. In another alternative, the 3' end may be conjugated with an aminoalkyl group, e.g., a 3'C5-aminoalkyl dT. In an additional alternative, the 3' end may be conjugated with an abasic site, e.g., an apurinic or apyrimidinic site. In some cases, the 5' end is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent. In some cases, the 5' end is conjugated with an abasic site, e.g., an apurinic or apyrimidinic site.

[0098] In some embodiments, the oligonucleotide molecule comprises one or more synthetic nucleotide analogs described herein. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more synthetic nucleotide analogs described herein. In some embodiments, the synthetic nucleotide analog comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof. In some cases, the oligonucleotide molecule may be 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE). or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more synthetic nucleotide analogs selected from 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof.In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methyl modified nucleotides described herein. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides described herein. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiol phosphonate nucleotides described herein.

[0099] In some cases, the oligonucleotide molecule comprises at least one of about 5% to about 100% modifications, about 10% to about 100% modifications, about 20% to about 100% modifications, about 30% to about 100% modifications, about 40% to about 100% modifications, about 50% to about 100% modifications, about 60% to about 100% modifications, about 70% to about 100% modifications, about 80% to about 100% modifications, or about 90% to about 100% modifications. In some cases, the oligonucleotide molecule comprises 100% modifications.

[0100] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications.

[0101] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications.

[0102] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications.

[0103] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications.

[0104] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications.

[0105] In some cases, the oligonucleotide molecules contain at least one of about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0106] In some cases, the oligonucleotide molecules contain at least one of about 10% to about 30% modifications and about 20% to about 30% modifications.

[0107] In some cases, the oligonucleotide molecules contain about 10% to about 20% modifications.

[0108] In some cases, the oligonucleotide molecules contain between about 15% and about 90%, between about 20% and about 80%, between about 30% and about 70%, or between about 40% and about 60% modifications.

[0109] In additional cases, the oligonucleotide molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0110] In some embodiments, the oligonucleotide molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 modifications.

[0111] In some cases, the oligonucleotide molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 modified nucleotides.

[0112] In some cases, about 5 to about 100% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 5% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 10% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 15% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 20% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 25% of the oligonucleotide molecules comprise a synthetic nucleotide analogue described herein. In some cases, about 30% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 35% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 40% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 45% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 50% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 55% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 60% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 65% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 70% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 75% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein. In some cases, about 80% of the oligonucleotide molecules contain a synthetic nucleotide analog described herein.In some cases, about 85% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 90% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 95% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 96% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 97% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 98% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 99% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some cases, about 100% of the oligonucleotide molecules contain a synthetic nucleotide analogue described herein. In some embodiments, the synthetic nucleotide analog comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof.

[0113] In some embodiments, the oligonucleotide molecule comprises from about 1 to about 25 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 1 modification, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 2 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 3 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 4 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 5 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 6 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 7 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 8 modifications, wherein the modification comprises a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 9 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 10 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 11 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 12 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 13 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 14 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein.In some embodiments, the oligonucleotide molecule comprises about 15 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 16 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 17 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 18 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 19 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 20 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 21 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 22 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 23 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 24 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 25 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 26 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 27 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 28 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 29 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein.In some embodiments, the oligonucleotide molecule comprises about 30 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 31 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 32 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 33 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 34 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 35 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 36 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 37 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 38 modifications, wherein the modifications comprise a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 39 modifications, wherein the modifications comprise synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 40 modifications, wherein the modifications comprise synthetic nucleotide analogs described herein.

[0114] In some embodiments, the oligonucleotide molecule is assembled from two separate polynucleotides, where one polynucleotide comprises the sense strand of the oligonucleotide molecule and the other polynucleotide comprises the antisense strand of the oligonucleotide molecule. In other embodiments, the sense strand is connected to the antisense strand via a linker molecule, which in some cases is a polynucleotide linker or a non-nucleotide linker.

[0115] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides in the sense strand comprise 2'-O-methylpyrimidine nucleotides and the purine nucleotides in the sense strand comprise 2'-deoxypurine nucleotides. In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides present in the sense strand comprise 2'-deoxypurine nucleotides.

[0116] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense strand are 2'-O-methyl purine nucleotides.

[0117] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense strand comprise 2'-deoxy purine nucleotides.

[0118] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and at least one of the sense strand and the antisense strand comprises a plurality (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, etc.) of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, at least 2, 3, 4, 5, 6, or 7 of the plurality of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are contiguous. In some embodiments, the contiguous 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, the contiguous 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the sense strand of the oligonucleotide molecule comprises at least four, at least five, or at least six consecutive 2'-O-methyl modified nucleotides at its 5'-end and / or 3'-end, or both. Optionally, in such embodiments, the sense strand of the oligonucleotide molecule comprises at least one, at least two, at least three, or at least four 2'-deoxy-2'-fluoro modified nucleotides at the 3'-end of the at least four, at least five, or at least six consecutive 2'-O-methyl modified nucleotides at the 5'-end of the polynucleotide, or at the 5'-end of the at least four, at least five, or at least six consecutive 2'-O-methyl modified nucleotides at the 3'-end of the polynucleotide. Also optionally, such at least two, at least three, or at least four 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides.

[0119] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein at least one of the sense strand and the antisense strand has a 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, at least one of the sense strand and the antisense strand has a 2'-O-methyl modified nucleotide located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand is a purine nucleotide. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand is a pyrimidine nucleotide.

[0120] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein one of the sense strand and the antisense strand has at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides located at its 5'-end, and the other strand has at least two consecutive 2'-O-methyl modified nucleotides located at its 5'-end. In some embodiments, when a strand has at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides located at its 5'-end, the strand also contains at least two or at least three consecutive 2'-O-methyl modified nucleotides at the 3'-end of the at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, one of the sense strand and the antisense strand has at least two, at least three, at least four, at least five, at least six, or at least seven consecutive 2'-O-methyl modified nucleotides linked to 2'-deoxy-2'-fluoro modified nucleotides at its 5'-end and / or 3'-end. In some embodiments, one of the sense strand and the antisense strand has at least four, at least five nucleotides that alternate between 2'-O-methyl modified nucleotides and 2'-deoxy-2'-fluoro modified nucleotides.

[0121] In some embodiments, an oligonucleotide molecule, such as an siRNA, has a formula as exemplified in Formula III: [ka] In the formula, each nucleotide represented by N is independently A, U, C, or G, or a modified nucleotide base such as those provided herein. The N1 nucleotide of the sense strand and the antisense strand represents the 5'-end of each strand. For clarity, Formula III uses N1, N2, N3, etc. in both the sense and antisense strands, but the nucleotide bases do not need to be, and are not intended to be, the same. The siRNA depicted in Formula III is complementary to a target sequence.

[0122] For example, in some embodiments, the sense strand comprises 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones at N1 and N2, N3, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23, N24, N25, N26, N27, N28 12 , and N 17 2'-fluoro-modified nucleotides, as well as N4, N5, N6, and N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 , and N 19 contains 2'O-methyl modified nucleotides.

[0123] In some embodiments, the antisense strand comprises a vinyl phosphonate moiety attached to N1, a 2' fluoro-modified nucleotide with a phosphorothioate (PS)-modified backbone at N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N2 10 , N 11 , N 12 , N 13 , N 15 , N 16 , N 17 , N 18 , and N 19 2'O-methyl modified nucleotide, N 14 2' fluoro-modified nucleotides, as well as N 20and N 21 It contains 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones.

[0124] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a terminal cap moiety at the 5'-terminus, the 3'-terminus, or both the 5'-terminus and the 3'-terminus of the sense strand, hi other embodiments, the terminal cap moiety is an inverted deoxy abasic moiety.

[0125] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

[0126] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'-terminus and the 5'-terminus. The antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages and / or terminal cap molecules at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus, whether present on the same or different strands.

[0127] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand has from about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'-terminus and the 5'-terminus. and the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, an end-cap molecule at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages and / or 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus, whether present on the same or different strands.

[0128] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioates, phosphorodithioates, phosphonates, phosphoramidates, or methyl groups at the 3' end, the 5' end, or both the 3' and 5' ends of the sense strand and / or the antisense strand. The sense strand comprises sylphosphoramidate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the sense strand. In some embodiments, the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally an end cap molecule at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of the antisense strand.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus, whether present on the same or different strands.

[0129] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'-terminus and the 5'-terminus. and optionally, the antisense strand comprises about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with about one to about five or more, e.g., about 1, 2, 3, 4, 5, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages and / or 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus, whether present on the same or different strands.

[0130] In some embodiments, the oligonucleotide molecules described herein are chemically modified short interfering nucleic acid molecules having about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoamidate internucleotide linkages in each strand of the oligonucleotide molecule. In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and the antisense strand comprises a phosphate backbone modification at the 3'-end of the antisense strand. Alternatively and / or additionally, the oligonucleotide molecule comprises a sense strand and an antisense strand, and the sense strand comprises a phosphate backbone modification at the 5'-end of the antisense strand. In some cases, the phosphate backbone modification is phosphorothioate. In some cases, the phosphate backbone modification is phosphorodithioate. In some cases, the phosphate backbone modification is phosphonate. In some cases, the phosphate backbone modification is a phosphoramidate. In some cases, the phosphate backbone modification is a mesyl phosphoramidate. In some embodiments, the sense or antisense strand has three consecutive nucleosides coupled via two phosphorothioate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides coupled via two phosphorodithioate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides coupled via two phosphonate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides coupled via two phosphoramidate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides coupled via two mesyl phosphoramidate backbones.

[0131] In another embodiment, the oligonucleotide molecules described herein comprise a 2'-5' internucleotide linkage. In some cases, the 2'-5' internucleotide linkage(s) are at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In additional cases, the 2'-5' internucleotide linkage(s) are present at various other positions within one or both strands of the sequence, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, including all, of the pyrimidine nucleotide internucleotide linkages in one or both strands of the oligonucleotide molecule, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, including all, of the pyrimidine nucleotide internucleotide linkages in one or both strands of the oligonucleotide molecule, constitute a 2'-5' internucleotide linkage.

[0132] In some embodiments, the oligonucleotide molecule is a single-stranded molecule that mediates RNAi activity in a cell or a reconstituted in vitro system, wherein the oligonucleotide molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the oligonucleotide molecule are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the oligonucleotide molecule are 2'-deoxy purine nucleotides. and a terminal cap modification which may be present at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense oligonucleotide molecule, wherein the oligonucleotide molecule optionally further comprises about one to about four (e.g., about one, two, three, or four) terminal 2'-deoxynucleotides at the 3'-terminus of the oligonucleotide molecule, wherein the terminal nucleotides comprise one or more (e.g., one, two, three, or four) phosphorothioate or mesyl phosphoramidate internucleotide linkages, and the oligonucleotide molecule optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0133] In some cases, one or more of the synthetic nucleotide analogs described herein are resistant to nucleases, such as ribonucleases (e.g., RNase H), deoxyribonucleases (e.g., DNase), or exonucleases (e.g., 5'-3' exonucleases and 3'-5' exonucleases), when compared to naturally occurring polynucleic acid molecules and endonucleases. In some cases, synthetic nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are resistant to ribonucleases (e.g., RNase In some cases, 2'-O-methyl modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, 2'-O-aminopropyl modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some cases, 2'-deoxy modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, LNA modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, ENA modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant).In some cases, HNA-modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, morpholinos are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, PNA-modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, methylphosphonate-modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the thiol phosphonate-modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the oligonucleotide molecule comprising a 2'-fluoro N3-P5'-phosphoramidite is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the 5' conjugates described herein inhibit 5'-3' exonucleolytic cleavage. In some cases, the 3' conjugates described herein inhibit 3'-5' exonucleolytic cleavage.

[0134] In some embodiments, one or more of the synthetic nucleotide analogues described herein have increased binding affinity for their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. One or more synthetic nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, or 2'-fluoro N3-P5'-phosphoramidite, have increased binding affinity for their mRNA target compared to equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-O-methyl modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-O-aminopropyl modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-deoxy modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules.In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, LNA modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, ENA modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, PNA-modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, HNA-modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, morpholino-modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, methylphosphonate nucleotide-modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, thiolphosphonate nucleotide-modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, oligonucleotide molecules comprising 2'-fluoroN3-P5'-phosphoramidites have increased binding affinity to mRNA targets compared to comparable natural polynucleic acid molecules.In some cases, increased affinity is manifested by a lower Kd, an increased melting temperature (Tm), or a combination thereof.

[0135] In some embodiments, the oligonucleotide molecules described herein are chirally pure (or stereochemically pure) polynucleic acid molecules or polynucleic acid molecules comprising a single enantiomer. In some cases, the oligonucleotide molecules comprise L-nucleotides. In some cases, the oligonucleotide molecules comprise D-nucleotides. In some cases, the oligonucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of its mirror enantiomer. In some cases, the oligonucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of a racemic mixture.

[0136] In some embodiments, the oligonucleotide molecules described herein are further modified to include an aptamer conjugate portion.In some cases, the aptamer conjugate portion is a DNA aptamer conjugate portion.In some cases, the aptamer conjugate portion is an alphamer, which includes an aptamer portion that recognizes a specific cell surface target and a portion that presents a specific epitope for binding to circulating antibodies.

[0137] In additional embodiments, the oligonucleotide molecules described herein are modified to increase their stability. In some embodiments, the oligonucleotide molecule is RNA (e.g., siRNA). In some cases, the oligonucleotide molecule is modified with one or more of the above-mentioned modifications to increase its stability. In some cases, the oligonucleotide molecule is modified at the 2' hydroxyl position with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, or with a locked or bridged ribose conformation (e.g., LNA or ENA). In some cases, the oligonucleotide molecule is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the oligonucleotide molecule also contains morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to enhance its stability. In some cases, the oligonucleotide molecule is a chirally pure (or stereochemically pure) oligonucleotide molecule. In some cases, the chirally pure (or stereochemically pure) oligonucleotide molecule is modified to enhance its stability. Suitable modifications of RNA to enhance stability for delivery will be apparent to those skilled in the art.

[0138] In some embodiments, the oligonucleotide molecule comprises a 2' modification. In some embodiments, the nucleotides at positions 3, 7, 8, 9, 12, and 17 from the 5' end of the sense strand of the oligonucleotide molecule are not modified with a 2'O-methyl modification. In some embodiments, the nucleotides at positions 3, 7, 8, 9, 12, and 17 from the 5' end of the sense strand of the oligonucleotide molecule are modified with a 2'-fluoro modification. In some embodiments, the nucleotides at positions 2 and 14 from the 5' end of the antisense strand of the oligonucleotide molecule are not modified with a 2'O-methyl modification. In some embodiments, the nucleotides at positions 2 and 14 from the 5' end of the antisense strand of the oligonucleotide molecule are modified with a 2'-fluoro modification. In some embodiments, any of the nucleotides may further comprise a 5'-phosphorothioate group modification. In some embodiments, the nucleotides at positions 1 and 2 from the 5' end of the sense strand of the oligonucleotide molecule are modified with a 5'-phosphorothioate group modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 1, 2, 20, and 21 from the 5' end of the antisense strand are modified with a 5'-phosphorothioate group modification. In some embodiments, the 5' end of the sense or antisense strand of the oligonucleotide molecule can further comprise a vinyl phosphonate modification. In some embodiments, the nucleotide of the oligonucleotide molecule at position 1 from the 5' end of the antisense strand is modified with a vinyl phosphonate modification.

[0139] In some cases, the oligonucleotide molecule is a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some cases, the oligonucleotide molecule may be constructed from two separate polynucleotides, one strand being the sense strand and the other being the antisense strand, in which case the antisense and sense strands are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to the nucleotide sequence of the other strand, e.g., when the antisense and sense strands form a double-stranded or double-stranded structure, e.g., the double-stranded region is about 19, 20, 21, 22, 23, or more base pairs), the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the oligonucleotide molecule can be constructed from a single oligonucleotide, where the self-complementary sense and antisense regions of the oligonucleotide molecule are linked by a nucleic acid-based or non-nucleic acid-based linker(s).

[0140] In some cases, oligonucleotide molecule is the polynucleotide of double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure, and has self-complementary sense and antisense region, wherein antisense region comprises the nucleotide sequence complementary to the nucleic acid sequence of separate target nucleic acid molecule or its part, and sense region comprises the nucleic acid sequence corresponding to target nucleic acid sequence or its part.In other cases, oligonucleotide molecule is the circular single-stranded polynucleotide of stem, which comprises two or more loop structures and self-complementary sense and antisense region, wherein antisense region comprises the nucleic acid sequence complementary to the nucleic acid sequence of target nucleic acid molecule or its part, and sense region comprises the nucleic acid sequence corresponding to target nucleic acid sequence or its part, and circular polynucleotide is processed in vivo or in vitro to produce the active oligonucleotide molecule that can mediate RNAi. In additional cases, oligonucleotide molecules also include single-stranded polynucleotides comprising a nucleic acid sequence complementary to a nucleic acid sequence of a target nucleic acid molecule or a portion thereof (e.g., such oligonucleotide molecules need not be present within the oligonucleotide molecule of a nucleic acid sequence corresponding to the target nucleic acid sequence or a portion thereof), where the single-stranded polynucleotide further comprises a terminal phosphate group, such as a 5'-phosphate or 5',3'-diphosphate.

[0141] In some cases, an asymmetric hairpin is a linear oligonucleotide molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region, where the sense region has fewer nucleotides than the antisense region (to the extent that the sense region has sufficient complementary nucleotides to base pair with the antisense region to form a duplex with a loop). For example, an asymmetric hairpin oligonucleotide molecule comprises an antisense region having a length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 19 to about 22 nucleotides), a loop region comprising about 4 to about 8 nucleotides, and a region of about 3 to about 18 nucleotides that is complementary to the antisense region. In some cases, the asymmetric hairpin oligonucleotide molecule also comprises a chemically modified 5'-terminal phosphate group. In additional cases, the loop portion of the asymmetric hairpin oligonucleotide molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0142] In some embodiments, an asymmetric duplex is an oligonucleotide molecule having two separate strands, including a sense region and an antisense region, where the sense region has fewer nucleotides than the antisense region (to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region to form a duplex). For example, an asymmetric duplex oligonucleotide molecule includes an antisense region of sufficient length (e.g., about 19 to about 22 nucleotides) to mediate RNAi in a cell or in vitro system, and a region of about 3 to about 19 nucleotides that is complementary to the antisense region.

[0143] In some cases, the term "universal base" refers to a nucleotide base analogue that base pairs with each of the natural DNA / RNA bases with little discrimination. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole, as are well known in the art.

[0144] In some embodiments, the dsRNA agent is 5' phosphorylated or contains a phosphoryl analog at the 5' end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate (HO2(O)P--O-5'); 5'-diphosphate ((HO)2(O)P--OP(HO)(O)--O-5'); 5'-triphosphate ((HO)2(O)P--O-(HO)(O)P--OP(HO)(O)--O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O--5'); 5'-adenosine cap (Appp), and any modifications. Modified or unmodified nucleotide cap structures (N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'), 5'-phosphorothiolate ((HO)2(O)P--S-5'); phosphorodithioate [--O2PS2-]; phosphonate [--PO(OH) 2-]; phosphoramidates [--O=P(OH)2--]; mesyl phosphoramidate (CH3)(SO2)(N)P(O)2--O-5'; oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R=alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenyl phosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)--O-5'-). In some embodiments, modifications can be placed on the antisense strand of a dsRNA agent.

[0145] Other modifications and modification patterns can be found, for example, in U.S. Patent No. 10,233,448, which is incorporated herein by reference. Other modifications and modification patterns can be found, for example, in Anderson et al., Nucleic Acids Research 2021, 49(16), 9026-9041, which is incorporated herein by reference. Other modifications and modification patterns can be found, for example, in PCT Publication No. WO2021 / 030778, which is incorporated herein by reference. Other modifications and modification patterns can be found, for example, in PCT Publication No. WO2021 / 030763, which is incorporated herein by reference.

[0146] In some embodiments, the sequence of the oligonucleotide molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence of CD40. In some embodiments, the target sequence of CD40 is a nucleic acid sequence of about 10-50 base pairs, about 15-50 base pairs, 15-40 base pairs, 15-30 base pairs, or 15-25 base pairs in length in CD40, wherein the first nucleotide of the target sequence begins with any nucleotide of the CD40 mRNA transcript within the coding region or the 5' or 3' untranslated region (UTR). For example, the first nucleotide of the target sequence can be selected to start at nucleic acid position (nal, a number starting from the 5' end of the full length of CD40 mRNA, e.g., the first nucleotide at the 5' end is nal 1) 1, nal 2, nal 3, nal 4, nal 5, nal 6, nal 7, nal 8, nal 9, nal 10, nal 11, nal 12, nal 13, nal 14, nal 15, nal 16, nal 17, or any other nucleic acid position within the coding or non-coding region (5' or 3' untranslated region) of CD40 mRNA. In some embodiments, the first nucleotide of the target sequence is nal10 to nal15, nal10 to nal20, nal50 to nal60, nal55 to nal65, nal75 to nal85, nal95 to nal105, nal135 to nal145, nal155 to nal165, nal225 to nal235, nal265 to nal275, nal275 to nal245, nal245 to nal255, nal285 to nal335, nal335 to nal345, nal385 to nal395, nal515 to nal525, nal665 to nal675, nal675 to nal685, nal695 to nal705, nal705 to nal715, nal875 to nal 885, nal 885~nal 895, nal 895~nal 905, nal 1035~nal 1045, nal 1045~nal 1055, nal 1125~nal 1135, nal 1135~nal 1145, nal1145~nal 1155, nal 1155~nal 1165, nal 1125~nal 1135, nal 1155~nal 1165, nal 1225~nal 1235, nal 1235~nal 1245, nal 1275~nal 1245, nal 1245~nal 1255, nal 1265~nal 1275, nal 1125~nal 1135, nal 1155~nal 1165, nal 1225~nal 1235, nal 1235~nal 1245, nal 1275~nal 1245, nal 1245~nal 1255, nal 1265~nal 1275, nal 1275~nal 1285, nal 1335~nal 1345, nal 1345~nal 1355, nal 1525~nal 1535, nal 1535~nal 1545, nal 1605~nal 1615, nal 1615~c.1625, nal 1625~nal 1635, nal 1635~1735, nal 1735~1835, nal 1835~1935, nal.1836~1856, nal 1935~2000, nal 2000~2100, nal 2100~2200, nal 2200~2260, nal 2260~2400, nal 2400~2500, nal 2500~2600, nal 2600~2700, nal The sequence may be selected to start at or between positions within the ranges of 2700-2800, 2800-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2860, etc. In some embodiments, the sequence of CD40 mRNA is provided as NCBI Reference Sequence: NM_001250.6 Homo sapiens CD40 molecule (CD40), transcript variant 1, mRNA:

[0147] In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA, hybridizes to the target RNA, and inhibits its expression by RNA interference. The target RNA can be any RNA expressed in a cell. In another embodiment, the cell is a tumor cell, liver cell, muscle cell, immune cell, cardiac cell, or cell of the central nervous system. In some embodiments, the antisense strand of the dsRNA agent is at least 99%, at least 98%, at least 97%, at least 96%, 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA. In some embodiments, the target RNA is CD40 RNA. In some embodiments, the siRNA molecule is an siRNA that reduces CD40 mRNA expression. In some embodiments, the siRNA molecule is an siRNA that reduces CD40 mRNA expression and does not reduce the expression of other RNAs by more than 50% in the assays described herein at a concentration of 200 nM or less as described herein.

[0148] In some embodiments, siRNA is linked to protein such as FN3 domain.SiRNA can be linked to multiple FN3 domains that bind to the same target protein or different target proteins.In some embodiments, linker is linked to sense strand, which is used to facilitate the linking of sense strand to FN3 domain.

[0149] In some embodiments, (X1) n -(X2) q -(X3) yProvided herein are compositions having the formula: -L-X4, where X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is an FN3 domain or a half-life extending molecule, L is a linker, and X4 is a nucleic acid molecule, such as, but not limited to, an siRNA molecule, where n, q, and y are each independently 0 or 1. In some embodiments, X1, X2, and X3 bind to different target proteins. In some embodiments, y is 0. In some embodiments, n is 1, q is 0, and y is 0. In some embodiments, n is 1, q is 1, and y is 0. In some embodiments, n is 1, q is 1, and y is 0. In some embodiments, n is 1, q is 1, and y is 1. In some embodiments, X3 increases the half-life of the overall molecule compared to the molecule without X3. In some embodiments, the half-life extending moiety is an FN3 domain that binds to albumin. Examples of such FN3 domains include, but are not limited to, those described in U.S. Patent Application Publication No. 2017 / 0348397 and U.S. Patent No. 9,156,887, which are incorporated herein by reference in their entireties. An FN3 domain may incorporate other subunits, for example, through covalent interactions. In some embodiments, an FN3 domain further comprises a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and / or domains, aliphatic chains that bind to serum proteins, transferrin and its fragments and analogs, and an Fc region. The amino acid sequence of the human Fc region is well known and includes IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE Fc regions. In some embodiments, an FN3 domain may incorporate a second FN3 domain that binds to a molecule that extends the half-life of the overall molecule, such as, but not limited to, any of the half-life extending moieties described herein. In some embodiments, the second FN3 domain binds to albumin, albumin variants, albumin binding proteins and / or domains, and fragments and analogs thereof.

[0150] In some embodiments, provided herein are compositions having the formula (X1)-(X2)-L-(X4), where X1 is a first FN3 domain, X2 is a second FN3 domain, L is a linker, and X4 is a nucleic acid molecule. In some embodiments, X4 is an siRNA molecule. In some embodiments, X1 is an FN3 domain that binds to CD71. In some embodiments, X2 is an FN3 domain that binds to CD71. In some embodiments, X1 and X2 do not bind to the same target protein. In some embodiments, X1 and X2 bind to the same target protein but at different binding sites on the protein. In some embodiments, X1 and X2 bind to the same target protein. In some embodiments, X1 and X2 are FN3 domains that bind to CD71. In some embodiments, the composition does not include (e.g., is free of) a compound or protein that binds to ASGPR.

[0151] In some embodiments, C—(X1) n -(X2) q [L-X4]-(X3) y wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension molecule, L is a linker, X4 is an oligonucleotide molecule, and C is a polymer; and wherein n, q, and y are each independently 0 or 1.

[0152] In some embodiments, (X1) n -(X2) q [L-X4]-(X3) y -C, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension molecule, L is a linker, X4 is an oligonucleotide molecule, and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0153] In some embodiments, C—(X1) n -(X2) q [L-X4]L-(X3) y wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension molecule, L is a linker, X4 is an oligonucleotide molecule, and C is a polymer; and wherein n, q, and y are each independently 0 or 1.

[0154] In some embodiments, (X1) n -(X2) q [L-X4]L-(X3) y -C, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension molecule, L is a linker, X4 is an oligonucleotide molecule, and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0155] In some embodiments, compositions or conjugates are provided having a formula of A1-B1, where A1 is C-L1-X s and B1 has the formula X AS -L2-F1, wherein C is a polymer such as PEG, L1 and L2 are each independently a linker; X S is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule, X AS is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule, F1 is a polypeptide comprising at least one FN3 domain; X S and X AS form a double-stranded oligonucleotide molecule to form the composition / complex.

[0156] In some embodiments, a composition or complex is provided having a formula of A1-B1, where A1 is X s and B1 has the formula X AS -L2-F1.

[0157] In some embodiments, compositions or conjugates are provided having a formula of A1-B1, where A1 is C-L1-X s and B1 has the formula X AS It has the formula:

[0158] In some embodiments, the sense strand is a sense strand provided herein. In some embodiments, the antisense strand is an antisense strand provided herein. In some embodiments, the sense and antisense strands form a double-stranded siRNA molecule targeting CD40. In some embodiments, the double-stranded oligonucleotide is about 21-23 nucleotide base pairs in length. In certain embodiments, C is optional.

[0159] In some embodiments, compositions or conjugates are provided having a formula of A1-B1, where A1 is F1-L1-X s and B1 has the formula X AS -L2-C, wherein F1 is a polypeptide comprising at least one FN3 domain; L1 and L2 are each independently a linker; C is a polymer such as PEG, X S is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule, X AS is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule, X S and X AS forms a double-stranded oligonucleotide molecule to form the composition / complex. In certain embodiments, C is optional.

[0160] In some embodiments, a composition or complex is provided having a formula of A1-B1, where A1 is X s and B1 has the formula X AS -L2-C.

[0161] In some embodiments, compositions or conjugates are provided having a formula of A1-B1, where A1 is F1-L1-X s and B1 has the formula X AS It has the formula:

[0162] In some embodiments, A1 and B1 interact with each other via hydrogen bonding. In some embodiments, A1 and B1 interact with each other via Watson-Crick base pairing.

[0163] In some embodiments, the composition describes a polymer (polymer moiety C, or simply C). In some embodiments, C can be a molecule that extends the half-life of the molecule. In some embodiments, the polymer is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of two- or three-dimensional monomers. In some cases, the polymer comprises a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some cases, the at least one polymer includes, but is not limited to, alpha-, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane, and mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound, as well as to block copolymers. In some cases, a block copolymer is a polymer in which at least a portion of the polymer is constructed from monomers of another polymer. In some cases, the polymer comprises polyalkylene oxide. In some cases, the polymer comprises PEG. In some cases, the polymer comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0164] In some embodiments, C is a PEG moiety. In some embodiments, the PEG moiety is conjugated at the 5' end of the oligonucleotide molecule, while the linking moiety is conjugated at the 3' end of the oligonucleotide molecule. In some embodiments, the PEG moiety is conjugated at the 3' end of the oligonucleotide molecule, while the linking moiety is conjugated at the 5' end of the oligonucleotide molecule. In some embodiments, the PEG moiety is conjugated to an internal site of the oligonucleotide molecule. In some embodiments, the PEG moiety, the linking moiety, or a combination thereof, is conjugated to an internal site of the oligonucleotide molecule. In some embodiments, the conjugation is direct conjugation. In some embodiments, the conjugation is by native ligation.

[0165] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some embodiments, a polydisperse material comprises a disperse distribution of different molecular weights of material, characterized by average weight (weight average) size and dispersity. In some embodiments, a monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight given represents the average molecular weight of the polyalkylene oxide, e.g., PEG, molecules.

[0166] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 83 00, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0167] In some embodiments, C is a polyalkylene oxide (e.g., PEG) having a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 260 0, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and its molecular weight is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, In some embodiments, the molecular weight of C is about 200 Da. In some embodiments, the molecular weight of C is about 300 Da. In some embodiments, the molecular weight of C is about 400 Da. In some embodiments, the molecular weight of C is about 500 Da. In some embodiments, the molecular weight of C is about 600 Da. In some embodiments, the molecular weight of C is about 700 Da. In some embodiments, the molecular weight of C is about 800 Da. In some embodiments, the molecular weight of C is about 900 Da. In some embodiments, the molecular weight of C is about 1000 Da. In some embodiments, the molecular weight of C is about 1100 Da. In some embodiments, the molecular weight of C is about 1200 Da. In some embodiments, the molecular weight of C is about 1300 Da.In some embodiments, the molecular weight of C is about 1400 Da. In some embodiments, the molecular weight of C is about 1450 Da. In some embodiments, the molecular weight of C is about 1500 Da. In some embodiments, the molecular weight of C is about 1600 Da. In some embodiments, the molecular weight of C is about 1700 Da. In some embodiments, the molecular weight of C is about 1800 Da. In some embodiments, the molecular weight of C is about 1900 Da. In some embodiments, the molecular weight of C is about 2000 Da. In some embodiments, the molecular weight of C is about 2100 Da. In some embodiments, the molecular weight of C is about 2200 Da. In some embodiments, the molecular weight of C is about 2300 Da. In some embodiments, the molecular weight of C is about 2400 Da. In some embodiments, the molecular weight of C is about 2500 Da. In some embodiments, the molecular weight of C is about 2600 Da. In some embodiments, the molecular weight of C is about 2700 Da. In some embodiments, the molecular weight of C is about 2800 Da. In some embodiments, the molecular weight of C is about 2900 Da. In some embodiments, the molecular weight of C is about 3000 Da. In some embodiments, the molecular weight of C is about 3250 Da. In some embodiments, the molecular weight of C is about 3350 Da. In some embodiments, the molecular weight of C is about 3500 Da. In some embodiments, the molecular weight of C is about 3750 Da. In some embodiments, the molecular weight of C is about 4000 Da. In some embodiments, the molecular weight of C is about 4250 Da. In some embodiments, the molecular weight of C is about 4500 Da. In some embodiments, the molecular weight of C is about 4600 Da. In some embodiments, the molecular weight of C is about 4750 Da. In some embodiments, the molecular weight of C is about 5000 Da. In some embodiments, the molecular weight of C is about 5500 Da. In some embodiments, the molecular weight of C is about 6000 Da. In some embodiments, the molecular weight of C is about 6500 Da. In some embodiments, the molecular weight of C is about 7000 Da. In some embodiments, the molecular weight of C is about 7500 Da.In some embodiments, the molecular weight of C is about 8000 Da. In some embodiments, the molecular weight of C is about 10,000 Da. In some embodiments, the molecular weight of C is about 12,000 Da. In some embodiments, the molecular weight of C is about 20,000 Da. In some embodiments, the molecular weight of C is about 35,000 Da. In some embodiments, the molecular weight of C is about 40,000 Da. In some embodiments, the molecular weight of C is about 50,000 Da. In some embodiments, the molecular weight of C is about 60,000 Da. In some embodiments, the molecular weight of C is about 100,000 Da.

[0168] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, where the discrete PEG is a polymeric PEG containing more than one repeating ethylene oxide unit. In some embodiments, the discrete PEG (dPEG) contains 2-60, 2-50, or 2-48 repeating ethylene oxide units. In some embodiments, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 2 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 3 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 4 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 5 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 6 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 7 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 8 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 9 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 10 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 11 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 12 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 13 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 14 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 15 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 16 or more repeating ethylene oxide units.In some embodiments, dPEG contains about 17 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 18 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 19 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 20 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 22 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 24 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 26 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 28 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 30 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 35 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 40 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 42 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 48 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 50 or more repeating ethylene oxide units. In some embodiments, the dPEG is synthesized stepwise from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some cases, the dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0169] In some embodiments, C is an albumin-binding domain. In some embodiments, the albumin-binding domain specifically binds to serum albumin, e.g., human serum albumin (HSA), and extends the half-life of the domain or another therapeutic agent with which the albumin-binding domain is associated or linked. In some embodiments, the human serum albumin-binding domain comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the human serum albumin-binding domain comprises a cysteine ​​(Cys) linked to the C-terminus or N-terminus of the domain. The addition of the N-terminal Met and / or C-terminal Cys can facilitate expression and / or conjugation to another molecule, which may be another half-life-enhancing molecule, e.g., PEG, an Fc region, etc.

[0170] In some embodiments, the albumin binding domain comprises the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 shown in Table 1. In some embodiments, the albumin binding domain (protein) is isolated. In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the albumin binding domain is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. or 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, with the proviso that the protein has a substitution corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T, or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid. In some embodiments, the isolated albumin binding domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions compared to the amino acid sequence of SEQ ID NO: ... 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitutions are at a position corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.In some embodiments, the provided FN3 domains contain a cysteine ​​residue at at least one residue position corresponding to residues 6, 11, 22, 25, 26, 52, 53, 61, 88, or 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, or 93 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, or at the C-terminus. Although the positions are listed consecutively, each position may also be selected individually. In some embodiments, the cysteine ​​is at position 6, 53, or 88. In some embodiments, further examples of albumin binding domains can be found in U.S. Patent No. 10,925,932, which is incorporated herein by reference in its entirety. In some embodiments, further examples of albumin binding domains are described in U.S. Patent Nos. 8,969,289, 9,540,424, 10,221,438, 10,934,572, 10,442,851, 11,203,630, 10,766,946, 11,434,275, and U.S. Publication Nos. 2022 / 0204589 and 2023 / 0145413, each of which is incorporated herein by reference in its entirety. [Table 1]

[0171] In some embodiments, C can also be an endoporter, INF-7, TAT, polyarginine, polylysine, or an amphipathic peptide. These moieties can be used in place of or in addition to other half-life extending moieties provided herein. In some embodiments, C can be a molecule that delivers the complex into a cell, endosome, or ER, and the molecule is selected from the peptides listed in Table 2. [Table 2]

[0172] In some embodiments, L1 connects polymer C to sense strand X S or to link the polypeptide of F1 to the sense strand X S In some embodiments, L1 has the formula: [ka] In the formula, X S , X AS , and F1 are as defined above.

[0173] In some embodiments, n=0-20. In some embodiments, R and R1 are independently methyl. In some embodiments, R and R1 are independently present or both absent. In some embodiments, X and Y are independently S. In some embodiments, X and Y are independently present or absent. In some embodiments, the peptide is an enzymatically cleavable peptide, such as, but not limited to, Val-Cit, Val-Ala, etc.

[0174] In some embodiments, L2 binds the polypeptide of F1 to the antisense strand X AS or polymer C to link to antisense strand X AS is any linker that can be used to link

[0175] In some embodiments, L2 has the formula of the complex: [ka] In the formula, X AS , and F1 are as defined above.

[0176] In some embodiments, n=0-20. In some embodiments, R and R1 are independently methyl. In some embodiments, R and R1 are independently present or both absent. In some embodiments, X and Y are independently S. In some embodiments, X and Y are independently present or absent. In some embodiments, the peptide is an enzymatically cleavable peptide, such as, but not limited to, Val-Cit, Val-Ala, etc.

[0177] In some embodiments, the linker is covalently attached to F1 via a cysteine ​​residue present on F1, which can be exemplified as follows: [ka] In the formula, X S is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule, and X AS is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule, F1 is a polypeptide containing at least one FN3 domain, and wherein X S and X AS form a double-stranded siRNA molecule.

[0178] In some embodiments, A1-B1 have the formula: [ka] wherein C is a polymer such as PEG, endopoietin, INF-7, TAT, polyarginine, polylysine, an amphipathic peptide, or as provided herein, and F1 is a polypeptide comprising at least one FN3 domain. The sense and antisense strands are represented by the "N" designation, where each nucleotide represented by N is independently A, U, C, or G, or a modified nucleobase such as those provided herein. The N1 nucleotide of the sense and antisense strands represents the 5'-end of each strand. For clarity, Formula III utilizes N1, N2, N3, etc. in both the sense and antisense strands, but the nucleotide bases need not be, and are not intended to be, the same. The siRNA depicted in Formula III is complementary to the target sequence. For example, in some embodiments, the sense strand contains 2'O-methyl modified nucleotides with phosphorothioate (PS)-modified backbones at N1 and N2, N3, N7, N8, N9, N 12 , and N 17 2'-fluoro-modified nucleotides, as well as N4, N5, N6, and N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 , and N 19 contains 2'O-methyl modified nucleotides.

[0179] In some embodiments, the antisense strand comprises a vinyl phosphonate moiety attached to N1, a 2' fluoro-modified nucleotide with a phosphorothioate (PS)-modified backbone at N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23, N24, N25, N26, N27, N28, N29, N30, N31, N32, N33, N34, N35, N36, N37, N38, N39, N40, N41, N42, N43, N44, N45, N46, N47, N48, N49, N50, N51, N52, N53, N54, N5 10 , N 11 , N 12 , N 13 , N 15 , N 16 , N 17 , N 18 , and N 19 2'O-methyl modified nucleotide, N 14 2' fluoro-modified nucleotides, as well as N 20 and N 21It contains 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones.

[0180] In some embodiments, the formula: [ka] Compounds having the formula:

[0181] In some embodiments, the formula: [ka] wherein F1 is a polypeptide comprising at least one FN3 domain and is conjugated to a linker. The linkers illustrated above are non-limiting examples, and other types of linkers can be used.

[0182] In some embodiments, F1 is (X1) n -(X2) q -(X3) y wherein X1 is a first FN3 domain, X2 is a second FN3 domain, and X3 is a third FN3 domain or a half-life extension, and wherein n, q, and y are each independently 0 or 1, with the proviso that at least one of n, q, and y is 1. In some embodiments, n, q, and y are each 1. In some embodiments, n and q are each 1 and y is 0. In some embodiments, n and y are 1 and q is 0.

[0183] In some embodiments, X1 is a CD71-binding FN3 domain as provided herein. In some embodiments, X2 is a CD71-binding FN3 domain. In some embodiments, X1 and X2 are different CD71-binding FN3 domains. In some embodiments, the binding domains are the same. In some embodiments, X3 is an FN3 domain that binds human serum albumin. In some embodiments, X3 is an Fc domain without effector function that extends the half-life of the protein. In some embodiments, X1 is a first CD71-binding FN3 domain, X2 is a second CD71-binding FN3 domain, and X3 is an albumin-binding FN3 domain. Other examples of such polypeptides are provided herein and below. In some embodiments, C-(X1) n -(X2) q -(X3) y Provided herein are compositions having the formula: -L-X4, where C is a polymer such as PEG, endoporter, INF-7, TAT, polyarginine, polylysine, an amphipathic peptide, or a peptide shown in Table 2; X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1.

[0184] In some embodiments, (X1) n -(X2) q -(X3) y Provided herein are compositions having the formula -L-X4-C, where X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension, L is a linker, X4 is a nucleic acid molecule, and C is a polymer, and where n, q, and y are each independently 0 or 1.

[0185] In some embodiments, X-L-(X) n -(X2) q -(X3) ywherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension molecule, L is a linker, and X4 is a nucleic acid molecule, and wherein n, q, and y are each independently 0 or 1.

[0186] In some embodiments, C-X-L-(X) n -(X2) q -(X3) y wherein C is a polymer; X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension; L is a linker; and X4 is a nucleic acid molecule; and wherein n, q, and y are each independently 0 or 1.

[0187] In some embodiments, X-L-(X) n -(X2) q -(X3) y -C, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extension, L is a linker, X4 is a nucleic acid molecule, and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0188] In some embodiments, CD40-binding siRNA molecules comprise a pair of sequences that may follow the sequence: sense strand (5'-3')nsnsnnnnNfNfNfnnnnnnnnsnsa or (5'-3')nsnsnnnnNfNfNfnnnnnnnnna, and antisense strand (5'-3')UfsNfsnnNfnnnnnnNfnNfnnnsusu, where (n) is 2'-O-Me (methyl), (Nf) is 2'-F (fluoro), and (s) is a phosphorothioate backbone modification. Each nucleotide in both the sense and antisense strands may be modified at the ribosugar and nucleobase positions, independently or in combination.

[0189] In some embodiments, the siRNA molecule comprises a sequence pair from Table 3A, Table 3B, Table 4A, or Table 4B. In some embodiments, Tables 5A and 5B illustrate non-limiting examples of sequence pairs where the sense strand comprises a linker molecule. In some embodiments, any siRNA molecule provided herein can comprise a linker molecule disclosed herein.

[0190] In some embodiments, the siRNA molecule comprises a sense strand comprising the nucleic acid sequence of SEQ ID NO: 1890 and an antisense strand comprising the nucleic acid sequence of SEQ ID NO: 2290. In some embodiments, the siRNA molecule comprises a linker at the 3' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the sequence pair H11 set forth in Table 5B.

[0191] In some embodiments, the siRNA molecule comprises a sense strand comprising the nucleic acid sequence of SEQ ID NO: 1893 and an antisense strand comprising the nucleic acid sequence of SEQ ID NO: 2293. In some embodiments, the siRNA molecule comprises a linker at the 3' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the sequence pair K11 listed in Table 5B.

[0192] In some embodiments, the siRNA molecule comprises a sense strand comprising the nucleic acid sequence of SEQ ID NO: 1941 and an antisense strand comprising the nucleic acid sequence of SEQ ID NO: 2051. In some embodiments, the siRNA molecule comprises a linker at the 5' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises sequence pair O10 listed in Table 5B.

[0193] In some embodiments, the siRNA molecule comprises a sense strand comprising the nucleic acid sequence of SEQ ID NO: 1942 and an antisense strand comprising the nucleic acid sequence of SEQ ID NO: 2052. In some embodiments, the siRNA molecule comprises a linker at the 5' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises sequence pair P10 listed in Table 5B.

[0194] In some embodiments, the siRNA molecule comprises a sense strand comprising the nucleic acid sequence of SEQ ID NO: 1944 and an antisense strand comprising the nucleic acid sequence of SEQ ID NO: 2054. In some embodiments, the siRNA molecule comprises a linker at the 5' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the sequence pair R10 set forth in Table 5B. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10]

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

[0195] In some embodiments, the polynucleotides exemplified above include those that do not contain 2'-O methylvinylphosphonate uridine as the 5' nucleotide on the antisense strand of the siRNA.

[0196] In some embodiments, the polynucleotide is as provided herein. In some embodiments, the polynucleotide comprises a first strand and a second strand of the duplex portion. In some embodiments, the polynucleotide comprises a sense strand and an antisense strand. In some embodiments, the polynucleotide comprises a sequence exemplified in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, or Table 5B. In some embodiments, the polynucleotide comprises a sequence shown in Table 3A, Table 4A, or Table 5A, but without base modifications. In some embodiments, the polynucleotide comprises an siRNA pair provided herein. In some embodiments, the siRNA pair is not conjugated to an FN3 domain.

[0197] In some embodiments, the oligonucleotide molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, the oligonucleotide molecules are chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the oligonucleotide molecule and the target nucleic acid. Alternatively, the oligonucleotide molecules are biologically produced using an expression vector into which the oligonucleotide molecule has been subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted oligonucleotide molecule is in an antisense orientation relative to the target polynucleic acid molecule of interest).

[0198] In some embodiments, the oligonucleotide molecules are synthesized by tandem synthesis, where both strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize to allow purification of the duplex.

[0199] In some cases, oligonucleotide molecules are also assembled from two separate nucleic acid strands or fragments, one fragment comprising the sense region of the molecule and the other fragment comprising the antisense region of the molecule.

[0200] In some cases, the chemical modification of the internucleotide linkage of oligonucleotide molecules with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate or mesylphosphoramidate improves the stability of linkage.Excessive modification can sometimes cause toxicity or reduced activity.Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages can be minimized in some cases.In such cases, reducing the concentration of these linkages can reduce toxicity, increase the effectiveness of these molecules, and make them more specific.

[0201] As described herein, in some embodiments, any nucleic acid molecule disclosed herein can be modified to include a linker at the 5'-end of the sense strand of the dsRNA. In some embodiments, any nucleic acid molecule disclosed herein can be modified to include a vinyl phosphonate or modified vinyl phosphonate at the 5'-end of the antisense strand of the dsRNA. In some embodiments, any nucleic acid molecule disclosed herein can be modified to include a linker at the 3'-end of the sense strand of the dsRNA. In some embodiments, any nucleic acid molecule disclosed herein can be modified to include a vinyl phosphonate at the 3'-end of the antisense strand of the dsRNA. A linker can be used to link the dsRNA to the FN3 domain. The linker can be, for example, covalently attached to a cysteine ​​residue on the FN3 domain, whether naturally occurring or substituted as described herein, as described, for example, in U.S. Patent No. 10,196,446, the entire contents of which are incorporated herein by reference.

[0202] In some embodiments, the siRNA pair of A1-W6 and B7-G11 shown in Tables 3A and 4A provided above comprises a linker at the 3'-end of the sense strand. In some embodiments, the siRNA pair of A1-W6 and B7-G11 shown in Tables 3A and 4A provided above comprises a vinyl phosphonate at the 5'-end of the sense strand.

[0203] Non-limiting examples of linker (L) structures are illustrated in Tables 6A and 6B below. [Table 9] [Table 10]

[0204] Other linkers can also be used, such as linkers formed by click chemistry, amide coupling, reductive amination, oxime, and enzymatic coupling, e.g., transglutaminase and sorting conjugation. The linkers provided herein are exemplary in nature, and other linkers made by other methods can also be used. For example, linkers connected via phosphate groups can be phosphorothioates or phosphorodithioates.

[0205] When connected to an siRNA, the structure L-(X4) can be represented by either of the following formulas: [ka]

[0206] Although specific siRNA sequences are exemplified herein with specific modified nucleobases, sequences without such modifications are also provided herein. That is, the sequence can include the sequence shown in the table provided herein without any modification. In some embodiments, unmodified siRNA sequences can further include a linker at the 5'-end of the sense strand of dsRNA. In some embodiments, nucleic acid molecules can be modified to include a vinyl phosphonate at the 5'-end of the antisense strand of dsRNA. In some embodiments, nucleic acid molecules can be modified to include a linker at the 3'-end of the sense strand of dsRNA. In some embodiments, nucleic acid molecules can be modified to include a vinyl phosphonate at the 3'-end of the antisense strand of dsRNA. The linker can be as provided herein.

[0207] In some embodiments, an FN3 protein is provided comprising a polypeptide that binds to CD71. In some embodiments, the polypeptide comprises an FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849. In some embodiments, the polypeptide that binds to CD71 comprises the sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849. The sequence of the CD71 protein to which the polypeptide can bind can be, for example, SEQ ID NO: 3 or 4. In some embodiments, the CD71-binding FN3 domain specifically binds to CD71.

[0208] In some embodiments, the FN3 domain that binds to CD71 is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon 27 sequence of SEQ ID NO: 2 (LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT), optionally with substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 2).

[0209] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849.

[0210] In some embodiments, a protein is provided comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 360. SEQ ID NO: 360 is a consensus sequence based on the sequences of SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, and SEQ ID NO: 364. The sequence of SEQ ID NO: 360 is MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 X 13 VKGGX14 X 15 SX 16 PLX 17 AX 18 FTT, Here, X8, X9, X 17 , and X 18 are each independently any amino acid other than methionine or proline; X1 is selected from D, F, Y, or H; X2 is selected from Y, G, A, or V; X3 is selected from I, T, L, A, or H; X4 is selected from S, Y, or P; X5 is selected from Y, G, Q, or R; X6 is selected from G or P; X7 is selected from A, Y, P, D, or S; X 10 is selected from W, N, S, or E; X 11 is selected from L, Y, or G; X 12 is selected from D, Q, H, or V; X 13 is selected from G or S, X 14 is selected from R, G, F, L, or D, X 15 is selected from W, S, P, or L, and X 16 is selected from T, V, M, or S.

[0211] In some embodiments, X1 is selected from D, F, Y, or H; X2 is selected from G, A, or V; X3 is selected from T, L, A, or H; X4 is selected from Y or P; X5 is selected from G, Q, or R, X6 is selected from G or P; X7 is selected from Y, P, D, or S, X 10 is selected from W, N, S, or E; X 11 is selected from L, Y, or G; X 12 is selected from Q, H, or V; X 13 is selected from G or S, X 14 is selected from G, F, L, or D; X 15 is selected from S, P, or L, and X 16 is selected from V, M, or S.

[0212] In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 are as set forth in sequence SEQ ID NO: 361. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 are as set forth in sequence SEQ ID NO: 362. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 are as set forth in sequence SEQ ID NO: 363. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 is as shown in the sequence of SEQ ID NO:364.

[0213] In some embodiments, X8, X9, X 17 , and X 18 are independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 17 , and X 18 are not independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 17 , and X 18 are independently alanine. In some embodiments, X, X, X 17 , and X 18 are independently arginine. In some embodiments, X, X, X 17 , and X 18 are independently asparagine. In some embodiments, X, X, X 17 , and X 18 are independently aspartic acid. 17 , and X 18 are independently cysteine. In some embodiments, X, X, X 17 , and X 18 are independently glutamine. In some embodiments, X, X, X 17 , and X 18 are independently glutamic acid. In some embodiments, X, X, X 17 , and X 18 are independently glycine. In some embodiments, X, X, X 17 , and X 18 are independently histidine. In some embodiments, X, X, X 17 , and X18 are independently isoleucine. In some embodiments, X, X, X 17 , and X 18 are independently leucine. In some embodiments, X, X, X 17 , and X 18 are independently lysine. In some embodiments, X, X, X 17 , and X 18 are independently phenylalanine. In some embodiments, X, X, X 17 , and X 18 are independently serine. In some embodiments, X, X, X 17 , and X 18 are independently threonine. In some embodiments, X, X, X 17 , and X 18 are independently tryptophan. In some embodiments, X, X, X 17 , and X 18 are independently tyrosine. In some embodiments, X, X, X 17 , and X 18 is, independently, valine.

[0214] In some embodiments, the sequence is as set forth in SEQ ID NO: 361, except that X8, X9, X 17 , and X 18 The positions corresponding to positions X8, X9, and X9 may be any other amino acid listed above (except that in some embodiments, X8 is not V, X9 is not T, and X 17 is not S, but X 18 is not I).

[0215] In some embodiments, the sequence is as set forth in SEQ ID NO: 362, except that X8, X9, X 17 , and X 18 The positions corresponding to positions X8, X9, and X9 may be any other amino acid listed above (except that in some embodiments, X8 is not V, X9 is not T, and X 17 is not S, but X 18 is not I).

[0216] In some embodiments, the sequence is as set forth in SEQ ID NO: 363, except that X8, X9, X 17 , and X 18 The positions corresponding to positions X8, X9, and X9 may be any other amino acid listed above (except that in some embodiments, X8 is not V, X9 is not T, and X 17 is not S, but X 18 is not I).

[0217] In some embodiments, the sequence is as set forth in SEQ ID NO: 364, except that X8, X9, X 17 , and X 18 The positions corresponding to positions X8, X9, and X9 may be any other amino acid listed above (except that in some embodiments, X8 is not V, X9 is not T, and X 17 is not S, but X 18 is not I).

[0218] In some embodiments, the protein comprises a polypeptide comprising an amino acid sequence that is at least 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 360. In some embodiments, the protein is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360. In some embodiments, the protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360. In some embodiments, the protein is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360.

[0219] The sequences of SEQ ID NOs: 361 to 364 are listed in Table 7 below. [Table 11]

[0220] Percent identity can be determined by aligning the two sequences using BlastP, available on the NCBI website, using default parameters.

[0221] As provided herein, in some embodiments, the FN3 domain that binds to CD71 binds to human mature CD71 or the human mature CD71 extracellular domain. In some embodiments, the human mature CD71 is SEQ ID NO: 3 and the human mature CD71 extracellular binding domain is SEQ ID NO: 4, each of which is shown in Table 8 below. [Table 12]

[0222] As provided herein, FN3 domains can bind to CD71 protein. Even if not explicitly stated, it is also provided that a domain can specifically bind to CD71 protein. Thus, for example, an FN3 domain that binds to CD71 also encompasses FN3 domain proteins that specifically bind to CD71. These molecules can be used, for example, for therapeutic and diagnostic applications, as well as imaging. In some embodiments, polynucleotides encoding the FN3 domains disclosed herein, or their complementary nucleic acids, vectors, host cells, and methods of making and using them, are provided. In some embodiments, isolated FN3 domains that bind to or specifically bind to CD71 are provided.

[0223] In some embodiments, the FN3 domain has a molecular weight of about 1×10 as determined by surface plasmon resonance or Kinexa methods performed by one skilled in the art. -7 Less than M, e.g., about 1 x 10-8 Less than M, approximately 1 x 10 -9 Less than M, approximately 1 x 10 -10 Less than M, approximately 1 x 10 -11 Less than M, approximately 1 x 10 -12 Less than M or approximately 1 x 10 -13 A dissociation constant (K D ) can bind to CD71. The measured affinity of a particular FN3 domain-antigen interaction can vary when measured under different conditions (e.g., osmolality, pH). Therefore, affinity and other antigen binding parameters (e.g., K D , K. on , K. off ) is measured using standardized solutions of protein scaffold and antigen, and standardized buffers such as those described herein.

[0224] In some embodiments, the FN3 domain is capable of binding to CD71 with a signal at least 5-fold higher than that obtained for a negative control in a standard solution ELISA assay.

[0225] In some embodiments, an FN3 domain that binds to or specifically binds to CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, an FN3 domain that binds to or specifically binds to CD71 comprises a cysteine ​​(Cys) linked to the C-terminus of the FN3 domain. The addition of an N-terminal Met and / or a C-terminal Cys may facilitate expression and / or conjugation to extend half-life and provide other functions of the molecule.

[0226] The FN3 domain can also include cysteine ​​substitutions, such as those described in U.S. Patent No. 10,196,446 (incorporated herein by reference in its entirety). Briefly, in some embodiments, the polypeptides provided herein comprise a cysteine ​​substitution at a position selected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the FN3 domain based on SEQ ID NO:1 or SEQ ID NO:1 in U.S. Patent No. 10,196,446 (incorporated herein by reference in its entirety); LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2311), and at least one cysteine ​​substitution at an equivalent position in the relevant FN3 domain.

[0227] In some embodiments, the substitution is at residue 6. In some embodiments, the substitution is at residue 8. In some embodiments, the substitution is at residue 10. In some embodiments, the substitution is at residue 11. In some embodiments, the substitution is at residue 14. In some embodiments, the substitution is at residue 15. In some embodiments, the substitution is at residue 16. In some embodiments, the substitution is at residue 20. In some embodiments, the substitution is at residue 30. In some embodiments, the substitution is at residue 34. In some embodiments, the substitution is at residue 38. In some embodiments, the substitution is at residue 40. In some embodiments, the substitution is at residue 41. In some embodiments, the substitution is at residue 45. In some embodiments, the substitution is at residue 47. In some embodiments, the substitution is at residue 48. In some embodiments, the substitution is at residue 53. In some embodiments, the substitution is at residue 54. In some embodiments, the substitution is at residue 59. In some embodiments, the substitution is at residue 60. In some embodiments, the substitution is at residue 62. In some embodiments, the substitution is at residue 64. In some embodiments, the substitution is at residue 70. In some embodiments, the substitution is at residue 88. In some embodiments, the substitution is at residue 89. In some embodiments, the substitution is at residue 90. In some embodiments, the substitution is at residue 91. In some embodiments, the substitution is at residue 93.

[0228] Cysteine ​​substitution at a certain position in a domain or protein involves replacing an existing amino acid residue with a cysteine ​​residue.In some embodiments, instead of substitution, cysteine ​​is inserted into the sequence adjacent to the above position.Other examples of cysteine ​​modification can be found, for example, in US Patent Application Publication No. 2017 / 0362301, the entire contents of which are incorporated herein by reference.Sequence alignment can be performed using BlastP, for example, using default parameters on the NCBI website.

[0229] In some embodiments, a cysteine ​​residue is inserted at any position within the domain or protein.

[0230] In some embodiments, the FN3 domain that binds to CD71 is internalized into the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a detectable label or a therapeutic agent into the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a cytotoxic agent into the cell. The cytotoxic agent may act as a therapeutic agent. In some embodiments, internalization of the FN3 domain may facilitate delivery of any detectable label, therapeutic agent, and / or cytotoxic agent disclosed herein into the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of an oligonucleotide into the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a cell of the central nervous system. In some embodiments, the cell is a cardiac cell. In some embodiments, the therapeutic agent is an siRNA molecule provided herein. The CD71-binding FN3 domain conjugated to a detectable label can be used to assess CD71 expression on a sample, such as tumor tissue, in vivo or in vitro. The CD71-binding FN3 domain conjugated to a detectable label can be used to assess CD71 expression on a sample, such as blood, immune, or muscle cells, in vivo or in vitro.

[0231] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849.

[0232] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 365. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 366. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 367. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 368. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 369. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 371. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 372. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 373. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 374. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 375. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 376. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 377. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 378. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 379. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 380. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 381. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 382.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 383. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 384. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 385. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 386. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 387. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 388. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 389. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 390. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 391. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 392. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 393. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 394. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 395. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 396. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 397. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 398. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 399. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 400.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 401. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 402. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 405. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 407. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 409. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 410. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 412. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 414. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 415. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 416. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 418. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 422. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 423.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 424. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 425. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 427. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 429. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 430. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 431. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 432. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 433. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 434. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 435. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 436. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 437. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 438. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 439. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 440. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 441.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 443. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 444. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 445. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 446. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 447. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 448. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 449. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 450. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 451. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 452. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 453. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 455. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 456. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 457. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 458. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 459. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 461.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 463. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 464. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 465. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 467. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 469. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 471. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 473. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 475. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 479.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 481. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 483. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 484. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 485. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 487. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 489. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 490. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 491. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 492. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 493. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 495. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 497. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 499. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 501. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 503. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 504. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 505.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 507. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 508. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 509. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 510. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 511. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 512. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 513. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 514. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 515. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 516. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 517. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 518. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 519. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 520. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 521. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 522. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 523.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 524. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 525. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 527. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 528. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 529. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 530. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 531. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 532. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 533. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 534. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 535. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 536. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 537. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 538. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 539. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 540. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 541. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 542. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 543. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 544. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 545. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 546.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 547. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 548. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 549. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 550. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 551. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 552. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 553. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 554. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 555. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 556. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 557. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 558. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 559. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 560. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 561. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 562. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 563. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 564.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 565. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 566. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 567. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 568. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 569. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 570. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 571. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 572. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 573. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 574. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 575. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 576. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 577. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 579. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 580. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 581. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 582.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 583. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 584. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 585. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 586. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 587. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 588. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 589. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 593. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 594. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 595. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 596. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 597. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 598. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 599. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 600. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 601. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 602. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 603. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 604. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 605.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 606. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 612. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 613. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 615. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 616. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 619. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 620. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 621. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 622. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 623.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 624. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 625. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 626. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 627. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 628. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 629. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 630. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 631. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 633. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 634. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 635. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 636. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 637. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 638. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 639. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 640. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 641. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 642. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 643. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 644. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 663. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 664.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 665. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 667. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 668. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 669. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 670. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 671. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 672. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1395. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1396. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1397. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1398. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1399. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1400. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1401. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1402. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1403. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1404. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1405. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1406. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1407. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1408. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1409. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1410. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1411. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1412. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1413. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1414. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1415. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1416. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1417. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1418. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1419. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1420. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1421.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1422. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1423. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1424. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1425. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1426. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1427. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1428. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1429. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1430. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1431. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1432. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1433. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1434. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1435. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1436. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1437. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1438. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1439. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1440. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1441. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1442. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1443. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1444. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1445. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1446. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1447. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1448. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1449. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1450. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1451. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1452. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1453. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1454. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1455. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1456. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1457.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1458. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1459. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1460. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1461. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1462. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1463. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1464. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1465. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1466. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1467. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1468. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1469. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1470. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1471. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1472. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1473. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1474. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1475. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1476. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1477. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1478. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1479. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1480. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1481. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1482. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1483. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1484. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1485. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1486. ​​In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1487. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1488. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1489. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1490. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1491. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1492. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1493.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1494. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1495. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1496. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1497. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1498. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1499. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1500. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1501. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1502. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1503. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1504. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1505. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1506. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1507. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1508. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1509. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1510. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1511. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1512. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1513. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1514. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1515. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1516. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1517. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1518. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1519. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1520. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1521. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1522. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1523. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1524. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1525. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1526.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1527. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1528. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1529. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1530. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1531. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1532. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1533. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1534. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1535. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1536. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1537. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1538. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1539. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1540. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1541. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1542. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1543. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1544. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1545. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1546. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1547. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1548. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1549. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1550. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1551. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1552. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1553. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1554. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1555. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1556. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1557. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1558. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1559.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1560. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1561. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1562. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1563. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1564. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1565. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1566. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1567. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1568. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1569. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1570. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1571. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1572. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1573. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1574. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1575. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1576. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1577.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1578. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1579. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1580. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1581. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1582. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1583. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1584. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1585. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1586. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1587. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1588. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1589. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1590. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1591. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1592. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1593. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1594. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1595. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1596. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1597. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1598. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1599. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1600. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1601. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1602. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1603.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1604. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1605. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1606. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1607. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1608. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1609. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1610. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1611. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1612. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1613. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1614. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1615. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1616. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1617. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1618. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1619. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1620. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1621.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1622. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1623. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1624. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1625. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1626. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1627. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1628. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1629. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1630. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1631. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1632. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1633. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1634. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1635. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1636. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1637. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1638. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1639. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1640. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1641. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1642. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1643. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1644. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1645. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1646. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1647.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1648. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1649. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1650. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1651. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1652. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1653. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1654. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1655. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1656. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1657. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1658. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1659. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1660. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1661. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1662. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1663. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1664. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1665. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1666. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1667. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1668. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1669. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1670. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1671. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1672. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1673. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1674. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1675. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1676. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1677. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1678. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1679. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1680.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1681. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1682. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1683. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1684. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1685. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1686. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1687. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1688. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1689. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1690. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1691. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1692. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1693. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1694. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1695. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1696. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1697. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1698. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1699. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1700. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1701. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1702. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1703. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1704. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1705. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1706. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1707. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1708. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1709. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1710. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1711. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1712. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1713.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1714. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1715. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1716. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1717. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1718. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1719. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1720. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1721. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1722. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1723. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1724. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1725. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1726. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1727. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1728. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1729. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1730. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1731.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1732. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1733. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1734. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1735. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1736. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1737. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1738. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1739. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1740. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 1741. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 1742. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1743. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1744. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1745. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1746. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1747. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1748. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1749. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1750. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1751. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1752. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1753. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1754. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1755. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1756. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1757. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1758. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1759. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1760.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1761. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1762. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1763. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1764. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1765. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1766. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1767. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1768. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1769. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1770. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1771. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1772. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1773. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1774. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1775. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1776. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1777. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1778. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1779. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1780. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1781. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1782. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1783. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1784. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1785. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1786. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1787. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1788. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1789. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1790. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1791. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1792. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1793.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1794. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1795. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1796. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1797. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1798. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1799. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1800. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1801. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1802. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1803. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1804. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1805. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1806. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1807. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1808. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1809. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1810. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1811.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1812. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1813. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1814. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1815. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1816. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1817. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1818. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1819. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1820. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 1821. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 1822. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1823. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1824. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1825. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1826. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1827. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1828. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1829. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1830. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1831. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1832. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1833. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1834. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1835. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1836. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1837. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1838. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1839. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1840.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1841. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1842. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1843. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1844. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1845. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1846. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1847. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1848. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1849.

[0233] In some embodiments, the isolated FN3 domain that binds to CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

[0234] In some embodiments, the isolated FN3 domain that binds to CD71 comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of the amino acid sequences set forth in SEQ ID NOs: 365-644, 663-672, or 1395-1849. Percent identity can be determined by aligning the two sequences using BlastP, available on the NCBI website, using default parameters. The sequences of FN3 domains that bind to CD71 can be found, for example, in Table 9. These sequences are shown with an N-terminal methionine. The domain sequences can also be used without an N-terminal methionine. Simply to avoid duplication of nearly identical sequences, a table of such sequences is not provided; however, one skilled in the art can readily envision the sequences provided herein without an N-terminal methionine, and the present disclosure should be understood and interpreted to include such sequences. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8]

Table 13-9

Table 13-10

Table 13-11

Table 13-12

Table 13-13

Table 13-14

Table 13-15

Table 13-16

Table 13-17

Table 13-18

Table 13-19

Table 13-20

Table 13-21

Table 13-22

Table 13-23

Table 13-24

[0235] Without being bound by any particular theory, in some embodiments, FN3 domains linked to nucleic acid molecules can be used to target delivery of therapeutic agents to cells expressing one or more binding partners of the FN3 domain, resulting in intracellular accumulation of the nucleic acid molecule therein, allowing the siRNA molecule to properly interact with the cellular machinery to inhibit target gene expression, improving efficacy and, in some embodiments, avoiding toxicity that can occur with non-targeted administration of the same siRNA molecule.

[0236] The FN3 domains described herein that bind to specific target proteins can be produced as monomers, dimers, or multimers, e.g., to increase the valency and thus the affinity of target molecule binding, or as a means to generate bispecific or multispecific scaffolds that simultaneously bind two or more different target molecules. Dimers and multimers can be generated by linking monospecific, bispecific, or multispecific protein scaffolds, e.g., by including an amino acid linker, e.g., a linker containing polyglycine, glycine and serine, or alanine and proline.

[0237] Therefore, as provided herein, different FN3 domains linked to siRNA molecules can also be conjugated or linked to other FN3 domains that bind to different targets.The linker can be a flexible linker.The linker can be a short peptide sequence such as those described herein.For example, the linker can be a G / S or G / A linker, etc.As provided herein, the linker can be, for example, a linker as shown in Table 10. [Table 14]

[0238] In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 645. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 646. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 647. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 648. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 649. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 650. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 651. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 652. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 653. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 654. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 655. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 656. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 657. In some embodiments, the FN3 domains comprising two FN3 domains connected by a linker have the amino acid sequence of SEQ ID NO: 658.In some embodiments, the FN3 domain that constitutes two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 659. In some embodiments, the FN3 domain that constitutes two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 660. In some embodiments, the FN3 domain that constitutes two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 661. In some embodiments, the FN3 domain that constitutes two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 645-661.

[0239] Dimers and multimers may be linked together in the N to C direction. The use of naturally occurring and synthetic peptide linkers to link polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Patent No. 5,856,456). The linkers described in this paragraph may also be used to link the domains provided herein and in the formulas provided above.

[0240] Half-life extension moiety The FN3 domain may also incorporate other subunits, for example, via covalent interactions, in some embodiments. In some embodiments, the FN3 domain further comprises a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and / or domains, aliphatic chains or chains that bind to serum proteins, transferrin and fragments and analogs thereof, and Fc regions. The amino acid sequences of human Fc regions are well known and include IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE Fc regions. In some embodiments, the FN3 domain binds to albumin, albumin variants, albumin-binding proteins and / or domains, and fragments and analogs thereof, thereby extending the half-life of the overall molecule.

[0241] In some embodiments, the albumin binding domain comprises the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the albumin binding domain (protein) is isolated. In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the albumin binding domain is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. or 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, with the proviso that the protein has a substitution corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T, or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid. In some embodiments, the isolated albumin binding domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions compared to the amino acid sequence of SEQ ID NO: ... 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitutions are at a position corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.In some embodiments, the provided FN3 domains contain a cysteine ​​residue at at least one residue position corresponding to residues 6, 11, 22, 25, 26, 52, 53, 61, 88, or 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, or 90 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, or at the C-terminus. Although the positions are listed consecutively, each position may also be selected individually. In some embodiments, the cysteine ​​is at a position corresponding to 6, 53, or 88. In some embodiments, further examples of albumin binding domains can be found in US Pat. No. 10,925,932, which is incorporated herein by reference.

[0242] All or a portion of the antibody constant region may be attached to the FN3 domain to confer antibody-like properties, particularly properties associated with the Fc region, such as Fc effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptor; BCR), and may be further modified by modifying the residues of the Fc responsible for these activities (for a review, see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

[0243] Additional moieties may be incorporated into the FN3 domain for desired properties, such as polyethylene glycol (PEG) molecules, e.g., PEG 5000 or PEG 20,000, fatty acids and fatty acid esters of different chain lengths (e.g., lauric acid, myristic acid, stearic acid, arachidate, behenic acid, oleic acid, arachidonic acid, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc.), polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides). These moieties may be directly fused to the protein scaffold coding sequence or produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods may be used to attach moieties to recombinantly produced molecules disclosed herein.

[0244] A PEG moiety can be added to the FN3 domain t, for example, by incorporating a cysteine ​​residue at the C-terminus of the molecule, or by engineering a cysteine ​​at a residue position away from the binding face of the molecule and attaching a PEG group to the cysteine ​​using well-known methods.

[0245] The functionality of FN3 domains incorporating additional moieties can be compared using several well-known assays. For example, changes in properties due to the incorporation of an Fc domain and / or Fc domain variant can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn receptors, or in well-known cell-based assays measuring, for example, ADCC or CDC, or in assays evaluating the pharmacokinetic properties of the molecules disclosed herein in in vivo models.

[0246] The compositions provided herein can be prepared by preparing an FN3 protein and a nucleic acid molecule and linking them together. Techniques for linking proteins to nucleic acid molecules are known, and any method can be used. For example, in some embodiments, a nucleic acid molecule is modified with a linker, such as the linkers provided herein, and then the protein is mixed with the linker-containing nucleic acid molecule to form a composition. For example, in some embodiments, an FN3 domain is conjugated to an siRNA via a cysteine ​​using thiol-maleimide chemistry. In some embodiments, a cysteine-containing FN3 domain can be reduced in a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) in, for example, phosphate-buffered saline (or any other suitable buffer) to generate a free thiol. In some embodiments, the free thiol-containing FN3 domain is then mixed with a maleimide-linked modified siRNA duplex and incubated under conditions to form a ligated complex. In some embodiments, the mixture is incubated at room temperature (RT) for 0 to 5 hours, or for about 1, 2, 3, 4, or 5 hours. The reaction can be quenched, for example, with N-ethylmaleimide. In some embodiments, the conjugate can be purified using affinity chromatography and ion exchange. Other methods can also be used; this is just one non-limiting embodiment.

[0247] Methods for making FN3 proteins are known, and any method can be used to produce the protein. Examples are provided in the references incorporated herein by reference.

[0248] In some embodiments, the FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NOs: 365-644 or 663-672, wherein a histidine tag is added to the N- or C-terminus of the polypeptide to facilitate purification. In some embodiments, the histidine tag (His-tag) comprises six histidine residues (SEQ ID NO: 662). In further embodiments, the His-tag is connected to the FN3 domain by at least one glycine residue, or about two to about four glycine residues. Thus, after purification of the FN3 domain and cleavage of the His-tag from the polypeptide, one or more glycines may remain at the N- or C-terminus. In some embodiments, when the His-tag is removed from the N-terminus, all glycines are also removed. In some embodiments, when the His-tag is removed from the C-terminus, one or more of the glycines are retained.

[0249] In some embodiments, an FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NOs: 365-644 or 663-672, wherein the N-terminal methionine is retained after purification of the FN3 domain. In some embodiments, an FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NOs: 365-644 or 663-672, wherein the N-terminal methionine is not retained after purification of the FN3 domain.

[0250] For example, as described herein, in some embodiments, the amino acid sequence of SEQ ID NO:570 without methionine can be as follows: LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYAEPRPDGEAILLQVPGSCRSYDLTGLKPGTEYSVLIHGVKGGLLSSPLTAIFTT (SEQ ID NO: 2310)

[0251] As provided herein, FN3 domain can be linked to siRNA molecule.Although certain FN3 domain is shown with methionine, it should be understood that FN3 domain can be linked to siRNA without N-terminal methionine.In addition, those skilled in the art will understand that the numbering of the position of cysteine ​​residue provided herein will be shifted to the next lower residue without N-terminal methionine.

[0252] For example, the amino acid sequence of the FN3 domain can be one provided herein, including, but not limited to, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical to the amino acid sequence of SEQ ID NO:570 or SEQ ID NO:2310, and can be linked to an siRNA pair provided herein. In some embodiments, the siRNA pair comprises a sense strand and an antisense strand. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:2110, or a modified version thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2220, or a modified version thereof. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:2113, or a modified version thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2223, or a modified version thereof. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:2111, or a modified version thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2221, or a modified version thereof. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1890, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2290. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1893, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2293. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1941, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2051. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1942, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2052. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1944, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2054. In some embodiments, the sense strand and antisense strand pair (siRNA pair) are as provided herein.

[0253] kit In some embodiments, kits are provided that include the compositions described herein. The kits can be used for therapeutic purposes and as diagnostic kits. In some embodiments, the kits include an FN3 domain conjugated to a nucleic acid molecule.

[0254] Use of conjugates The compositions provided herein can be used to diagnose, monitor, regulate, treat, alleviate symptoms of, help prevent the occurrence of, or reduce the severity of a human disease or a particular pathological condition in a cell, tissue, organ, body fluid, or generally within a host.

[0255] In some embodiments, the FN3 domain can facilitate delivery to activated lymphocytes, dendritic cells, or other immune cells for the treatment of immune diseases. Thus, in some embodiments, the FN3 domain that binds to CD71 is targeted to immune cells. In some embodiments, the FN3 domain that binds to CD71 is targeted to B cells. In some embodiments, the FN3 domain that binds to CD71 is targeted to T cells. In some embodiments, the FN3 domain that binds to CD71 is targeted to dendritic cells. In some embodiments, the FN3 domain that binds to CD71 is targeted to monocytes. In some embodiments, the FN3 domain that binds to CD71 does not have an anti-proliferative effect on immune cells. For example, in some embodiments, the FN3 domain that binds to CD71 does not have an anti-proliferative effect on B cells, T cells, dendritic cells, monocytes, or any combination thereof.

[0256] In some embodiments, methods are provided for treating an autoimmune disease in a subject in need thereof. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds to CD71. In some embodiments, the polypeptide is an FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises an amino acid sequence, e.g., SEQ ID NOs: 361-644 or 663-672, or a polypeptide provided herein linked or conjugated to a therapeutic agent. In some embodiments, the method comprises a method for treating an autoimmune disease in a subject, the method comprising administering to the subject an FN3 domain that binds to CD71, wherein the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide, e.g., siRNA, ASO, or an FN3 domain that binds to another target).

[0257] In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Schaeublen's syndrome, myositis, lupus nephritis, neuroinflammatory diseases such as multiple sclerosis, or prevention of solid organ transplant rejection.

[0258] In some embodiments, methods for reducing the expression of a target gene in a cell are provided. In some embodiments, the method comprises delivering a composition or pharmaceutical composition provided herein to the cell. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the target gene is CD40. However, the target gene can be any target gene, as evidence provided herein demonstrates that siRNA molecules can be efficiently delivered when conjugated to an FN3 domain. In some embodiments, an siRNA targeting CD40 is linked to an FN3 domain. In some embodiments, the FN3 polypeptide (domain) is one that binds to CD71. In some embodiments, the FN3 polypeptide is as provided herein or as provided in PCT Application No. PCT / US20 / 55509, U.S. Application No. 17 / 070,337, PCT Application No. PCT / US20 / 55470, or U.S. Application No. 17 / 070,020, each of which is incorporated herein by reference in its entirety. In some embodiments, the siRNA is not conjugated to an FN3 domain. In some embodiments, the method of reducing expression of a target gene results in about a 99%, 90-99%, 50-90%, or 10-50% reduction in expression of the target gene.

[0259] In some embodiments, methods of reducing CD40 expression are provided. In some embodiments, the reduced expression is CD40 mRNA expression (amount). In some embodiments, the methods of reducing CD40 expression result in about a 99%, 90-99%, 50-90%, or 10-50% reduction in CD40 expression. In some embodiments, the reduced expression is CD40 protein expression (amount). In some embodiments, the reduced protein is CD40 protein. In some embodiments, the reduction in CD40 protein occurs in immune cells. In some embodiments, the reduction in CD40 protein occurs in B cells. In some embodiments, the reduction in CD40 protein occurs in T cells. In some embodiments, the reduction in CD40 protein occurs in dendritic cells. In some embodiments, the method comprises delivering to a cell an siRNA molecule provided herein that targets CD40. In some embodiments, the siRNA is conjugated to an FN3 domain. In some embodiments, the FN3 domain is an FN3 domain that binds to CD71. In some embodiments, the FN3 domain is as provided herein. In some embodiments, the FN3 domain is a dimer of two FN3 domains that bind to CD71. In some embodiments, the FN3 domains are the same. In some embodiments, the two FN3 domains are different, i.e., bind to different regions or amino acid residues of CD71, i.e., different epitopes. In some embodiments, the method comprises administering to a subject (patient) a CD40-targeting siRNA molecule as provided herein. In some embodiments, the CD40-targeting siRNA molecule administered to the subject is conjugated or linked to an FN3 domain. In some embodiments, the FN3 domain is an FN3 domain that binds to CD71. In some embodiments, the FN3 domain is as provided herein. In some embodiments, the FN3 domain is a dimer of two FN3 domains that bind to CD71. In some embodiments, the FN3 domains are the same.In some embodiments, the two FN3 domains are different, i.e., bind to different regions or amino acid residues of CD71, i.e., different epitopes. In some embodiments, the CD71-binding domain is a polypeptide as provided herein.

[0260] In some embodiments, a method of delivering an siRNA molecule to a cell in a subject is provided. In some embodiments, the method includes administering to the subject a pharmaceutical composition comprising a composition provided herein. In some embodiments, the cell is a CD71-positive cell. The term "positive cell" with respect to a protein refers to a cell that expresses the protein. In some embodiments, the protein is expressed on the cell surface. In some embodiments, the cell is a tumor cell, a liver cell, an immune cell, a cardiac cell, a muscle cell, a cell of the CNS, or a cell within the blood-brain barrier. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the siRNA molecule downregulates expression of a target gene in the cell. In some embodiments, the target gene is CD40.

[0261] In some embodiments, methods are provided for reducing one or more serum cytokines in a subject. In some embodiments, the methods include administering an siRNA molecule. In some embodiments, the siRNA molecule downregulates expression of a target gene in the cell. In some embodiments, the target gene is CD40. In some embodiments, the one or more cells are CD71-positive cells. In some embodiments, the one or more cells are immune cells. In some embodiments, the cells are B cells. In some embodiments, the cells are dendritic cells. In some embodiments, the cells are T cells. In some embodiments, the one or more serum cytokines include IFN-γ, IL-6, TNF-α, IL-12, IP-10, and / or RANTES, or any combination thereof. In some embodiments, the one or more serum cytokines include IFN-γ. In some embodiments, the one or more serum cytokines include IL-6. In some embodiments, the one or more serum cytokines include TNF-α. In some embodiments, the one or more serum cytokines include IL-12. In some embodiments, the one or more serum cytokines include IP-10. In some embodiments, the one or more serum cytokines include RANTES.

[0262] In some embodiments, methods of reducing or inhibiting cell migration are provided. In some embodiments, the methods include contacting a cell with an siRNA molecule. In some embodiments, the siRNA molecule downregulates expression of a target gene in the cell. In some embodiments, the target gene is CD40. In some embodiments, the cell is a CD71-positive cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the methods include reducing or inhibiting cell migration from blood to a tissue. In some embodiments, the methods include reducing or inhibiting cell migration from blood to a lymphoid organ tissue. In some embodiments, the methods include selectively reducing or inhibiting migration of B cells and / or dendritic cells, but not reducing or inhibiting migration of T cells.

[0263] In some embodiments, methods of inhibiting margination are provided. In some embodiments, the methods include contacting a cell with an siRNA molecule. In some embodiments, the siRNA molecule downregulates expression of a target gene in the cell. In some embodiments, the target gene is CD40. In some embodiments, the cell is a CD71-positive cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the methods include reducing or inhibiting cell margination from the interior of a blood vessel toward the blood vessel wall. In some embodiments, the methods include selectively reducing or inhibiting margination of B cells and / or dendritic cells, but not reducing or inhibiting margination of T cells.

[0264] In some embodiments, the compositions or pharmaceutical compositions provided herein can be administered alone or in combination with other therapeutic agents, i.e., simultaneously or sequentially.

[0265] "Treating" and "treatment" refer to therapeutic and prophylactic treatment, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder (such as the onset or spread of cancer). In some embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, remission or palliation of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0266] A "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount of the compositions provided herein may vary depending on factors such as the individual's condition, age, sex, and weight. Exemplary indicators of an effective amount are an improvement in the patient's health, a decrease or shrinkage of tumor size, a halt or slowdown in tumor growth, and / or a lack of metastasis of cancer cells to other locations in the body.

[0267] Administration / Pharmaceutical Compositions In some embodiments, provided herein is a pharmaceutical composition comprising a composition provided herein and a pharmaceutically acceptable carrier. For therapeutic applications, the composition may be prepared as a pharmaceutical composition containing an effective amount of a domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which an active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and coloring agents. The concentration of the molecules disclosed herein in such pharmaceutical formulations can vary widely, i.e., less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight, and is selected primarily based on the required dose, fluid volume, viscosity, etc., according to the particular mode of administration selected. Suitable vehicles and formulations (including other human proteins, e.g., human serum albumin) are described in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092 (see especially pp. 958-989).

[0268] The mode of administration for therapeutic applications of the compositions disclosed herein can be any suitable route that delivers an agent to a recipient, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or pulmonary administration; transmucosal (oral, intranasal, intravaginal, rectal) administration using tablet, capsule, solution, powder, gel, particle formulations; and containment in a syringe, implantable device, osmotic pump, cartridge, micropump; or other means recognized by one of skill in the art as being well known in the art. Site-specific administration can be achieved, for example, by intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

[0269] The pharmaceutical composition may be provided as a kit comprising a container containing the pharmaceutical composition as described herein. The pharmaceutical composition may be provided, for example, in the form of a single or multiple dose injectable solution or as a sterile powder to be reconstituted before injection. Alternatively, such a kit may include a dry powder disperser, liquid aerosol generator, or nebulizer for administering the pharmaceutical composition. Such a kit may further include written information regarding the indications and usage of the pharmaceutical composition.

[0270] Enumerated Embodiments Embodiments provided herein also include, but are not limited to, the following:

[0271] 1. A composition comprising an siRNA molecule, including a sense strand and an antisense strand, that targets the CD40 gene, such as those provided herein.

[0272] 2. The composition of embodiment 1, wherein said si...

Claims

1. A composition comprising an siRNA molecule comprising a sense strand and an antisense strand targeting the CD40 gene, the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1890, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2290; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1893, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2293; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1941, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2051; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1942, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2052; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1944, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2054; the sense strand comprises the nucleotide sequence of SEQ ID NO: 2110, or a modified version thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2220, or a modified version thereof; the sense strand comprises the nucleotide sequence of SEQ ID NO: 2113, or a modified version thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2223, or a modified version thereof; the sense strand comprises the nucleotide sequence of SEQ ID NO: 2111, or a modified version thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2221, or a modified version thereof; Or the composition comprising a sense strand and antisense strand pair (siRNA pair) provided herein.

2. The composition of claim 1 , wherein the siRNA molecule further comprises a linker covalently attached to the sense strand or the antisense strand.

3. The composition of claim 2 , wherein the linker is attached to the 5′ or 3′ end of the sense strand or the antisense strand.

4. The composition of claim 1 , wherein the siRNA molecule further comprises a vinyl phosphonate modification on the sense strand or the antisense strand.

5. The composition of claim 4 , wherein the vinyl phosphonate modification is attached to the 5′ or 3′ end of the sense strand or the antisense strand.

6. 2. The composition of claim 1, wherein the sense strand comprises a nucleic acid sequence selected from SEQ ID NOs: 1890, 1893, 1941, 1942, 1944, 46-178, 312-331, 1850, 1851, 1891, 1892, 1894-1928, 1932-1940, 1943, 1945-1959, 2298, 2302, 2304, 352-356, 673-805, 939-958, 2070, 2071, 2110-2148, 2152-2179, 2300, 2306, and 2308, or a modified version thereof.

7. 2. The composition of claim 1, wherein the antisense strand comprises a nucleic acid sequence selected from SEQ ID NOs: 2290, 2293, 2051, 2052, 2054, 179-311, 332-351, 1960, 1961, 2000-2038, 2042-2050, 2053, 2055-2069, 2291, 2292, 2294-2297, 2299, 2303, 2305, 356-359, 806-938, 959-978, 2180, 2181, 2220-2258, 2262-2289, 2301 and 2309, or a modified version thereof.

8. The siRNA molecules are A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, M1, N1, O1, P1, Q1, R1, S1, T1, U1, V1, W1, X1, Y1, Z1, A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, M2, N2, O2, P2, Q2, R2, S2, T2, U2, V2, W2, X2, Y2, Z2, A3, B3, C3, D3, E3, F3, G3, H3, I3, J3, K3, L3, M3, N3, O3, P3, Q3, R3, S3, T3, U3, V3, W3, X3, Y3, Z3, A4, B4, C4, D4, E4, F4, G4, H4, I4, J4, K4, L4, M4, N4, O4, P4, Q4, R4, S4, T4, U4, V4, W4 , , G6, H6, I6, J6, K6, L6, M6, N6, O6, P6, Q6, R6, S6, T6, U6, V6, W6, B7, C7, P8, Q8, R8, S8, T8, U8, V8, W8, X8, Y8, Z8, A9, B9, C9, D9, E 9, F9, G9, H9, I9, J9, K9, L9, M9, N9, O9, P9, Q9, R9, S9, T9, U9, V9, W9, X9, Y9, Z9, A10, B10, F10, G10, H10, I10, J10, K10, L10, M10, 10. The composition of any one of the preceding claims, comprising an siRNA pair (sense and antisense strands) of N10, O10, P10, Q10, R10, S10, T10, U10, V10, W10, X10, Y10, Z10, A11, B11, C11, D11, E11, F11, G11, H11, I11, J11, K11, L11, M11, N11, O11, P11, Q11, R11, or those set forth in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, or Table 5B.

9. 9. The composition of claim 8, wherein the composition comprises an siRNA pair shown in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, or Table 5B having a linker and / or vinyl phosphonate modification provided herein.

10. The composition of claim 1 , wherein the siRNA molecule has the formula depicted in Formula III: 【Chemical 1】 wherein each nucleotide represented by N is as set forth in SEQ ID NOs:2110 and 2220, SEQ ID NOs:2113 and 2223, SEQ ID NOs:2111 and 2221, or includes a sense strand and antisense strand pair (an siRNA pair) provided herein, or a modified nucleotide base thereof (e.g., as provided herein).

11. The sense strand is N １ and N ２ 2'O-methyl modified nucleotides with phosphorothioate (PS)-modified backbones at N ３ , N ７ , N ８ , N ９ , N １２ , and N １７ 2'-fluoro modified nucleotides, and N ４ , N ５ , N ６ , N １０ , N １１ , N １３ , N １４ , N １５ , N １６ , N １８ , and N １９ The composition of claim 10, wherein the nucleotide sequence is a 2'O-methyl modified nucleotide.

12. The antisense strand is N １ vinyl phosphonate moieties with phosphorothioate (PS) modified backbones attached to N ２ 2' fluoro-modified nucleotide having a PS-modified backbone at N ３ , N ４ , N ５ , N ６ , N ７ , N ８ , N ９ , N １０ , N １１ , N １２ , N １３ , N １５ , N １６ , N １７ , N １８ , and N １９ 2'O-methyl modified nucleotide, N １４ 2' fluoro-modified nucleotides, and N ２０ and N ２１ The composition of claim 10 or 11, comprising a 2'O-methyl modified nucleotide having a PS-modified backbone at

13. The antisense strand is N １ 11. The composition of claim 10, comprising a vinyl phosphonate moiety bonded to:

14. 11. The composition of claim 10, wherein the siRNA molecule is conjugated to a linker having the formula: 【Chemistry 2】

15. 2. The composition of claim 1, wherein the siRNA molecule has the formula shown in the following formula: 【Chemistry 3】 (In the formula, F １ is a polypeptide comprising at least one FN3 domain.

16. The composition of claim 1, further comprising one or more FN3 domains conjugated to the siRNA molecule.

17. 17. The composition of claim 16, wherein the one or more FN3 domains comprise an FN3 domain conjugated to the siRNA molecule via a cysteine ​​in the FN3 domain.

18. The composition of claim 16 or 17, wherein the one or more FN3 domains comprise an FN3 domain that binds to CD71.

19. 19. The composition of claim 18, wherein the FN3 domain that binds to CD71 comprises an amino acid sequence at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or identical to a sequence selected from any one of SEQ ID NOs: 570, 672, 1848, 1773, 1849, 1767, 360-569, 571-644, 663-67, 1395-1772, 1774-1766, 1768-1847, and 2310.

20. 19. The composition of claim 18, wherein the FN3 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:570, SEQ ID NO:2310, or SEQ ID NO:

672.

21. 19. The composition of claim 18, wherein the FN3 domain comprises an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 570 or SEQ ID NO: 2310.

22. 19. The composition of claim 18, wherein the FN3 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 570, with the proviso that the residue at position 54 is a cysteine, or at least 95% identical to the amino acid sequence of SEQ ID NO: 2310, with the proviso that the residue at position 53 is a cysteine.

23. A pharmaceutical composition comprising the composition of any one of claims 1 to 22.

24. A kit comprising the composition of any one of claims 1 to 22.

25. 23. A method of treating an immune disease in a subject in need thereof, comprising administering to said subject a composition according to any one of claims 1 to 22.

26. 26. The method of claim 25, wherein the immune disease is rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Schaeublen's syndrome, inflammatory myositis, lupus nephritis, pemphigus vulgaris, multiple sclerosis, or prophylaxis of solid organ transplant rejection.

27. Use of a composition according to any one of claims 1 to 22 in the preparation of a pharmaceutical composition or medicament for treating an immune disease, such as an autoimmune disease.

28. 28. The use of claim 27, wherein the immune disease is rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Schaeublen's syndrome, inflammatory myositis, lupus nephritis, pemphigus vulgaris, multiple sclerosis, or the prevention of solid organ transplant rejection.

29. Use of a composition according to any one of claims 1 to 22 for treating an immune disease, such as an autoimmune disease.

30. 30. The use of claim 29, wherein the immune disease is rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Schaeublen's syndrome, inflammatory myositis, lupus nephritis, pemphigus vulgaris, multiple sclerosis, or the prevention of solid organ transplant rejection.

31. 23. A method of reducing the expression of CD40 in a cell, said method comprising contacting said cell with the composition of any one of claims 1 to 22.

32. 23. A method of delivering an siRNA molecule to immune cells in a subject, comprising administering to the subject a pharmaceutical composition comprising the composition of any one of claims 1 to 22, or a composition comprising an siRNA that targets an immune-specific cellular gene target.

33. 33. The method of claim 32, wherein the immune cell is a B cell, a T cell, or a dendritic cell.

34. 34. The method of claim 32 or 33, wherein the target gene is CD40.

35. 23. A method of delivering a CD40-targeting siRNA molecule to CD71-positive immune cells in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the composition of any one of claims 1 to 22, wherein the siRNA molecule downregulates expression of CD40 in the CD71-positive immune cells.

36. 23. A method of reducing one or more cytokines in a subject, the method comprising administering to the subject a composition according to any one of claims 1 to 22.

37. 37. The method of claim 36, wherein the one or more cytokines are selected from IFN-γ, IL-6, TNF-α, IL-12, IP-10, RANTES, and any combination thereof.

38. 1. A method for reducing or inhibiting migration of an immune cell population from blood to a tissue, comprising contacting said cell population with a composition comprising a CD40-targeting siRNA molecule.

39. The method of claim 38, wherein the composition is a composition according to any one of claims 1 to 22.

40. The immune cell population expresses CD40 (CD40 ＋ 39. The method of claim 38, comprising:

41. 41. The method of any one of claims 38 to 40, wherein the immune cell population comprises dendritic cells, B cells, or a combination thereof.

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