FN3 domain-siRNA conjugates for enzyme replacement therapy

Abstract

The present disclosure relates to compositions, such as siRNA molecules and FN3 domains conjugated thereto, and methods of making and using the same, in combination with enzyme replacement therapy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 330,797, filed April 14, 2022, which is incorporated by reference in its entirety.

[0002] Reference to Electronic Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The Sequence Listing is named "ROO-030US_SL.xml", was created on Jun. 20, 2023, and is 2,074,533 bytes in size.

[0003] The present embodiments relate to siRNA molecules, which may be conjugated fibronectin type III domains (FN3), and methods of using such molecules in combination with enzyme replacement therapy. [Background technology]

[0004] Therapeutic nucleic acids include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense, antagomir, antimir, microRNA mimics, supermir, 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). The therapeutic applications of RNAi are very broad, since siRNA and miRNA constructs can be synthesized with any nucleotide sequence against a target protein. Thus far, siRNA constructs have shown the ability to specifically downregulate target proteins in both in vitro and in vivo models. In addition, siRNA constructs are currently being evaluated in clinical studies and approved for various diseases.

[0005] However, two problems currently faced by siRNA constructs are, firstly, their susceptibility to nuclease digestion in plasma, and secondly, when administered systemically as free siRNA or miRNA, their limited ability to access the intracellular compartments where they can bind to RISC (RNA-induced silencing complex). Certain delivery systems, such as lipid nanoparticles formed from cationic lipids with other lipid components such as cholesterol and PEG lipids, carbohydrates (such as GalNac trimers), etc., have been used to facilitate the cellular uptake of oligonucleotides. However, these have not been shown to successfully deliver siRNA efficiently and effectively to its intended target in tissues other than the liver.

[0006] Compositions and methods for delivering siRNA to its intended cellular target are still needed. Further, what is needed is a method of using such molecules for optimized properties for clinical use of an FN3 domain capable of specifically binding to CD71, and for novel therapeutic agents that enable intracellular access via receptor-mediated endocytosis of CD71. The present embodiments meet these and other needs. SUMMARY OF THE INVENTION

[0007] In some embodiments, a method of treating a glycogen storage disease in a subject in need thereof is provided. In some embodiments, the method comprises administering a composition comprising one or more FN3 domains linked to an siRNA molecule comprising a sense strand and an antisense strand provided herein, and enzyme replacement therapy (ERT).

[0008] In some embodiments, the glycogen storage disease is selected from the group consisting of Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Cori disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Tarui disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes mellitus / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucosan body disease.

[0009] In some embodiments, the ERT comprises one or more enzymes selected from the group consisting of glucosidase alpha acid (GAA), glycogen debranching enzyme (AGL), glycogen branching enzyme (BGE1), muscle glycogen phosphorylase (PYGM), muscle phosphofructokinase (PFKM), aldolase A (ALDOA), malin, laforin, glycogen synthase (GYS2), glucose-6-phosphatase (G6PC / SLC37A4), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1), phosphoglycerate mutase (PGAM2), muscle lactate dehydrogenase (LDHA), glucose transporter (GLUT2), β-enolase (ENO3), and glycogenin-1 (GYG1).

[0010] In some embodiments, siRNA conjugated to an FN3 domain that binds to the CD71 protein is provided.

[0011] In some embodiments, an FN3 domain is provided that comprises the amino acid sequence of any of the FN3 domains provided herein. In some embodiments, the FN3 domain binds to CD71. In some embodiments, the FN3 domain specifically binds to CD71.

[0012] In some embodiments, the composition comprises two FN3 domains connected by a linker, such as a flexible linker. In some embodiments, the two FN3 domains bind to different targets. In some embodiments, the first FN3 domain binds to CD71. In some embodiments, the second FN3 domain binds to a different target that is not CD71.

[0013] In some embodiments, oligonucleotides such as dsRNA or siRNA molecules are provided herein.In some embodiments, oligonucleotides have sequences provided herein, with or without modifications provided herein.In some embodiments, oligonucleotides are provided in compositions, such as pharmaceutical compositions.In some embodiments, oligonucleotides are conjugated to polypeptides.

[0014] In some embodiments, compositions are provided that include one or more FN3 domains conjugated to a siRNA molecule.

[0015] In some embodiments, (X1) n -(X2) q -(X3) y -L-X4, where 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 n, q, and y are each independently 0 or 1.

[0016] In some embodiments, C-(X1) n -(X2) q -(X3) yA composition having the formula -L-X4, wherein C is a polymer or albumin binding domain (ABD), X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extending molecule, L is a linker, X4 is an oligonucleotide molecule, and n, q, and y are each independently 0 or 1, is provided.

[0017] In some embodiments, (X1) n -(X2) q -(X3) y A composition having the formula -L-X4-C, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extending molecule, L is a linker, X4 is an oligonucleotide molecule, C is a polymer or albumin binding domain (ABD), and n, q, and y are each independently 0 or 1, is provided.

[0018] In some embodiments, X4-L-(X1) n -(X2) q -(X3) y A composition having the formula, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extending molecule, L is a linker, X4 is an oligonucleotide molecule, and n, q, and y are each independently 0 or 1, is provided.

[0019] In some embodiments, C-X4-L-(X1) n -(X2) q -(X3) ywherein C is a polymer or an albumin binding domain (ABD), 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 an oligonucleotide molecule, and n, q, and y are each independently 0 or 1.

[0020] 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 an oligonucleotide molecule, C is a polymer or an albumin binding domain (ABD), and n, q, and y are each independently 0 or 1.

[0021] 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, L is a linker, X4 is an oligonucleotide molecule, C is a polymer or an albumin binding domain (ABD), and n, q, and y are each independently 0 or 1.

[0022] 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, L is a linker, X4 is an oligonucleotide molecule, C is a polymer or an albumin binding domain (ABD), and n, q, and y are each independently 0 or 1.

[0023] In some embodiments, pharmaceutical compositions are provided that include one or more of the compositions provided herein.

[0024] In some embodiments, provided is a use of a composition provided herein, or any of the uses provided herein in the preparation of a pharmaceutical composition or medicament for treating a condition.

[0025] In some embodiments, methods are provided that selectively reduce GYS1 mRNA and protein in skeletal muscle and administer the enzymes provided herein. In certain embodiments, GYS1 mRNA and protein are not reduced or not significantly reduced in the liver and / or kidney.

[0026] In some embodiments, an isolated polynucleotide encoding the FN3 domain described herein is provided.

[0027] In some embodiments, a vector is provided that comprises a polynucleotide described herein.

[0028] In some embodiments, a host cell is provided that comprises a vector described herein.

[0029] In some embodiments, a method for producing an FN3 domain is provided. In some embodiments, the method comprises culturing a host cell comprising a vector encoding or expressing the FN3 domain. In some embodiments, the method further comprises purifying the FN3 domain. In some embodiments, the FN3 domain binds to CD71.

[0030] In some embodiments, a pharmaceutical composition is provided that includes a CD71-binding FN3 domain linked to an oligonucleotide molecule and a pharma- ceutically acceptable carrier. In some embodiments, a kit is provided that includes one or more of the FN3 domains, with or without an oligonucleotide molecule. [Brief description of the drawings]

[0031] [Figure 1] 1 is a flow chart depicting the properties evaluated and considered for siRNA screening. [Diagram 2] Graph of RNA sequencing experiment identifying changes in the transcriptome after transfecting cells with siRNA vs. HHH. The arrow identifies a significant decrease in GYS1 transcript. [Diagram 3] We provide the results of a target binding assay using over 6,000 receptors in a proteomic array, and the data indicate that CD71 is the exclusive binding target of the FN3 domain. [Figure 4A] 1 shows knockdown of GYS1 mRNA in mouse gastrocnemius muscle using three different FN3 domain-siRNA conjugates compared to vehicle alone. [Figure 4B] 1 shows knockdown of GYS1 protein in mouse gastrocnemius muscle using three different FN3 domain-siRNA conjugates compared to vehicle alone. [Diagram 5]Using three different FN3 domain-siRNA conjugates, in comparison to siRNA against a different target (AHA-1), we show that GYS1 knockdown is highly specific to skeletal muscle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] 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.

[0033] "Fibronectin type III (FN3) domain" (FN3 domain) refers to a domain that occurs frequently in proteins including fibronectins, tenascins, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci. USA, 1992, 89:8990-8994; Meinke et al., J Bacteriol., 1993, 175:1910-1918; Watanabe et al., J Biol Chem., 1990, 265:15659-15665). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains, as described, for example, 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 tenascin (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 associated with variable heavy chains (V H ) and / or light chain (V L ) and therefore is not an antibody.

[0034] As used herein, "autoimmune disease" refers to medical conditions and pathologies in which an individual's immune response is directed against the individual's own components, 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 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 I), depression, diabetes mellitus (including type 1 and / or type 2 diabetes), epididymitis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, and inflammatory bowel disease (IBD). , immune responses to recombinant preparations, such as, but not limited to, factor VII in hemophilia, juvenile idiopathic arthritis, systemic lupus erythematosus, lupus nephritis, male infertility, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, oncology, osteoarthritis, pain, primary myxedema, pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, sympathetic ophthalmia, T-cell lymphoma, T-cell acute lymphoblastic leukemia, testicular anti-central T-cell lymphoma, thyroiditis, transplant rejection, ulcerative colitis, autoimmune uveitis, and vasculitis. Autoimmune diseases include, but are not limited to, conditions in which the affected tissue is the primary target and, in some cases, the secondary target. Such conditions include, but are not limited to, AIDS, atopic allergies, bronchial asthma, eczema, leprosy, schizophrenia, hereditary depression, tissue and organ transplants, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus phenomenon, anaphylaxis, and alcohol and drug addiction.

[0035] As used herein, a "capture agent" refers to a substance that binds to a particular type of cell and allows the cell to be isolated from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulation reagents, molecules that bind to specific cell types, etc.

[0036] As used herein, a "sample" refers to a collection of fluids, cells, or tissues, such as those isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are tissue biopsies, fine needle aspirates, surgically removed tissues, organ cultures, cell cultures, and cell and organ culture media, including blood, serum and serum fluids, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell or organ conditioned media and lavage fluids.

[0037] "Substituting" or "substituted" or "mutating" or "mutated" refers to altering, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

[0038] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, eg, a substitution, insertion, or deletion.

[0039] "Specifically binds" or "specific binding" means a binding density of about 1 × 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 about 1 x 10 -13 The dissociation constant (K D) refers to the ability of an FN3 domain to bind to its target, e.g., CD71. Alternatively, "specific binding" refers to the ability of an FN3 domain to bind to its target (e.g., CD71) at least 5-fold more than a negative control in a standard solution ELISA assay. Specific binding can also be demonstrated using a proteomic array as described herein and shown in FIG. 3. 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 (homologs), such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee).

[0040] A "library" refers to a collection of variants. A library can be composed of polypeptide or polynucleotide variants.

[0041] "Stability" refers to the ability of a molecule to remain folded under physiological conditions so as to retain at least one of its normal functional activities, e.g., binding to a given antigen, such as CD71.

[0042] "CD71" refers to the human CD71 protein having the amino acid sequence of SEQ ID NO: 2 or 5. In some embodiments, SEQ ID NO: 2 is the full-length human CD71 protein. In some embodiments, SEQ ID NO: 5 is the extracellular domain of human CD71.

[0043] "Tencon" refers to a synthetic fibronectin type III (FN3) domain having the consensus sequence set forth in SEQ ID NO:1 (LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT) and described in U.S. Patent Publication No. 2010 / 0216708, which is incorporated by reference in its entirety.

[0044] "Cancer cells" or "tumor cells" refer to cancerous, precancerous, or transformed cells, either in vivo, ex vivo, and in tissue culture, that have spontaneous or induced phenotypic changes that do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and incorporation of new genomic nucleic acid, or incorporation of exogenous nucleic acid, but it can also occur naturally or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is demonstrated, for example, by in vitro, in vivo, and ex vivo morphological changes, cellular immortalization, aberrant growth control, foci formation, proliferation, malignancy, tumor-specific marker levels, invasiveness, tumor growth or suppression in suitable animal hosts, such as nude mice (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

[0045] "Dendritic cells" refers to a type of antigen-presenting cell (APC) that forms an important role in the adaptive immune system. The main function of dendritic cells is to present antigens to T lymphocytes and secrete cytokines that can further regulate immune responses directly or indirectly. Dendritic cells have the ability to induce primary immune responses in inactive or resting naive T lymphocytes.

[0046] "Immune cell" refers to a cell of the immune system classified as a lymphocyte (T cell, B cell, and NK cell), neutrophil, or monocyte / macrophage, which are any type of white blood cell.

[0047] Muscle cells are cells that make up the various muscle tissues and are classified as skeletal, smooth, and cardiac muscle cells. Skeletal muscle cells are multinucleated and linear. Smooth muscle cells are mononucleated and non-linear. Cardiomyocytes are mononucleated, linear, and found only in the heart. As used herein, "muscle cells" refers to skeletal and smooth muscle cells, not cardiac muscle cells. As used herein, "cardiac cells" refers only to cardiac muscle cells.

[0048] "Vector" refers to a polynucleotide that can replicate in a biological system or can be transferred between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers that function to facilitate the replication or maintenance of these polynucleotides in biological systems. Examples of such biological systems may include cells, viruses, animals, plants, and reconstituted biological systems utilizing biological elements that can replicate the vector. The polynucleotides that comprise the vector may be DNA or RNA molecules, or hybrids thereof.

[0049] An "expression vector" refers to a vector that can be utilized in a biological system, or a reconstituted biological system, to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0050] "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.

[0051] "Polypeptide" or "protein" refers to a molecule containing at least two amino acid residues linked by a peptide bond to form the polypeptide. Small polypeptides, less than about 50 amino acids, may be referred to as "peptides."

[0052] "Valency" refers to the presence of a specific number of binding sites specific to an antigen within a molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence, respectively, of one, two, four, and six binding sites specific to an antigen within a molecule.

[0053] A "subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except where noted, the terms "patient" and "subject" are used interchangeably.

[0054] "Isolated" refers to a homogenous population of a molecule (e.g., a synthetic polynucleotide or a polypeptide such as an FN3 domain) that has been substantially separated and / or purified from other components of the system in which the molecule is produced, e.g., recombinant cells, and 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 includes an FN3 domain that has been isolated to a greater degree 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.

[0055] "Enzyme replacement therapy" or "ERT" refers to the use of a pharmaceutical composition administered to a subject that contains one or more enzymes. In some embodiments, the enzyme is administered to supplement or replace a missing or deficient enzyme in a subject in some aspects. Typically, the one or more enzymes involved in the therapy are administered to the subject by intravenous infusion of a pharmaceutical composition that contains one or more enzymes. As described herein, enzyme replacement therapy can be administered with a composition that contains an FN3 domain that can be linked to an additional therapeutic agent, such as an oligonucleotide (e.g., siRNA, mRNA, cDNA, antisense oligonucleotide, etc.). In some embodiments, administration of enzyme replacement therapy can be before, during, or after administration of a composition that contains an FN3 domain linked to an additional therapeutic agent provided herein.

[0056] In some embodiments, compositions are provided that include a polypeptide, such as a polypeptide that includes an FN3 domain, that can be linked to an oligonucleotide molecule. The oligonucleotide molecule can be, for example, a siRNA molecule, an antisense oligonucleotide, an mRNA, etc.

[0057] Thus, in some embodiments, the siRNA 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 in length from 12 to 40 nucleotides. 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.

[0058] In some embodiments, the sense strand and the antisense strand typically form a double-stranded dsRNA. The double-stranded region of the dsRNA agent can be 12-40 nucleotide pairs in length. For example, the double-stranded region can be 14-40 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotide pairs in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.

[0059] In some embodiments, the dsRNA comprises one or more overhang regions and / or capping groups of the dsRNA agent at the 3' or 5' end or both ends of the strand. The overhang can be 1-10 nucleotides in length, 1-6 nucleotides in length, e.g., 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, 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 twisted. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence. The first and second strands can also be linked, for example, by additional bases to form a hairpin, or by other non-basic linkers.

[0060] In some embodiments, the nucleotides in the overhang region of the dsRNA agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications such as, for example, 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 for either end on either strand. This overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0061] The 5'- or 3'-overhang in 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 mesyl phosphoramidate between the two nucleotides, and the 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, the 3' overhang is present in the antisense strand.In one embodiment, the 3' overhang is present in the sense strand.

[0062] dsRNA agent may only comprise a single overhang, which can enhance the interference activity of dsRNA without affecting its 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 may also have a blunt end, which is 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 wishing to be bound by theory, such an asymmetric blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are favorable for the insertion of the guide strand into RISC.For example, the single overhang comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0063] In some embodiments, a dsRNA agent can also have two blunt ends on either end of the dsRNA duplex.

[0064] 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, which can include one or more of one or both of non-linked phosphate oxygen and / or linked phosphate oxygen, modification of the moiety of ribose sugar, such as the moiety of 2 hydroxyls in ribose sugar, large substitution of phosphate moiety with "dephosphorylation" linker, modification or substitution of naturally occurring base, and substitution or modification of ribose-phosphate backbone.

[0065] In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, by the modifications described herein. Modifications may include, for example, modifications known in the art, such as the use of deoxyribonucleotides, the use of 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modified in place of the ribosugar of the nucleobase, and the use of phosphate group modifications, such as phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate modifications at the 2' position of the ribose sugar. The overhang does not need to be homologous to the target sequence.

[0066] In some embodiments, each residue of 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. A strand may contain more than one modification. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0067] In some embodiments, at least two different modifications are present, typically in the sense and antisense strands. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides or others.

[0068] In one embodiment, the sense strand and the antisense strand each contain two different modified nucleotides selected from 2'-fluoro, 2'-O-methyl, or 2'-deoxy.

[0069] The dsRNA agent may further contain at least one phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate, or methylphosphonate internucleotide linkage. The phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate, or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or the antisense strand, or both, at any position in the strand. For example, the internucleotide linkage modification may occur on all nucleotides on the sense strand and / or the antisense strand, and each internucleotide linkage modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift compared to the alternating pattern of internucleotide linkage modifications on the antisense strand.

[0070] In some embodiments, dsRNA agent comprises phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesyl phosphoramidate or methyl phosphonate internucleotide bond modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesyl phosphoramidate or methyl phosphonate internucleotide bond between two nucleotides.Internucleotide bond modification can also be performed to link overhang nucleotide with terminal pairing nucleotide in double-stranded region. For example, at least 2, 3, 4 or all of the overhanging nucleotides can be linked via phosphorothioate, phosphorodithioate, phosphorodithioate, phosphoramidate, mesyl phosphoramidate or methylphosphonate internucleotide bond, and optionally there can be additional phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesyl phosphoramidate or methylphosphonate internucleotide bond that connects the overhanging nucleotide with the paired nucleotide adjacent to the overhanging nucleotide.For example, there can be at least two phosphorothioate internucleotide bonds between the three nucleotides at the end, two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.In some embodiments, these three nucleotides at the end can be at the 3' end of the antisense strand.

[0071] In some embodiments, dsRNA composition is linked by modified base or nucleoside analogue as described in US Patent No. 7,427,672 (herein incorporated by reference).In some embodiments, modified base or nucleoside analogue is referred to as linker or L in the formula described herein.

[0072] 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 optionally having a substituent; R 1 and R 2 are the same or different, each of which is a hydrogen atom, a protecting group for a hydroxyl 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(R 4 )R 5 (In the formula, R 4 and R 5 are the same or different), each of which 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 an alkyl group having 1 to 5 carbon atoms; R 3 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 substituent of a functional molecular unit, 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.

[0073] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein R 1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by 1 to 3 aryl groups, a methyl group substituted by 1 to 3 aryl groups having an aryl ring substituted by a lower alkyl, lower alkoxy, halogen, or cyano group, or a silyl group.

[0074] In some embodiments, the modified base or nucleoside analog has the structure shown in 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.

[0075] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein R 2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by 1 to 3 aryl groups, a methyl group substituted by 1 to 3 aryl groups having an aryl ring substituted by 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.

[0076] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein R 2 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(OC 2 H 4 CN)(N(i-Pr) 2 ), --P(OCH 3 )(N(i-Pr) 2 ), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenyl phosphate group.

[0077] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein R 3is 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 by 1 to 3 aryl groups, a lower aliphatic or aromatic sulfonyl group such as a methanesulfonyl group or a p-toluenesulfonyl group, an aliphatic acyl group having 1 to 5 carbon atoms such as an acetyl group, or an aromatic acyl group such as a benzoyl group.

[0078] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, wherein R 3 The substituent of the functional molecule unit as a functional molecule is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or intranuclear transport signal peptide.

[0079] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, where 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 α groups: α group: 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 group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0080] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I and salts thereof, where 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, 2-amino-6-fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, -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 having an amino group protected with a protecting group for nucleic acid synthesis -1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 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., uracilinyl), 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 with the amino group protected with a protecting group for nucleic acid synthesis.

[0081] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I, and salts thereof, where m is 0 and n is 1.

[0082] In some embodiments, the modified base or nucleoside analog is a DNA or RNA oligonucleotide analog containing one or more types of unit structures of nucleoside analogs having the structure shown in Formula II, or one or more pharmacologically acceptable salts thereof, provided that the bond between each nucleoside in the oligonucleotide analog is the same phosphodiester bond [--OP(O 2 - )O--] plus one or more phosphorothioate bonds [--OP(O)(S - )O--], phosphorodithioate bond [--O 2 P.S. 2 --], phosphonate bond [--PO(OH) 2 --], phosphoramidate bond [--O=P(OH) 2 --], or mesyl phosphoramidate bond [--OP(O)(N)(SO 2 )(CH 3 )O--], and when two or more of one or more types of these structures are included, the bases may be the same or different between these structures: [ka] In the formula, the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group optionally having a substituent; R 3represents 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 substituent of a functional molecular unit, 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.

[0083] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein R 1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by 1 to 3 aryl groups, a methyl group substituted by 1 to 3 aryl groups having an aryl ring substituted by a lower alkyl, lower alkoxy, halogen, or cyano group, or a silyl group.

[0084] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein R 1 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.

[0085] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein R 2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by 1 to 3 aryl groups, a methyl group substituted by 1 to 3 aryl groups having an aryl ring substituted by 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.

[0086] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein R 2 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(OC 2 H 4 CN)(N(i-Pr) 2 ), --P(OCH 3 )(N(i-Pr) 2 ), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenyl phosphate group.

[0087] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein R 3 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 by 1 to 3 aryl groups, a lower aliphatic or aromatic sulfonyl group such as a methanesulfonyl group or a p-toluenesulfonyl group, an aliphatic acyl group having 1 to 5 carbon atoms such as an acetyl group, or an aromatic acyl group such as a benzoyl group.

[0088] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein R 3 The substituent of the functional molecule unit as a functional molecule is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or intranuclear transport signal peptide.

[0089] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, where 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 α groups: α group: 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 group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0090] In some embodiments, the oligonucleotide analog or a pharma- ceutically 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, 2-amino-6-fluoropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis; 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) having an amino group protected by a protecting group for nucleic acid synthesis; 2-amino-6-hydroxypurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis; -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, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl with amino groups protected with protecting groups for nucleic acid synthesis 1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group with amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxypurine-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. uracilyl), 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 with the amino group protected with a protecting group for nucleic acid synthesis.

[0091] In some embodiments, the oligonucleotide analog or a pharma- ceutical acceptable salt thereof has the structure shown in Formula II, wherein m is 0 and n is 1.

[0092] In some embodiments, the compositions described herein further comprise a polymer (polymer moiety C). In some cases, the polymer is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of monomers in two or three dimensions. 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, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalat (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, mixtures refer to the use of different polymers within the same compound, not just in relation to block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some cases, the polymer comprises a polyalkylene oxide. In some cases, the polymer comprises PEG. In some cases, the polymer comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0093] In some cases, C is a PEG moiety. In some cases, 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 cases, 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 cases, the PEG moiety is conjugated to an internal site of the oligonucleotide molecule. In some cases, the PEG moiety, the linking moiety, or a combination thereof is conjugated to an internal site of the oligonucleotide molecule. In some cases, the conjugation is direct conjugation. In some cases, the conjugation is performed via native ligation.

[0094] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, a polydisperse material comprises a disperse distribution of different molecular weights of material, characterized by average weight (weight average) size and dispersity. In some cases, 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.

[0095] 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.

[0096] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and 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, 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 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, 850 In some embodiments, the C has a molecular weight of about 00, 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, the C has a molecular weight of about 200 Da. In some embodiments, the C has a molecular weight of about 300 Da. In some embodiments, the C has a molecular weight of about 400 Da. In some embodiments, the C has a molecular weight of about 500 Da. In some embodiments, the C has a molecular weight of about 600 Da. In some cases, the molecular weight of C is about 700 Da. In some cases, the molecular weight of C is about 800 Da. In some cases, the molecular weight of C is about 900 Da. In some cases, the molecular weight of C is about 1000 Da. In some cases, the molecular weight of C is about 1100 Da. In some cases, the molecular weight of C is about 1200 Da. In some cases, the molecular weight of C is about 1300 Da. In some cases, the molecular weight of C is about 1400 Da.In some cases, the molecular weight of C is about 1450 Da. In some cases, the molecular weight of C is about 1500 Da. In some cases, the molecular weight of C is about 1600 Da. In some cases, the molecular weight of C is about 1700 Da. In some cases, the molecular weight of C is about 1800 Da. In some cases, the molecular weight of C is about 1900 Da. In some cases, the molecular weight of C is about 2000 Da. In some cases, the molecular weight of C is about 2100 Da. In some cases, the molecular weight of C is about 2200 Da. In some cases, the molecular weight of C is about 2300 Da. In some cases, the molecular weight of C is about 2400 Da. In some cases, the molecular weight of C is about 2500 Da. In some cases, the molecular weight of C is about 2600 Da. In some cases, the molecular weight of C is about 2700 Da. In some cases, the molecular weight of C is about 2800 Da. In some cases, the molecular weight of C is about 2900 Da. In some cases, the molecular weight of C is about 3000 Da. In some cases, the molecular weight of C is about 3250 Da. In some cases, the molecular weight of C is about 3350 Da. In some cases, the molecular weight of C is about 3500 Da. In some cases, the molecular weight of C is about 3750 Da. In some cases, the molecular weight of C is about 4000 Da. In some cases, the molecular weight of C is about 4250 Da. In some cases, the molecular weight of C is about 4500 Da. In some cases, the molecular weight of C is about 4600 Da. In some cases, the molecular weight of C is about 4750 Da. In some cases, the molecular weight of C is about 5000 Da. In some cases, the molecular weight of C is about 5500 Da. In some cases, the molecular weight of C is about 6000 Da. In some cases, the molecular weight of C is about 6500 Da. In some cases, the molecular weight of C is about 7000 Da. In some cases, the molecular weight of C is about 7500 Da. In some cases, the molecular weight of C is about 8000 Da. In some cases, the molecular weight of C is about 10,000 Da. In some cases, the molecular weight of C is about 12,000 Da. In some cases, the molecular weight of C is about 20,000 Da. In some cases, the molecular weight of C is about 35,000 Da. In some cases, the molecular weight of C is about 40,000 Da.In some cases, the molecular weight of C is about 50,000 Da. In some cases, the molecular weight of C is about 60,000 Da. In some cases, the molecular weight of C is about 100,000 Da.

[0097] In some embodiments, the polyalkylene oxide (e.g., PEG) is a separate PEG, and the separate PEG is a polymeric PEG containing more than one repeating ethylene oxide unit. In some cases, the separate PEG (dPEG) contains from 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some examples, 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 cases, the dPEG contains about 2 or more repeating ethylene oxide units. In some cases, the dPEG contains about 3 or more repeating ethylene oxide units. In some cases, the dPEG contains about 4 or more repeating ethylene oxide units. In some cases, the dPEG contains about 5 or more repeating ethylene oxide units. In some cases, the dPEG contains about 6 or more repeating ethylene oxide units. In some cases, the dPEG contains about 7 or more repeating ethylene oxide units. In some cases, the dPEG contains about 8 or more repeating ethylene oxide units. In some cases, the dPEG contains about 9 or more repeating ethylene oxide units. In some cases, the dPEG contains about 10 or more repeating ethylene oxide units. In some cases, the dPEG contains about 11 or more repeating ethylene oxide units. In some cases, the dPEG contains about 12 or more repeating ethylene oxide units. In some cases, the dPEG contains about 13 or more repeating ethylene oxide units. In some cases, the dPEG contains about 14 or more repeating ethylene oxide units. In some cases, the dPEG contains about 15 or more repeating ethylene oxide units. In some cases, the dPEG contains about 16 or more repeating ethylene oxide units. In some cases, the dPEG contains about 17 or more repeating ethylene oxide units. In some cases, the dPEG contains about 18 or more repeating ethylene oxide units. In some cases, the dPEG contains about 19 or more repeating ethylene oxide units. In some cases, the dPEG contains about 20 or more repeating ethylene oxide units. In some cases, the dPEG contains about 22 or more repeating ethylene oxide units.In some cases, dPEG contains about 24 or more repeating ethylene oxide units. In some cases, dPEG contains about 26 or more repeating ethylene oxide units. In some cases, dPEG contains about 28 or more repeating ethylene oxide units. In some cases, dPEG contains about 30 or more repeating ethylene oxide units. In some cases, dPEG contains about 35 or more repeating ethylene oxide units. In some cases, dPEG contains about 40 or more repeating ethylene oxide units. In some cases, dPEG contains about 42 or more repeating ethylene oxide units. In some cases, dPEG contains about 48 or more repeating ethylene oxide units. In some cases, dPEG contains about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials in a stepwise manner. In some cases, 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.

[0098] In some embodiments, C is an albumin binding domain. In certain aspects, the albumin binding domain specifically binds serum albumin, e.g., human serum albumin (HSA), to extend the half-life of the domain or another therapeutic agent to which the albumin binding domain is associated or attached. 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 extending molecule, such as PEG, Fc region, etc.

[0099] In some embodiments, the albumin binding domain comprises the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, as provided in Table 1 below. 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%, or 99% identical to or is an amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the albumin binding domain has a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. The amino acid sequence of any of the present invention may be 95%, 96%, 97%, 98%, or 99% identical to or with the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, provided that the protein has a substitution corresponding to position 10 of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. 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 when compared to the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.In some embodiments, the substitution is at a position corresponding to position 10 of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the provided FN3 domains comprise a cysteine ​​residue at at least one residue position corresponding to residue position 6, 11, 22, 25, 26, 52, 53, 61, 88, or position 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: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or at the C-terminus. Although the positions are listed in a series, each position can also be selected individually. In some embodiments, the cysteine ​​is at a position corresponding to position 6, 53, or 88. In some embodiments, additional examples of albumin binding domains can be found in U.S. Pat. No. 10,925,932, which is incorporated herein by reference. [Table 1-1] [Table 1-2]

[0100] In some embodiments, the dsRNA agent contains mismatch(es) with the target, within the duplex, or combinations 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., relative to the free energy of association or dissociation of a particular pairing; the simplest approach is to evaluate pairs on an individual pair basis, but affinity 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, e.g., non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical pairings (A:T, A:U, G:C), and base pairs containing universal bases are preferred over canonical pairings.

[0101] In some embodiments, a dsRNA agent can include a phosphorus-containing group at the 5' end of the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS 2 ), 5'-terminal vinyl phosphonate (5'-VP), 5'-terminal methyl phosphonate (MePhos), 5'-terminal mesyl phosphoramidate (5'MsPA), or 5'-deoxy-5'-C-malonyl. When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP can be either a 5'-E-VP isomer, such as trans-vinyl phosphate or cis-vinyl phosphate, or a mixture thereof. Representative structures of these modifications can be found, for example, in U.S. Pat. No. 10,233,448, which is incorporated herein by reference in its entirety.

[0102] 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 comprises H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, C 1 -C 10 The alkyl moiety may further include, but is not limited to, both straight and branched chain lengths. In some cases, the alkyl moiety further includes modifications. In some cases, the modifications include azo, keto, aldehyde, carboxyl, nitro, nitroso, nitrile, heterocyclic (e.g., imidazole, hydrazine, or hydroxylamino) groups, isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some cases, the alkyl moiety further includes additional heteroatoms such as O, S, N, 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 replaced by nitrogen, oxygen, or sulfur. In some cases, the heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0103] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl or 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]

[0104] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group containing a propyl linker connects 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 the cellular uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is shown below. [ka]

[0105] 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, thus forming a 2'-C, 4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. Exemplary depictions of the chemical structure of LNA are shown below. The depiction shown on the left highlights the chemical connectivity of the LNA monomer. The depiction shown on the right highlights the locked 3'-endo (3E) conformation of the furanose ring of the LNA monomer. [ka]

[0106] In some cases, the modification at the 2' hydroxyl group includes an ethylene nucleic acid (ENA), such as, for example, a 2'-4'-ethylene bridged nucleic acid, which locks the sugar conformation into a C3'-endo sugar puckering conformation. ENA is part of the bridged nucleic acid group of modified nucleic acids, which also includes LNA. Exemplary chemical structures of ENA and bridged nucleic acids are shown below. [ka]

[0107] 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).

[0108] In some embodiments, the nucleotide analogs are 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2 propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 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-azuridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiocyt ... Modified bases include, but are not limited to, uridine, 2-thiouridine and 4-thiouridine and other thio bases such as 2-thiocytidine, dihydrouridine, pseudouridine, queosine, 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 uracil and thymine, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moiety is, or is based on, mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles, as the case may be. 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 nebularine.

[0109] In some embodiments, the nucleotide analogue further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics natural nucleic acid structures by deviations from the normal 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, the ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In such cases, the backbone modification allows the removal of all positive and negative charges, making the morpholino a neutral molecule that can cross cell membranes without the aid of a cell delivery agent, as used by charged oligonucleotides. [ka]

[0110] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar rings or phosphate linkages, and the bases are attached to and appropriately arranged by oligoglycine-like molecules, thus eliminating the backbone charge. [ka]

[0111] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some cases, the modified internucleotide bond may be phosphorothioate, mesyl phosphoramidate, phosphorodithioate, methyl phosphonate, 5'-alkylene phosphonate, 5'-methyl phosphonate, 3'-alkylene phosphonate, boron trifluoride, 3'-5' or 2'-5' boranophosphate and selenophosphate, phosphotriester, thionoalkyl phosphotriester, phosphonic acid hydrogen bond, alkyl phosphonate, alkyl phosphonothioate, aryl phosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkyl phosphoramidate, aminoalkyl phosphoramidate, thionophosphoramidate, phosphoropiperazide. Examples of suitable antisense oligonucleotides include, but are not limited to, phosphoroanylthioates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethylhydrazo, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamidates, bonds with riboacetyl groups, aminoethylglycines, silyl or siloxane bonds, alkyl or cycloalkyl bonds, such as saturated or unsaturated and / or substituted and / or containing heteroatoms, with or without heteroatoms, of 1 to 10 carbons, bonds with morpholino structures, amides, polyamides in which the bases are directly or indirectly bound to the azanitrogen backbone, and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate bonds. Mesyl phosphoramidate antisense oligonucleotides (MsPA ASOs) are antisense oligonucleotides containing mesyl phosphoramidate bonds.

[0112] In some cases, the modification is a methyl or thiol modification, such as a methyl phosphonate, mesyl phosphoramidate, or thiol phosphonate modification. In some cases, the modified nucleotide includes, but is not limited to, a 2'-fluoro N3-P5'-phosphoramidite.

[0113] In some cases, the modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)).

[0114] In some embodiments, the one or more modifications optionally further include modifications of the ribose moiety, the phosphate backbone and the nucleoside, or modifications of the nucleotide analogue at the 3' or 5' end. For example, the 3' end optionally includes a 3' cationic group or by inverting the nucleoside at the 3' end with a 3'-3' bond. In another alternative, the 3' end is optionally conjugated with an amino alkyl group, e.g., a 3' C5-amino alkyl dT. In a further alternative, the 3' end is optionally conjugated with an abasic site, e.g., an apurinic or apyrimidinic site. In some cases, the 5' end is conjugated with an amino alkyl group, e.g., a 5'-O-alkyl amino substituent. In some cases, the 5' end is conjugated with an abasic site, e.g., an apurinic or apyrimidinic site.

[0115] In some embodiments, the oligonucleotide molecule comprises one or more of the 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, synthetic nucleotide analogs include 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, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations 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 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'-fluoro N3-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. 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. 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.

[0116] In some cases, the oligonucleotide molecule comprises at least one of about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification. In some cases, the oligonucleotide molecule comprises 100% modification.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] In some cases, the oligonucleotide molecule contains at least one of about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0123] In some cases, the oligonucleotide molecule contains at least one of about 10% to about 30% modifications and about 20% to about 30% modifications.

[0124] In some cases, the oligonucleotide molecules contain about 10% to about 20% modifications.

[0125] In some cases, the oligonucleotide molecule contains from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

[0126] In additional cases, the oligonucleotide molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0127] 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.

[0128] 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.

[0129] 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 include a synthetic nucleotide analog as described herein. In some cases, about 35% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 40% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 45% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 50% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 55% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 60% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 65% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 70% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 75% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein. In some cases, about 80% of the oligonucleotide molecules include a synthetic nucleotide analog as described herein.In some cases, about 85% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 90% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 95% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 96% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 97% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 98% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 99% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 100% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some embodiments, synthetic nucleotide analogs include 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, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0130] In some embodiments, the oligonucleotide molecule comprises from about 1 to about 25 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 1 modification, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 2 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 3 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 4 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 5 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 6 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 7 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 8 modifications, the modifications comprising a synthetic nucleotide analog described herein. In some embodiments, the oligonucleotide molecule comprises about 9 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 10 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 11 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 12 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 13 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 14 modifications, which include synthetic nucleotide analogs as described herein.In some embodiments, the oligonucleotide molecule comprises about 15 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 16 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 17 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 18 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 19 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 20 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 21 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 22 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 23 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 24 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 25 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 26 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 27 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 28 modifications, which include synthetic nucleotide analogs as described herein.In some embodiments, the oligonucleotide molecule comprises about 29 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 30 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 31 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 32 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 33 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 34 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 35 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 36 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 37 modifications, which include synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 38 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 39 modifications, which include synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 40 modifications, which include synthetic nucleotide analogs as described herein.

[0131] In some embodiments, the oligonucleotide molecule is assembled from two separate polynucleotides, one polynucleotide comprises the sense strand and the second 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 may be a polynucleotide linker or a non-nucleotide linker.

[0132] In some embodiments, the 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, the 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.

[0133] 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.

[0134] 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.

[0135] 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 has a plurality (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 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 nucleotides. 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, 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, at least four 2'-deoxy-2'-fluoro modified nucleotides at the 3'-end of at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at the 5'-end of the polynucleotide, or at least four, at least five, 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, at least four 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides.

[0136] 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 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.

[0137] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, one of which has at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides located at its 5'-terminus, while the other strand has at least two consecutive 2'-O-methyl modified nucleotides located at its 5'-terminus. In some embodiments, when a strand has at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides located at its 5'-terminus, the strand also comprises at least two, at least three consecutive 2'-O-methyl modified nucleotides at the 3'-terminus 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'-terminus and / or 3'-terminus. In some embodiments, one of the sense strand and the antisense strand has at least 4, at least 5 nucleotides with alternating 2'-O-methyl and 2'-deoxy-2'-fluoro modified nucleotides.

[0138] In some embodiments, an oligonucleotide molecule, such as a siRNA, is shown in Formula I: [ka] where each nucleotide represented by N is independently A, U, C, or G, or a modified nucleotide base such as those provided herein. 1 The nucleotides represent the 5' end of each strand. For clarity, Formula I represents N 1 , N 2 , N 3 etc., although the nucleotide bases need not, and are not intended to, be identical. The siRNA shown in Formula I will be complementary to the target sequence.

[0139] For example, in some embodiments, the sense strand is 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, as well as N 4 , N 5 , N 6 , N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 , and N 19 contains 2'O-methyl modified nucleotides.

[0140] In some embodiments, the antisense strand is 1 A vinyl phosphonate moiety bound to N 2 2' fluoro-modified nucleotide with phosphorothioate (PS) modified backbone at N 3 , N 4 , N5 , 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, as well as N 20 and N 21 contains 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones.

[0141] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and 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.

[0142] 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.

[0143] 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 mesylphosphoramidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2′-deoxy groups, 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 at both the 3′-terminus and the 5′-terminus of the sense strand. and 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 mesylphosphoramidate 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, a terminal cap molecule at the 3'-terminus, 5'-terminus, or both the 3'- 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 strand are chemically modified with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without a terminal cap molecule, at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus, present on the same or different strands.

[0144] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand has 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 mesyl phosphoramidate 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 at both the 3′-terminus and the 5′-terminus of the sense strand. At both ends, the antisense strand contains 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 mesylphosphoramidate 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, a terminal cap molecule at the 3'-end, 5'-end, or both the 3'- and 5'-ends 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 strand are chemically modified with 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 mesylphosphoramidate internucleotide linkages, and / or 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without a terminal cap molecule, at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0145] 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 phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages and / or at the 3'-terminus of the sense strand and / or the antisense strand. , 5'-terminus, or both the 3'- and 5'-terminus of the sense strand, 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, an end cap molecule at the 3'-terminus, 5'-terminus, or both the 3'- 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 mesylphosphoramidate 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 nucleotides, 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'- 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without a terminal cap molecule, at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0146] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand has 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 mesylphosphoramidate 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 at the 3′- and 5′-terminus of the sense strand. 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, phosphononate, phosphoramidate, or mesyl phosphoramidate internucleotide linkages, and / or one or more (e.g., The antisense strand may comprise 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'-end, 5'-end, or both the 3'- and 5'-ends 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 strand(s) are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoronucleotides, with about 1 to about 5, e.g., about 1, 2, 3, 4, 5, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate nucleotide linkages, and / or in the presence or absence of terminal cap molecules at the 3'-end, 5'-end, or both 3'- and 5'-ends, present in the same or different strands.

[0147] 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 mesylphosphoramidate 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 a phosphorothioate. In some cases, the phosphate backbone modification is a phosphorodithioate. In some cases, the phosphate backbone modification is a phosphonate. In some embodiments, the phosphate backbone modification is phosphoramidate. In some embodiments, the phosphate backbone modification is mesyl phosphoramidate. In some embodiments, the sense or antisense strand has three consecutive nucleosides linked through two phosphorothioate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides linked through two phosphorodithioate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides linked through two phosphonate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides linked through two phosphoramidate backbones. In some embodiments, the sense or antisense strand has three consecutive nucleosides linked through two mesyl phosphoramidate backbones.

[0148] In another embodiment, the oligonucleotide molecules described herein comprise 2'-5' internucleotide bond. In some cases, the 2'-5' internucleotide bond(s) are at the 3' end, the 5' end, or both the 3' end and the 5' end of one or both strands of sequence. In some cases, the 2'-5' internucleotide bond(s) are at various other positions within one or both strands of sequence, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, including all internucleotide bonds of pyrimidine nucleotides of one or both strands of the oligonucleotide molecule, comprise 2'-5' internucleotide bond, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, including all internucleotide bonds of purine nucleotides of one or both strands of the oligonucleotide molecule, comprise 2'-5' internucleotide bond.

[0149] In some embodiments, the oligonucleotide molecule is a single-stranded molecule that mediates RNAi activity in a cell or is reconstituted in an 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 alternating multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and any purine nucleotides present in the oligonucleotide molecule are 2'-deoxy purine nucleotides (e.g., all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides). all purine nucleotides are 2'-deoxy purine nucleotides, or alternating multiple purine nucleotides are 2'-deoxy purine nucleotides), and a terminal cap modification optionally present at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense sequence, the oligonucleotide molecule optionally further comprises about one to about four (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3'-terminus of the oligonucleotide molecule, the terminal nucleotides further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate or mesyl phosphoramidate internucleotide linkages, and the oligonucleotide molecule optionally further comprises a terminal phosphate group, such as a 5' terminal phosphate group.

[0150] In some cases, one or more of the synthetic nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNases, or exonucleases such as 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) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, can be resistant to nucleases, e.g., RNase resistant to ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some cases, 2'-O-methyl modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some cases, 2'O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some cases, 2'-O-aminopropyl modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant).In some cases, the 2'-deoxy modified oligonucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 2'-deoxy-2'-fluoro modified oligonucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 2'-ON-methylacetamide (2'-O-NMA) modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the LNA modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the ENA modified oligonucleotide molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant).In some cases, the HNA modified oligonucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the morpholino is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the PNA modified oligonucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the methylphosphonate nucleotide modified oligonucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the thiol phosphonate nucleotide 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.

[0151] 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 of the synthetic nucleotide analogues, 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) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites, have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleic acid molecules. In some cases, 2'-O-methyl modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some cases, 2'-O-aminopropyl modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some cases, 2'-deoxy modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent naturally occurring polynucleic acid molecule.In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, LNA modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, ENA modified oligonucleotide molecules have increased binding affinity to their mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, the PNA modified oligonucleotide molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some cases, the HNA modified oligonucleotide molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some cases, the morpholino modified oligonucleotide molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some cases, the methylphosphonate nucleotide modified oligonucleotide molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some cases, the thiol phosphonate nucleotide modified oligonucleotide molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some cases, the oligonucleotide molecule containing 2'-fluoro N3-P5'-phosphoramidite has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule.In some cases, increased affinity is manifested by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0152] In some embodiments, the oligonucleotide molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules that contain a single enantiomer. In some cases, the oligonucleotide molecules contain L-nucleotides. In some cases, the oligonucleotide molecules contain D-nucleotides. In some cases, the oligonucleotide molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some cases, the oligonucleotide molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of racemic mixture.

[0153] In some embodiments, the oligonucleotide molecules described herein are further modified to include an aptamer-conjugated portion.In some cases, the aptamer-conjugated portion is a DNA aptamer-conjugated portion.In some cases, the aptamer-binding portion is an alphamer that includes an aptamer portion that recognizes a specific cell surface target and a portion that presents a specific epitope for binding to circulating antibodies.

[0154] In additional embodiments, the oligonucleotide molecule described herein is modified to increase its stability. In some embodiments, the oligonucleotide molecule is RNA (e.g., siRNA). In some cases, the oligonucleotide molecule is modified by one or more of the above modifications to increase its stability. In some cases, the oligonucleotide molecule is modified at the 2' hydroxyl position, for example, by 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, or by locked or bridged ribose conformation (e.g., LNA or ENA). In some cases, the oligonucleotide molecule is modified by 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the oligonucleotide molecule also includes morpholino, PNA, HNA, methyl phosphonate nucleotide, thiol phosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability. In some cases, the oligonucleotide molecule is a chirally pure (or stereopure) oligonucleotide molecule. In some cases, the chirally pure (or stereopure) oligonucleotide molecule is modified to increase its stability. Suitable modifications to RNA to increase stability for delivery will be apparent to those skilled in the art.

[0155] In some embodiments, the oligonucleotide molecule comprises a 2' modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 3, 7, 8, 9, 12, and 17 from the 5' end of the sense strand are not modified with a 2'O-methyl modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 3, 7, 8, 9, 12, and 17 from the 5' end of the sense strand are modified with a 2'fluoro modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 2 and 14 from the 5' end of the antisense strand are not modified with a 2'O-methyl modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 2 and 14 from the 5' end of the antisense strand 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 of the oligonucleotide molecule at positions 1 and 2 from the 5' end of the sense strand 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 may 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.

[0156] In some cases, the oligonucleotide molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence that corresponds to a target nucleic acid sequence or a portion thereof. In some cases, the oligonucleotide molecule is assembled from two separate polynucleotides, one strand being a sense strand, and the other strand being an antisense strand and a sense strand that are self-complementary (e.g., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand, e.g., when the antisense strand and the sense strand form a duplex 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 that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence that corresponds to a target nucleic acid sequence or a portion thereof. Alternatively, the oligonucleotide molecule is assembled from a single oligonucleotide, the self-complementary sense and antisense regions of which are linked by a nucleic acid-based or non-nucleic acid-based linker(s).

[0157] In some cases, oligonucleotide molecule is a polynucleotide having double stranded, asymmetric double stranded, hairpin or asymmetric hairpin secondary structure with self-complementary sense and antisense regions, antisense region comprises nucleotide sequence complementary to nucleotide sequence in separate target nucleic acid molecule or a part thereof, and sense region comprises nucleotide sequence corresponding to target nucleic acid sequence or a part thereof.In other cases, oligonucleotide molecule is a circular single stranded polynucleotide having two or more loop structures, and a stem comprising self-complementary sense and antisense regions, antisense region comprises nucleotide sequence complementary to nucleotide sequence in target nucleic acid molecule or a part thereof, and sense region comprises nucleotide sequence corresponding to target nucleic acid sequence or a part thereof, and circular polynucleotide is processed either in vivo or in vitro to generate active oligonucleotide molecule that can mediate RNAi. In additional cases, the oligonucleotide molecule also includes a single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., where such an oligonucleotide molecule does not require the presence within the oligonucleotide molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), which single-stranded polynucleotide further includes a terminal phosphate group, e.g., a 5'-phosphate group, or a 5',3'-diphosphate group.

[0158] In some cases, an asymmetric hairpin is a linear oligonucleotide molecule that includes an antisense region, a loop portion that includes nucleotides or non-nucleotides, and a sense region that includes fewer nucleotides than the antisense region, to the extent that the sense region has sufficient complementary nucleotides to base pair with the antisense region and form a duplex with the loop. For example, an asymmetric hairpin oligonucleotide molecule includes an antisense region having a sufficient length (e.g., about 19 to about 22 nucleotides) to mediate RNAi in a cell or in vitro system, a loop region that includes about 4 to about 8 nucleotides, and a sense region that has about 3 to about 18 nucleotides complementary to the antisense region. In some cases, the asymmetric hairpin oligonucleotide molecule also includes a chemically modified 5'-terminal phosphate group. In additional cases, the loop portion of the asymmetric hairpin oligonucleotide molecule includes nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0159] 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 contains fewer nucleotides than the antisense region, to the extent that the sense region has sufficient complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex oligonucleotide molecule includes an antisense region having a sufficient length (e.g., about 19 to about 22 nucleotides) to mediate RNAi in a cell or in vitro system, and a sense region having about 3 to about 19 nucleotides complementary to the antisense region.

[0160] In some cases, universal bases refer to nucleotide base analogs that base pair with each of the natural DNA / RNA bases with little discrimination between them. 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.

[0161] In some embodiments, the dsRNA agent is 5' phosphorylated or contains a phosphoryl analog at the 5' prime end. 5' phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO 2 (O)P--O-5'); 5'-diphosphate ((HO) 2 (O)P--O--P(HO)(O)--O-5'); 5'-triphosphate ((HO) 2 (O)P--O--(HO)(O)P--O--P(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 modified or unmodified nucleotide cap structure (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'-phosphorothioate ((HO)2(O)P--S-5'); phosphorodithioate [--O 2 P.S. 2 --];phosphonate [--PO(OH) 2 --]; phosphoramidate [--O=P(OH) 2 --]; mesyl phosphoramidate (CH 3 )(SO 2 )(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)(NH 2)(O)P--O-5'), 5'-alkyl phosphonates (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'-alkylphosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)--O-5'-). In some embodiments, the modifications can be placed in the antisense strand of a dsRNA agent.

[0162] 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 the target sequence of GYS1. In some embodiments, the target sequence of GYS1 is a nucleic acid sequence of about 10-50 base pairs in length, about 15-50 base pairs in length, 15-40 base pairs in length, 15-30 base pairs in length, or 15-25 base pairs in length in GYS1, and the first nucleotide of the target sequence starts at any nucleotide in the GYS1 mRNA transcript in the coding region or in the 5' or 3' untranslated region (UTR). For example, the first nucleotide of the target sequence can be selected such that it begins at nucleic acid position (nal, a number starting from the 5' end of the full length of GYS1 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 GYS1 mRNA. In some embodiments, the first nucleotide of the target sequence is selected from the group consisting of nal 10 to nal 15, nal 10 to nal 20, nal 50 to nal 60, nal 55 to nal 65, nal 75 to nal 85, nal 95 to nal 105, nal 135 to nal 145, nal 155 to nal 165, nal 225 to nal 235, nal 265 to nal 275, nal 275 to nal 245, nal 245 to nal 255, nal 285 to nal 335, nal 335 to nal 345, nal 385 to nal 395, nal 515 to nal 525, nal 665 to nal 675, nal 675 to nal 685, nal 695 to nal 705, nal 705 to nal 715, 875~nal 885, nal 885~nal 895, nal 895~nal 905, nal 1035~nal 1045, nal 1045~nal 1055, nal 1125~nal 1135, nal1135~nal 1145, nal 1145~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 The sequence may be selected to begin at a position within or between 2600-2700, nal 2700-2800, nal 2800-2500, nal 2500-2600, nal 2600-2700, nal 2700-2800, nal 2800-2860, etc. In some embodiments, the sequence of GYS1 mRNA is provided as NCBI Reference Sequence: NM_002103.

[0163] In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA, hybridizes to it, and inhibits its expression via RNA interference. The target RNA can be any RNA expressed in a cell. In another embodiment, the cell is a tumor cell, a liver cell, a muscle cell, an immune cell, a dendritic cell, a cardiac cell, or a cell of the central nervous system. In another embodiment, 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 GYS1 RNA. In some embodiments, the siRNA molecule is an siRNA that reduces the expression of GYS1. In some embodiments, the siRNA molecule is an siRNA that reduces expression of GYS and does not reduce expression of other RNAs by more than 50% in the assays described herein at concentrations of 200 nM or less as described herein.

[0164] The siRNA can be targeted to any gene or RNA (eg, mRNA) transcript of interest.

[0165] Other modifications and modification patterns can be found, for example, in US Pat. No. 10,233,448, which is incorporated herein by reference.

[0166] Other modifications and modification patterns can be found, for example, in Anderson et al. (Nucleic Acids Research, 2021, 49(16), 9026-9041) (incorporated herein by reference).

[0167] Other modifications and modification patterns can be found, for example, in International Patent Application Publication No. WO2021 / 030778, which is incorporated herein by reference.

[0168] Other modifications and modification patterns can be found, for example, in International Patent Application Publication No. WO2021 / 030763, which is incorporated herein by reference.

[0169] In some embodiments, siRNA is linked to a 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, a linker is linked to the sense strand, which is used to facilitate the linking of the sense strand to the FN3 domain.

[0170] In some embodiments, (X1) n -(X2) q -(X3) yProvided herein are compositions having the formula of -L-X4, where X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extending molecule, L is a linker, X4 is a nucleic acid molecule, such as, but not limited to, an siRNA molecule, and 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, the third FN3 domain increases the half-life of the molecule as a whole compared to a molecule without X3. In some embodiments, the half-life extending moiety is an FN3 domain that binds albumin. Examples of such FN3 domains include, but are not limited to, those described in US Patent Publication No. 20170348397 and US Patent No. 9,156,887 (incorporated herein by reference in their entirety). FN3 domains may incorporate other subunits, for example, via covalent interactions. 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, 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 may incorporate a second FN3 domain that binds to a molecule that extends the half-life of the entire molecule, including, 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.

[0171] In some embodiments, compositions are provided herein having a formula of (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 a siRNA molecule. In some embodiments, X1 is a FN3 domain that binds to one of CD71. In some embodiments, X2 is a FN3 domain that binds to one of 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.

[0172] 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, L is a linker, X4 is an oligonucleotide molecule, C is a polymer, and n, q, and y are each independently 0 or 1.

[0173] In some embodiments, (X1) n -(X2) q [L-X4]-(X3) y Provided herein are compositions having the formula: -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 an oligonucleotide molecule, C is a polymer, and n, q, and y are each independently 0 or 1.

[0174] 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, L is a linker, X4 is an oligonucleotide molecule, C is a polymer, and n, q, and y are each independently 0 or 1.

[0175] In some embodiments, (X1) n -(X2) q [L-X4]L-(X3) y Provided herein are compositions having the formula: -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 an oligonucleotide molecule, C is a polymer, and n, q, and y are each independently 0 or 1.

[0176] In some embodiments, A 1 -B 1 A composition or complex having the formula: 1 CL 1 -X s and B 1 X AS -L 2 -F 1 and having the formula C is a polymer such as PEG; L 1 and L 2 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, F 1 is a polypeptide comprising at least one FN3 domain, In the formula, X S and X AS forms a double stranded oligonucleotide molecule to form the composition / complex.

[0177] In some embodiments, C can be a molecule that extends the half-life of a molecule. Examples of such moieties are described herein. 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 to a cell, endosome, or ER, and the molecule is selected from those peptides listed in Table 2 below. [Table 2]

[0178] In some embodiments, A 1 -B 1 A composition or complex having the formula: 1 X s and B 1 X AS -L 2 -F 1 It has the formula:

[0179] In some embodiments, A 1 -B 1 A composition or complex having the formula: 1 CL 1 -X s and B 1 X AS It has the formula:

[0180] In some embodiments, the sense strand is a sense strand provided herein.

[0181] In some embodiments, the antisense strand is an antisense strand provided herein.

[0182] In some embodiments, the sense and antisense strands form a double-stranded siRNA molecule targeting GYS1. In some embodiments, the double-stranded oligonucleotide is about 21-23 nucleotide base pairs in length. In certain embodiments, C is optional.

[0183] In some embodiments, A 1 -B 1 A composition or complex having the formula: 1 is F 1 -L 1 -X s and B 1 X AS -L 2 -C, F 1 is a polypeptide comprising at least one FN3 domain, L 1 and L 2 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, In the formula, X S and X AS forms a double stranded oligonucleotide molecule to form the composition / complex. In certain embodiments, C is optional.

[0184] In some embodiments, A 1 -B 1 A composition or complex having the formula: 1 X s and B 1 X AS -L2 -C.

[0185] In some embodiments, A 1 -B 1 A composition or complex having the formula: 1 F 1 -L 1 -X s B has the formula 1 X AS It has the formula:

[0186] In some embodiments, C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of monomers in two or three dimensions. In some cases, the polymer comprises a polysaccharide, lignin, rubber, or polyalkylene oxide (which may be, for example, polyethylene glycol). In some examples, 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 terephthalat (PET, PETG), polyethylene-BB terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane, as well as mixtures thereof. As used herein, mixture refers to the use of different polymers within the same compound, as well as in reference to block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some cases, the polymer comprises a polyalkylene oxide. In some cases, the polymer comprises PEG. In some cases, the polymer comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0187] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, a polydisperse material comprises a disperse distribution of different molecular weights of material, characterized by average weight (weight average) size and dispersity. In some cases, 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.

[0188] 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.

[0189] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and 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, 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 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, 850 In some embodiments, the C has a molecular weight of about 00, 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, the C has a molecular weight of about 200 Da. In some embodiments, the C has a molecular weight of about 300 Da. In some embodiments, the C has a molecular weight of about 400 Da. In some embodiments, the C has a molecular weight of about 500 Da. In some embodiments, the C has a molecular weight of about 600 Da. In some cases, the molecular weight of C is about 700 Da. In some cases, the molecular weight of C is about 800 Da. In some cases, the molecular weight of C is about 900 Da. In some cases, the molecular weight of C is about 1000 Da. In some cases, the molecular weight of C is about 1100 Da. In some cases, the molecular weight of C is about 1200 Da. In some cases, the molecular weight of C is about 1300 Da. In some cases, the molecular weight of C is about 1400 Da.In some cases, the molecular weight of C is about 1450 Da. In some cases, the molecular weight of C is about 1500 Da. In some cases, the molecular weight of C is about 1600 Da. In some cases, the molecular weight of C is about 1700 Da. In some cases, the molecular weight of C is about 1800 Da. In some cases, the molecular weight of C is about 1900 Da. In some cases, the molecular weight of C is about 2000 Da. In some cases, the molecular weight of C is about 2100 Da. In some cases, the molecular weight of C is about 2200 Da. In some cases, the molecular weight of C is about 2300 Da. In some cases, the molecular weight of C is about 2400 Da. In some cases, the molecular weight of C is about 2500 Da. In some cases, the molecular weight of C is about 2600 Da. In some cases, the molecular weight of C is about 2700 Da. In some cases, the molecular weight of C is about 2800 Da. In some cases, the molecular weight of C is about 2900 Da. In some cases, the molecular weight of C is about 3000 Da. In some cases, the molecular weight of C is about 3250 Da. In some cases, the molecular weight of C is about 3350 Da. In some cases, the molecular weight of C is about 3500 Da. In some cases, the molecular weight of C is about 3750 Da. In some cases, the molecular weight of C is about 4000 Da. In some cases, the molecular weight of C is about 4250 Da. In some cases, the molecular weight of C is about 4500 Da. In some cases, the molecular weight of C is about 4600 Da. In some cases, the molecular weight of C is about 4750 Da. In some cases, the molecular weight of C is about 5000 Da. In some cases, the molecular weight of C is about 5500 Da. In some cases, the molecular weight of C is about 6000 Da. In some cases, the molecular weight of C is about 6500 Da. In some cases, the molecular weight of C is about 7000 Da. In some cases, the molecular weight of C is about 7500 Da. In some cases, the molecular weight of C is about 8000 Da. In some cases, the molecular weight of C is about 10,000 Da. In some cases, the molecular weight of C is about 12,000 Da. In some cases, the molecular weight of C is about 20,000 Da. In some cases, the molecular weight of C is about 35,000 Da. In some cases, the molecular weight of C is about 40,000 Da.In some cases, the molecular weight of C is about 50,000 Da. In some cases, the molecular weight of C is about 60,000 Da. In some cases, the molecular weight of C is about 100,000 Da.

[0190] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, where the discrete PEG is a polymeric PEG that includes more than one repeating ethylene oxide unit. In some cases, the discrete PEG (dPEG) includes 2-60, 2-50, or 2-48 repeating ethylene oxide units. In some examples, the dPEG includes 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 cases, the dPEG includes about 2 or more repeating ethylene oxide units. In some cases, the dPEG includes about 3 or more repeating ethylene oxide units. In some cases, the dPEG includes about 4 or more repeating ethylene oxide units. In some cases, the dPEG includes about 5 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 6 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 7 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 8 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 9 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 10 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 11 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 12 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 13 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 14 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 15 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 16 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 17 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 18 or more repeating ethylene oxide units. In some cases, the dPEG contains about 19 or more repeating ethylene oxide units. In some cases, the dPEG contains about 20 or more repeating ethylene oxide units. In some cases, the dPEG contains about 22 or more repeating ethylene oxide units.In some cases, dPEG contains about 24 or more repeating ethylene oxide units. In some cases, dPEG contains about 26 or more repeating ethylene oxide units. In some cases, dPEG contains about 28 or more repeating ethylene oxide units. In some cases, dPEG contains about 30 or more repeating ethylene oxide units. In some cases, dPEG contains about 35 or more repeating ethylene oxide units. In some cases, dPEG contains about 40 or more repeating ethylene oxide units. In some cases, dPEG contains about 42 or more repeating ethylene oxide units. In some cases, dPEG contains about 48 or more repeating ethylene oxide units. In some cases, dPEG contains about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials in a stepwise manner. In some cases, 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.

[0191] In some embodiments, L 1 Polymer C is the sense strand X S or F 1 The polypeptide of the sense strand X S In some embodiments, L is any linker that can be used to link 1 has the following formula: [ka] In the formula, X S , X AS , and F 1 is as defined above.

[0192] In some embodiments, n is 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.

[0193] In some embodiments, L 2 The polypeptide of F1 is the antisense strand X AS or polymer C is linked to antisense strand X AS is any linker that can be used to link

[0194] In some embodiments, L 2 has the formula in the complex: [ka] In the formula, X AS and F 1 is as defined above.

[0195] In some embodiments, n is 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.

[0196] In some embodiments, the linker is covalently attached to F1 via a cysteine ​​residue present in F1, which can be depicted as follows: [ka] In some embodiments, A1-B1 has the formula: [ka] where C is a polymer such as PEG, an endoporator, INF-7, TAT, polyarginine, polylysine, an amphiphilic peptide, or a peptide listed in Table 2, as provided herein; and X is 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, and F 1 is a polypeptide comprising at least one FN3 domain, S and X AS form a double-stranded siRNA molecule. The sense and antisense strands are represented by the notation "N", where each nucleotide represented by N is independently A, U, C, or G, or a modified nucleobase such as those provided herein. The N of the sense and antisense strands 1 The nucleotides represent the 5' end of each strand. For clarity, Formula I represents N 1 , N 2 , N 3 etc., although the nucleotide bases need not, and are not intended to, be identical. The siRNA shown in Formula I will be complementary to the target sequence.

[0197] For example, in some embodiments, the sense strand is 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 172'-fluoro modified nucleotides, as well as N 4 , N 5 , N 6 , N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 , and N 19 contains 2'O-methyl modified nucleotides.

[0198] In some embodiments, the antisense strand is 1 A vinyl phosphonate moiety bound to N 2 2' fluoro-modified nucleotide with phosphorothioate (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, as well as N 20 and N 21 contains 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones.

[0199] In some embodiments, the compound has the formula: [ka] In the formula, F 1 comprises at least one FN3 domain and a linker L 1 A polypeptide conjugated to L 1 X S is concatenated to X Sis 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, and X S and X AS form a double-stranded siRNA molecule. The above linkers are non-limiting examples and other types of linkers can be used.

[0200] In some embodiments, F 1 is (X 1 ) n -(X 2 ) q -(X 3 ) y wherein X 1 is the first FN3 domain, and X 2 is the second FN3 domain, X 3 is a third FN3 domain or half-life extender, and 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 1 and y is 0. In some embodiments, n and y are 1 and q is 0.

[0201] In some embodiments, X 1 is a CD71 FN3 binding domain, such as those provided herein. In some embodiments, X 2 is a CD71 FN3 binding domain. In some embodiments, X1 and X 2 are different CD71 FN3 binding domains. In some embodiments, the binding domains are the same. In some embodiments, X 3 is an FN3 domain that binds to human serum albumin. In some embodiments, X 3 is an Fc domain that does not have an effector function that extends the half-life of the protein. 1 is the first CD71-binding domain, and X 2is the second CD71-binding domain, X 3 is an FN3 albumin binding domain. Examples of such polypeptides are provided herein and below. In some embodiments, C-(X 1 ) n -(X 2 ) q -(X 3 ) y -LX 4 Provided herein are compositions having the formula: 1 is the first FN3 domain, and X 2 is the second FN3 domain, X 3 is a third FN3 domain or a half-life extender, L is a linker, and X 4 is a nucleic acid molecule, and n, q, and y are each independently 0 or 1.

[0202] In some embodiments, (X1) n -(X2) q -(X3) y Provided herein is a composition having the formula of -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, C is a polymer, and n, q, and y are each independently 0 or 1.

[0203] In some embodiments, X-L-(X) n -(X2) q -(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, L is a linker, X4 is a nucleic acid molecule, and n, q, and y are each independently 0 or 1.

[0204] In some embodiments, C-X-L-(X1) 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, X4 is a nucleic acid molecule, and n, q, and y are each independently 0 or 1.

[0205] In some embodiments, X-L-(X) n -(X2) q -(X3) y Provided herein are compositions having the formula: -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, C is a polymer, and n, q, and y are each independently 0 or 1.

[0206] In some embodiments, the GYS1 siRNA pair can be according to the sequence of sense strand (5'-3')nsnsnnnnNfNfNfnnnnnnnnsnsa and antisense strand (5'-3')UfsNfsnnnNfnnnnnnnNfnNfnnnsusu, where (n) is 2'-O-Me (methyl), (Nf) is 2'-F (fluoro), and (s) is a phosphorothioate backbone modification. Each nucleotide of both the sense and antisense strands is modified independently or in combination at the ribosugar and nucleobase positions.

[0207] In some embodiments, the siRNA molecule comprises a sequence pair from Table 3A or 3B. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0208] Abbreviation key: (n / N = any nucleotide) mN = 2'-O-methyl residue, fN = 2'-F residue, * = phosphorothioate and (idT) = inverted Dt, (VP) 2'-O methylvinylphosphonate uridine. Brackets indicate individual bases. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0209] In some embodiments, the polynucleotides exemplified above include those that do not contain 2'-O methylvinylylphosphonate uridine as the 5' nucleotide on the antisense strand of the siRNA.

[0210] In some embodiments, the polynucleotide is as provided herein. In some embodiments, the polynucleotide comprises a first strand and a second strand to form a portion containing a double strand. In some embodiments, the polynucleotide comprises a sense strand and an antisense strand. In some embodiments, it comprises the sequences shown in Table 3A or 3B. In some embodiments, it comprises the sequences shown in Table 3A or 3B but does not include base modifications. In some embodiments, the pharmaceutical composition comprises the siRNA pair provided herein. In some embodiments, the siRNA pair is not conjugated to the FN3 domain.

[0211] 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, oligonucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to enhance the biological stability of the molecule or the physical stability of the double strand formed between the oligonucleotide molecule and the target nucleic acid. Alternatively, oligonucleotide molecules are biologically generated using an expression vector in which the oligonucleotide molecule is subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted oligonucleotide molecule is in an antisense orientation to the target polynucleic acid molecule of interest).

[0212] In some embodiments, the oligonucleotide molecule is synthesized by a tandem synthesis method, and both strands are synthesized as strands separated by a single continuous oligonucleotide fragment or a cleavable linker, and then cleaved to provide separate fragments or strands that hybridize and allow purification of the double strand.

[0213] In some cases, the oligonucleotide molecule is also assembled from two different nucleic acid strands or fragments, one fragment containing the sense region and the second fragment containing the antisense region of the molecule.

[0214] In some cases, chemical modification of oligonucleotide molecule internucleotide bonds with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate or mesyl phosphoramidate bonds improves stability.Excessive modification can cause toxicity or reduced activity.Therefore, when designing nucleic acid molecules, in some cases, the amount of these internucleotide bonds is minimized.In such cases, reducing the concentration of these bonds reduces toxicity and increases the efficacy and higher specificity of these molecules.

[0215] As described herein, in some embodiments, the nucleic acid molecule may be modified to include a linker at the 5' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule may 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, the nucleic acid molecule may be modified to include a linker at the 3' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule may be modified to include a vinyl phosphonate at the 3' end of the antisense strand of the dsRNA. A linker may be used to link the dsRNA to the FN3 domain. The linker may be covalently attached to a cysteine ​​residue on the FN3 domain that is naturally occurring or substituted as described herein and in, for example, U.S. Patent No. 10,196,446 (incorporated herein by reference in its entirety). Non-limiting examples of such modified strands of dsRNA are shown in Table 4 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0216] In some embodiments, the siRNA pairs of A-PPPP provided above comprise a linker at the 3'-end of the sense strand. In some embodiments, the siRNA pairs of A-PPPP provided above comprise a vinyl phosphonate at the 5'-end of the sense strand.

[0217] Abbreviation key: (n / N = any nucleotide) mN = 2'-O-methyl residue, fN = 2'-F residue, * = phosphorothioate and (idT) = inverted Dt, (VP) 2'-O methylvinylphosphonate uridine, BMPS = propylmaleimide.

[0218] The structure of the linker (L) is shown in Table 5 below. [Table 6]

[0219] Other linkers can also be used, for example, linkers formed by click chemistry, amide coupling, reductive amination, oxime, enzymatic coupling such as transglutaminase, and sortage conjugation. The linkers provided herein are exemplary in nature, and other linkers made by other such methods can also be used.

[0220] When attached to an siRNA, the structure, L-(X4), can be represented by the formula: [ka]

[0221] A particular siRNA sequence is shown herein with a particular modified nucleobase, but sequences without such modifications are also provided herein. That is, the sequences can include those exemplified in the tables provided herein that have no modifications. Unmodified siRNA sequences can still, in some embodiments, include a linker at the 5' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule can be modified to include a vinylphosphonate at the 5' end of the antisense strand of the dsRNA. In some embodiments, the nucleic acid molecule can be modified to include a linker at the 3' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule can be modified to include a vinylphosphonate at the 3' end of the antisense strand of the dsRNA. The linker can be as provided herein.

[0222] In some embodiments, provided is an FN3 protein comprising a polypeptide that binds to CD71. In some embodiments, the polypeptide comprises an FN3 domain that binds to CD71. In some embodiments, provided are polypeptides comprising the sequences of SEQ ID NOs: 273, 288-291, 301-310, 312-572, 592-599, or 708-710. In some embodiments, the polypeptide that binds to CD71 comprises the sequences of SEQ ID NOs: 301-301, 310, 312-572, 592-599, or 708-710. The sequence of the CD71 protein to which the polypeptide can bind can be, for example, SEQ ID NO: 2 or 3. In some embodiments, the FN3 domain that binds to CD71 binds specifically to CD71.

[0223] In some embodiments, the FN3 domain that binds to CD71 is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon27 sequence of SEQ ID NO: 4 (LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT), optionally with substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 4).

[0224] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0225] In some embodiments, the protein comprising the polypeptide comprises the amino acid sequence of SEQ ID NO: 273. SEQ ID NO: 273 is a consensus sequence based on the sequences of SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, and SEQ ID NO: 291. The sequence of SEQ ID NO: 273 is MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GEAIX 8 LX 9 VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 X 13 VKGGX 14 X 15 SX 16 PLX 17 AX 18 FTT, In the formula, X 8 , X 9 , X 17 , and X 18 are each independently any amino acid other than methionine or proline; X 1is selected from D, F, Y, or H; X 2 is selected from Y, G, A, or V; X 3 is selected from I, T, L, A, or H; X 4 is selected from S, Y, or P; X 5 is selected from Y, G, Q, or R; X 6 is selected from G or P; X 7 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.

[0226] In some embodiments, X 1 is selected from D, F, Y, or H; X 2 is selected from G, A, or V; X 3 is selected from T, L, A, or H; X 4 is selected from Y or P; X 5 is selected from G, Q, or R; X 6 is selected from G or P; X 7 is selected from Y, P, D, or S; X 10is 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.

[0227] In some embodiments, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 is as set forth in SEQ ID NO: 288. In some embodiments, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 is as set forth in SEQ ID NO: 289. In some embodiments, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 10 , X 11 , X 12 , X 13 , X14 , X 15 , and X 16 is as shown in sequence SEQ ID NO: 290. In some embodiments, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , 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:291.

[0228] In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 8 , X 9 , X 17 , and X 18 is not independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 8 , X 9 , X 17 , and X 18 is independently alanine. 8 , X 9 , X 17 , and X 18 is independently arginine. 8 , X 9 , X 17 , and X 18is independently asparagine. In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently aspartic acid. 8 , X 9 , X 17 , and X 18 is independently a cysteine. In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently glutamine. 8 , X 9 , X 17 , and X 18 is independently glutamic acid. 8 , X 9 , X 17 , and X 18 is independently glycine. 8 , X 9 , X 17 , and X 18 is independently histidine. In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently isoleucine. 8 , X 9 , X 17 , and X 18 is independently leucine. In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently lysine. In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently phenylalanine. 8 , X 9 , X 17 , and X 18is independently serine. 8 , X 9 , X 17 , and X 18 is independently threonine. 8 , X 9 , X 17 , and X 18 is independently tryptophan. In some embodiments, X 8 , X 9 , X 17 , and X 18 is independently tyrosine. In some embodiments, X 8 , X 9 , X 17 , and X 18 is, independently, valine.

[0229] In some embodiments, the sequence is 8 , X 9 , X 17 , and X 18 In some embodiments, X is an amino acid residue, except that the position corresponding to the 8 But it's not V, it's X. 9 But it's not T, it's X. 17 But it's not S, it's X. 18 is shown as shown in sequence SEQ ID NO:288, except that it is not I.

[0230] In some embodiments, the sequence is 8 , X 9 , X 17 , and X 18 In some embodiments, X is an amino acid residue, except that the position corresponding to the 8 But it's not V, it's X. 9 But it's not T, it's X. 17 But it's not S, it's X. 18 is shown as shown in sequence SEQ ID NO:289, except that it is not I.

[0231] In some embodiments, the sequence is8 , X 9 , X 17 , and X 18 In some embodiments, X is an amino acid residue, except that the position corresponding to the 8 But it's not V, it's X. 9 But it's not T, it's X. 17 But it's not S, it's X. 18 is shown as shown in sequence SEQ ID NO:290, except that it is not I.

[0232] In some embodiments, the sequence is 8 , X 9 , X 17 , and X 18 In some embodiments, X is an amino acid residue, except that the position corresponding to the 8 But it's not V, it's X. 9 But it's not T, it's X. 17 But it's not S, it's X. 18 is shown as shown in sequence SEQ ID NO:291, except that it is not I. In some embodiments, a protein, including a polypeptide, comprises 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:273. 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: 273. 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: 273. In some embodiments, the protein is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 273.

[0233] Percent identity can be determined using default parameters to align two sequences using BlastP, available from the NCBI website.

[0234] In some embodiments, fibronectin type III (FN3) domains are provided that bind or specifically bind to human CD71 protein (SEQ ID NO: 2 or 5). As provided herein, FN3 domains can bind to CD71 protein. It is also provided that, even if not explicitly stated, the domains can also specifically bind to CD71 protein. Thus, for example, FN3 domains that bind to CD71 also encompass 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, vectors, host cells encoding the FN3 domains disclosed herein or their complementary nucleic acids, as well as methods of making and using them, are provided.

[0235] In some embodiments, an isolated FN3 domain that binds or specifically binds to CD71 is provided.

[0236] In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker. 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 linker, etc.

[0237] In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker, such as those provided herein. Exemplary linkers include (GS) 2 , (SEQ ID NO: 720), (GGGS) 2 (SEQ ID NO:721), (GGGGS) 1-5 (SEQ ID NO: 722), (AP)1-20 ;(AP) 2 (SEQ ID NO: 723), (AP) 5 (SEQ ID NO: 724), (AP) 10 (SEQ ID NO: 725), (AP) 20 (SEQ ID NO:726), A(EAAAK) 5 AAA (SEQ ID NO: 727), or (EAAAK) 1-5 (SEQ ID NO: 728). In some embodiments, the linker comprises or is the amino acid sequence of EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 729), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 730), APAPAPAPAP (SEQ ID NO: 731), or EAAAK (SEQ ID NO: 732).

[0238] In some embodiments, the FN3 domain has a molecular weight of about 1×10 as determined by surface plasmon resonance or Kinexa methods, as practiced by one of skill in the art. -7 Less than M, e.g., about 1 x 10 -8 Less than M, approx. 1×10 -9 Less than M, approx. 1×10 -10 Less than M, approx. 1×10 -11 Less than M, approx. 1×10 -12 Less than M or about 1×10 -13 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., osmolarity, pH). Thus, 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.

[0239] In some embodiments, the FN3 domain is capable of binding to CD71 with a signal at least 5-fold greater than that obtained for a negative control in a standard solution ELISA assay.

[0240] In some embodiments, the FN3 domain that binds or specifically binds CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds 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.

[0241] The FN3 domain can also include cysteine ​​substitutions such as those described in U.S. Patent No. 10,196,446, which is incorporated herein by reference in its entirety. Briefly, in some embodiments, the polypeptides provided herein can include at least one 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 of U.S. Patent No. 10,196,446, or SEQ ID NO:4 as described herein, and at equivalent positions in related FN3 domains.

[0242] The amino acid sequence of SEQ ID NO:1 in U.S. Pat. No. 10,196,446 is LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (herein, sequence number 713).

[0243] 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.

[0244] Cysteine ​​substitution at a position in a domain or protein comprises 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 Publication No. 20170362301, which is incorporated herein by reference in its entirety.Sequence alignment can be performed using BlastP, for example, using default parameters on the NCBI website.

[0245] In some embodiments, a cysteine ​​residue is inserted at any position within the domain or protein.

[0246] 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 therapeutic agent to the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a cytotoxic agent to 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, and / or cytotoxic agent disclosed herein to the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of an oligonucleotide to 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 dendritic cell. In some embodiments, the cell is a cell of the central nervous system. In some embodiments, the cell is a cardiac cell.

[0247] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0248] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 301. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 302. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 305. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 306. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 310. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 312. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 313. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 314. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 315. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 316. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 317. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 318. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 319. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 320. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 321.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 322. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 323. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 324. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 325. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 326. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 327. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 328. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 329. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 330. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 331. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 332. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 333. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 334. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 336. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 337. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 338. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 339.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 340. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 341. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 342. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 345. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 347. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 348. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 349. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 350. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 351. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 352. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 353. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 354. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 355. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 356. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 357. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 358. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 359.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 360. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 361. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 362. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 363. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 364. 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 410. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 412. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 414. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 415. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 416. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 418. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 422. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 423. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 424. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 425. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, the isolated FN3 domain that binds to 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 CD71 comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, the isolated FN3 domain that binds 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 503. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 504. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 505. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 507. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 508. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 509. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 510. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 511. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 512. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 513. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 514. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 515. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 516. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 517. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 518. In some embodiments, the isolated FN3 domain that binds to 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: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: 708. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 709. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:710.

[0249] In some embodiments, the isolated FN3 domain that binds CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule. 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 of 273, 288 - 291, 301 - 310, 312 - 572, 592 - 599, or 708 - 710. Percent identity can be determined using default parameters for aligning two sequences using BlastP available from the NCBI website. The sequence of the FN3 domain that binds to CD71 can be found, for example, in Table 6 below.

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

[0250] As provided herein, in some embodiments, the FN3 domain that binds to CD71 binds to SEQ ID NO:2 (human mature CD71) or SEQ ID NO:5 (human mature CD71 extracellular domain), the sequences of each of which are provided in Table 7 below. [Table 8]

[0251] In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker. 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 a (GS) 2 , (SEQ ID NO: 720), (GGGS) 2(SEQ ID NO:721), (GGGGS) 5 (SEQ ID NO: 722), (AP) 2-20 , (AP) 2 (SEQ ID NO: 723), (AP) 5 (SEQ ID NO: 724), (AP) 10 (SEQ ID NO: 725), (AP) 20 (SEQ ID NO: 726), and A(EAAAK) 5 AAA (SEQ ID NO: 727) or (EAAAK) 1-5 (SEQ ID NO: 728). These are non-limiting examples and other linkers can be used. The number of GGGGS or GGGGA repeats can also be 1, 2, 3, 4, or 5. In some embodiments, the linker comprises one or more GGGGS repeats and one or more GGGGA repeats. In some embodiments, the linker comprises EAAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 729), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 730), APAPAPAPAP (SEQ ID NO: 731), or EAAAK (SEQ ID NO: 732).

[0252] In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:592. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:593. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:594. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:595. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:596. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:597. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:598. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of SEQ ID NO:599. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has an amino acid sequence of one of SEQ ID NOs: 592-599.

[0253] In some embodiments, the FN3 domain has a molecular weight of about 1×10, as determined by surface plasmon resonance or Kinexa methods, as applicable, as practiced by one of skill in the art. -7 Less than M, e.g., about 1 x 10 -8 Less than M, approx. 1×10 -9 Less than M, approx. 1×10 -10 Less than M, approx. 1×10 -11 Less than M, approx. 1×10 -12 Less than M or about 1×10 -13 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., osmolarity, pH). Thus, 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.

[0254] In some embodiments, the FN3 domain is capable of binding to its target protein with a signal at least 5-fold greater than that obtained for a negative control in a standard solution ELISA assay.

[0255] In some embodiments, the FN3 domain that binds or specifically binds to its target protein comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to its target protein comprises a cysteine ​​(Cys) linked to the C-terminus of the FN3 domain. The addition of the N-terminal Met and / or C-terminal Cys can facilitate expression and / or conjugation of the half-life extension molecule.

[0256] FN3 domains can also include cysteine ​​substitutions, such as those described in U.S. Patent No. 10,196,446, which is incorporated herein by reference in its entirety. Briefly, in some embodiments, a polypeptide comprising an FN3 domain can have an FN3 domain with residues substituted with cysteines, which can be referred to as a cysteine ​​engineered fibronectin type III (FN3) domain. In some embodiments, the FN3 domain comprises at least one 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 (LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT, SEQ ID NO: 713) of U.S. Patent No. 10,196,446, which is incorporated herein by reference in its entirety, and at equivalent positions in related FN3 domains. A cysteine ​​substitution at a position within a domain or protein comprises the replacement of an existing amino acid residue with a cysteine ​​residue. Other examples of cysteine ​​modifications can be found, for example, in US Patent Publication No. 20170362301, which is incorporated by reference in its entirety. Sequence alignment can be performed using BlastP, for example, using default parameters on the NCBI website. In some embodiments, the FN3 domain that binds to the target protein is internalized into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a detectable label or therapeutic agent to the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent to the cell. The cytotoxic agent can act as a therapeutic agent. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of any detectable label, therapeutic agent, and / or cytotoxic agent disclosed herein to the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell, a lung cell, a muscle cell, an immune cell, a dendritic cell, a cell of the CNS, or a cardiac cell. In some embodiments, the therapeutic agent is an siRNA molecule provided herein. The FN3 domain that binds to CD71 conjugated to a detectable label can be used to assess the expression of CD71 on a sample, such as a tumor tissue, in vivo or in vitro. FN3 domains that bind CD71 conjugated to a detectable label can be used to assess the expression of CD71 on blood, immune cells, muscle cells, or dendritic cells in vivo or in vitro.

[0257] As provided herein, the different FN3 domains linked to the siRNA molecule can also be conjugated or linked to another FN3 domain that binds to a different target. This allows the molecule to be multispecific (e.g., bispecific, trispecific, etc.), for example, to bind to a first target and another target. In some embodiments, the first FN3 binding domain is linked to another FN3 domain that binds to an antigen (tumor antigen) expressed by tumor cells.

[0258] In some embodiments, the FN3 domains can be linked together by a linker to form a bivalent FN3 domain. The linker can be a flexible linker. In some embodiments, the linker is a G / S linker. In some embodiments, the linker has 1, 2, 3, or 4 G / S repeats. The G / S repeat unit is four glycines followed by a serine, e.g., GGGGS. Other examples of linkers can also be provided and used herein.

[0259] In some embodiments, the linker is (GS) 2 , (SEQ ID NO: 720), (GGGS) 2 (SEQ ID NO: 721), (GGGGS) 5 (SEQ ID NO: 722), (AP) 2-20 , (AP) 2 (SEQ ID NO: 723), (AP) 5 (SEQ ID NO: 724), (AP) 10 (SEQ ID NO: 725), (AP) 20 (SEQ ID NO: 726), and A(EAAAK) 5 AAA(SEQ ID NO: 727) or (EAAAK) 1-5 (SEQ ID NO: 728) polypeptides. These are non-limiting examples and other linkers can also be used. The number of GGGGS or GGGGA repeats can also be 1, 2, 3, 4, or 5. In some embodiments, the linker includes one or more GGGGS repeats and one or more GGGGA repeats. In some embodiments, the linker includes one or more GGGGS repeats and one or more EAAAK repeats. In some embodiments, the linker includes one or more GGGGS repeats and one or more "AP" repeats. In some embodiments, the linker includes EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 729), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 730), APAPAPAPAP (SEQ ID NO: 731), or EAAAK (SEQ ID NO: 732).

[0260] Without being bound to any particular theory, in some embodiments, the FN3 domain linked to the nucleic acid molecule can be used in targeted delivery of therapeutic agents to cells that express one or more binding partners of the FN3 domain (e.g., tumor cells), resulting in intracellular accumulation of the nucleic acid molecule therein. This allows the siRNA molecule to properly interact with the cellular machinery to inhibit the expression of target genes, improving efficacy, and in some embodiments, also avoiding toxicity that may occur from non-targeted administration of the same siRNA molecule.

[0261] The FN3 domains described herein that bind to their specific target proteins can be generated as monomers, dimers, or multimers, for example, as a means of increasing the valency and thus avidity of target molecule binding, or as a means of generating bispecific or multispecific scaffolds that bind two or more different target molecules simultaneously. Dimers and multimers can be generated by linking monospecific, bispecific, or multispecific protein scaffolds, for example, by including an amino acid linker, such as a linker containing polyglycine, glycine and serine, or alanine and proline. Exemplary linkers include (GS) 2 , (SEQ ID NO: 720), (GGGS) 2 (SEQ ID NO:721), (GGGGS) 5 (SEQ ID NO: 722), (AP) 2-20 , (AP) 2 (SEQ ID NO: 723), (AP) 5 (SEQ ID NO: 724), (AP) 10 (SEQ ID NO: 725), (AP) 20 (SEQ ID NO: 726), and A(EAAAK) 5 AAA (SEQ ID NO: 727) or (EAAAK) 1-5 (SEQ ID NO: 728). In some embodiments, the linker comprises or is the amino acid sequence of EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 729), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 730), APAPAPAPAP (SEQ ID NO: 731), or EAAAK (SEQ ID NO: 732).

[0262] Dimers and multimers can be linked together in the N to C direction. The use of naturally occurring and synthetic peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature. See, for example, Hallewell et al. (J Biol Chem., 1989, 264:5260-5268), Alfthan et al. (Protein Eng., 1995, 8:725-731), Robinson & Sauer (Biochemistry, 1996, 35:109-116), and U.S. Patent No. 5,856,456. The linkers described in this paragraph can also be used to link the domains provided herein and in the formulas provided above.

[0263] Half-life extension moiety

[0264] 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, 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. The half-life of the entire molecule is extended.

[0265] In some embodiments, the albumin binding domain comprises the amino acid of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. 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%, or 99% identical to or is an amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the albumin binding domain has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 amino acid sequence identity. 5%, 96%, 97%, 98%, or 99% identical to or with an amino acid sequence thereof, provided that the protein has a substitution corresponding to position 10 of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. 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 when compared to the amino acid sequence of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.In some embodiments, the substitution is at a position corresponding to position 10 of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the provided FN3 domains comprise a cysteine ​​residue at at least one residue position corresponding to residue position 6, 11, 22, 25, 26, 52, 53, 61, 88, or position 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: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or at the C-terminus. Although the positions are listed in a series, each position can also be selected individually. In some embodiments, the cysteine ​​is at a position corresponding to position 6, 53, or 88. In some embodiments, additional examples of albumin binding domains can be found in U.S. Pat. No. 10,925,932, which is incorporated herein by reference.

[0266] All or a portion of an antibody constant region may be attached to an FN3 domain to confer antibody-like properties, in particular properties associated with the Fc region, such as complement activity, half-life, etc., and Fc effector functions, such as C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor; BCR), etc., may be further modified by modifying residues in the Fc involved in these activities (for review, see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

[0267] Additional moieties can be incorporated into the FN3 domain for desired properties, such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, such as laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanediic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, carbohydrates (dextran, cellulose, oligos or polysaccharides), etc. These moieties can be direct fusions with protein scaffold coding sequences or can be produced by standard cloning and expression techniques. Alternatively, well-known chemical conjugation methods can be used to attach the moiety to the recombinantly produced molecule disclosed herein.

[0268] A PEG moiety can be added to the FN3 domain by, for example, incorporating a cysteine ​​residue at the C-terminus of the molecule or engineering a cysteine ​​into a residue position away from the binding face of the molecule and attaching a PEG group to the cysteine ​​using well-known methods.

[0269] FN3 domains incorporating additional moieties can be compared for functionality by several well-known assays. For example, the altered properties resulting from the incorporation of the Fc domain and / or Fc domain variants can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn receptors, or using well-known cell-based assays that measure, for example, ADCC or CDC, or evaluate the pharmacokinetic properties of the molecules disclosed herein in in vivo models.

[0270] The compositions provided herein can be prepared by preparing FN3 protein and nucleic acid molecules 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, the nucleic acid molecule is modified with a linker, such as the linkers provided herein, and then the protein is mixed with the nucleic acid molecule containing the linker to form a composition. For example, in some embodiments, the FN3 domain is conjugated to an siRNA cysteine ​​using thiol-maleimide chemistry. In some embodiments, the cysteine-containing FN3 domain is reduced in phosphate buffered saline (or any other suitable buffer) using, for example, a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) to obtain a free thiol. Then, in some embodiments, the free thiol-containing FN3 domain is mixed with the maleimide-linked-modified siRNA duplex and incubated under conditions to form a linked complex. In some embodiments, the mixture is incubated at room temperature 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.

[0271] 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.

[0272] In some embodiments, the FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, and a histidine tag is added to the N-terminus or C-terminus of the polypeptide for ease of purification. In some embodiments, the histidine tag (His-tag) comprises six histidine residues. 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-terminus or C-terminus. In some embodiments, when the His-tag is removed from the N-terminus, all glycines are removed. In some embodiments, when the His-tag is removed from the C-terminus, one or more of the glycines are retained.

[0273] In some embodiments, the FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, and the N-terminal methionine is retained following purification of the FN3 domain.

[0274] kit

[0275] In some embodiments, a kit is provided that contains the compositions described herein.

[0276] The kits can be used for therapeutic applications and as diagnostic kits.

[0277] In some embodiments, the kit comprises an FN3 domain conjugated to a nucleic acid molecule.

[0278] In some embodiments, a kit for treating a glycogen storage disease is provided. In some embodiments, the kit includes a first container containing a pharmaceutical composition comprising one or more FN3 domains linked to an siRNA comprising a sense strand and an antisense strand, and a second container containing a pharmaceutical composition comprising an enzyme replacement therapy (ERT) for treating a glycogen storage disease. In some embodiments, the one or more FN3 domains include an FN3 domain that binds to CD71. In some embodiments, the siRNA targets Gys1. In some embodiments, the one or more FN3 domains include an FN3 domain that binds to CD71. In some embodiments, the glycogen storage disease is selected from the group consisting of Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Cori disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Tarui disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes mellitus / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucosan body disease. In some embodiments, the ERT comprises glucosidase alpha acid (GAA), glycogen debranching enzyme (AGL), glycogen branching enzyme (BGE1), muscle glycogen phosphorylase (PYGM), muscle phosphofructokinase (PFKM), aldolase A (ALDOA), malin, laforin, glycogen synthase (GYS2), glucose-6-phosphatase (G6PC / SLC37A4), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1), phosphoglycerate mutase (PGAM2), muscle lactate dehydrogenase (LDHA), glucose transporter (GLUT2), β-enolase (ENO3), and glycogenin-1 (GYG1), or a combination thereof. In some embodiments, the ERT comprises GAA, malin, laforin, or a combination thereof.In some embodiments, the first container contains a pharmaceutical composition comprising an FN3 domain that binds to CD71 conjugated to siRNA targeting Gys1, and the second container contains a pharmaceutical composition comprising the enzyme GAA.

[0279] Use of the conjugate FN3 domain

[0280] The compositions provided herein can be used to diagnose, monitor, regulate, treat, alleviate, prevent the occurrence thereof or relieve the symptoms thereof of a human disease or a specific pathology in a cell, tissue, organ, fluid, or generally in a host.

[0281] In some embodiments, a method of selectively reducing GYS1 mRNA and protein in skeletal muscle. In certain embodiments, the GYS1 mRNA and protein are not reduced in the liver and / or kidney.

[0282] In some embodiments, the reduction of GYS1 mRNA and protein persists for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or more than 5 weeks after administration of the conjugate described herein.

[0283] In some embodiments, the FN3 domain can promote delivery to CD71-positive tissues (e.g., skeletal muscle, smooth muscle) for the treatment of muscle diseases.

[0284] In some embodiments, the FN3 domain can promote delivery to activated lymphocytes, dendritic cells, or other immune cells for the treatment of immune diseases.

[0285] In some embodiments, the polypeptide that binds CD71 is directed to an immune cell. In some embodiments, the polypeptide that binds CD71 is directed to a dendritic cell. In some embodiments, a method of treating an autoimmune disease in a subject in need of treatment is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes mellitus, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, and immune thrombocytopenic purpura.

[0286] In some embodiments, methods of treating a subject having Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency) are provided, the methods comprising administering to the subject a composition provided herein. In some embodiments, the methods comprise administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent.

[0287] In some embodiments, methods are provided for treating a glycogen storage disease in a subject in need thereof, comprising administering a composition provided herein. In some embodiments, the glycogen storage disease is selected from the group consisting of Cori's or Forbes' disease (GSD3, glycogen debranching enzyme (AGL) deficiency), McArdle's disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), type II diabetes / diabetic nephropathy, aldolase A deficiency (GSD12), Lafora's disease, hypoxia, Andersen's disease (GSD4, glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke disease), Haas disease (GSD6, hepatic glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency, syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11), glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0288] In some embodiments, there is provided a use of any of the compositions provided herein in the preparation of a pharmaceutical composition or medicament for treating cancer, in some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia, myelodysplastic syndrome, gastric cancer, clear cell renal cell carcinoma, clear cell carcinoma of the breast, clear cell carcinoma of the endometrium, clear cell carcinoma of the ovary, clear cell carcinoma of the uterus, hepatocellular carcinoma, pancreatic cancer, prostate cancer, soft tissue cancer, Ewing's sarcoma, and non-small cell lung cancer.

[0289] In some embodiments, the CD71 cells are cells involved in CNS diseases, inflammatory / immune diseases such as MS, and infections of the brain. In some embodiments, the polypeptide that binds to CD71 is directed to the central nervous system. In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject in need of treatment is provided. 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 a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent. In some embodiments, the brain tumor is selected from the group consisting of non-malignant, benign, and malignant brain tumors. In some embodiments, the neurological condition is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Lafora's disease, Pompe's disease, adult polyglucosan body disease, stroke, spinal cord injury, ataxia, Bell's palsy, cerebral aneurysm, epilepsy, seizures, Guillain-Barre syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndromes, migraine headaches, encephalitis, sepsis, and myasthenia gravis.

[0290] In some embodiments, a method of treating a subject having cancer is provided, the method comprising administering to the subject a composition provided herein. 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 a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent.

[0291] In some embodiments, the subject has a solid tumor.

[0292] In some embodiments, the solid tumor is a melanoma.

[0293] In some embodiments, the solid tumor is lung cancer. In some embodiments, the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is squamous non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is non-squamous NSCLC. In some embodiments, the solid tumor is lung adenocarcinoma.

[0294] In some embodiments, the solid tumor is renal cell carcinoma (RCC).

[0295] In some embodiments, the solid tumor is mesothelioma.

[0296] In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).

[0297] In some embodiments, the solid tumor is colorectal cancer.

[0298] In some embodiments, the solid tumor is prostate cancer. In some embodiments, the solid tumor is castration-resistant prostate cancer.

[0299] In some embodiments, the solid tumor is gastric cancer.

[0300] In some embodiments, the solid tumor is ovarian cancer.

[0301] In some embodiments, the solid tumor is gastric cancer.

[0302] In some embodiments, the solid tumor is liver cancer.

[0303] In some embodiments, the solid tumor is pancreatic cancer.

[0304] In some embodiments, the solid tumor is thyroid cancer.

[0305] In some embodiments, the solid tumor is squamous cell carcinoma of the head and neck.

[0306] In some embodiments, the solid tumor is a carcinoma of the esophagus or gastrointestinal tract.

[0307] In some embodiments, the solid tumor is breast cancer.

[0308] In some embodiments, the solid tumor is fallopian tube cancer.

[0309] In some embodiments, the solid tumor is a brain tumor.

[0310] In some embodiments, the solid tumor is a urethral cancer.

[0311] In some embodiments, the solid tumor is a genitourinary cancer.

[0312] In some embodiments, the solid tumor is endometriosis.

[0313] In some embodiments, the solid tumor is cervical cancer.

[0314] In some embodiments, the solid tumor is a metastatic lesion of a cancer.

[0315] In some embodiments, the subject has a hematological malignancy.

[0316] In some embodiments, the hematological malignancy is lymphoma, myeloma, or leukemia. In some embodiments, the hematological malignancy is B-cell lymphoma. In some embodiments, the hematological malignancy is Burkitt's lymphoma. In some embodiments, the hematological malignancy is Hodgkin's lymphoma. In some embodiments, the hematological malignancy is non-Hodgkin's lymphoma.

[0317] In some embodiments, the hematological malignancy is a myelodysplastic syndrome.

[0318] In some embodiments, the hematological malignancy is acute myeloid leukemia (AML). In some embodiments, the hematological malignancy is chronic myeloid leukemia (CML). In some embodiments, the hematological malignancy is chronic myelomonocytic leukemia (CMML).

[0319] In some embodiments, the hematological malignancy is multiple myeloma (MM).

[0320] In some embodiments, the hematological malignancy is plasmacytoma.

[0321] In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the soft tissue cancer is Ewing's sarcoma.

[0322] In some embodiments, a method for treating cancer in a subject in need of cancer treatment is provided. In some embodiments, the method comprises administering to the subject any of the compositions provided herein. In some embodiments, the use of the compositions provided herein is provided in the preparation of a pharmaceutical composition or medicament for treating cancer. In some embodiments, the compositions can be used to treat cancer.

[0323] In some embodiments, a method of treating Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency) in a subject in need of such treatment is provided. In some embodiments, the method comprises administering to the subject any of the compositions provided herein. In some embodiments, a use of the compositions provided herein is provided in the preparation of a pharmaceutical composition or medicament for treating Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency). In some embodiments, the composition can be used to treat Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency).

[0324] In some embodiments, a method for treating a glycogen storage disease in a subject in need of such treatment is provided. In some embodiments, the method comprises administering to the subject any of the compositions provided herein. In some embodiments, the use of the compositions provided herein is provided in the preparation of a pharmaceutical composition or medicament for treating a glycogen storage disease. In some embodiments, the composition can be used to treat a glycogen storage disease.

[0325] In some embodiments, methods are provided for treating a glycogen storage disorder in a subject in need thereof, comprising administering a composition provided herein. In some embodiments, the glycogen storage disorder is selected from the group consisting of Cori's or Forbes' disease (GSD3, glycogen debranching enzyme (AGL) deficiency), McArdle's disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), type II diabetes / diabetic nephropathy, aldolase A deficiency (GSD12), Lafora's disease, hypoxia, Andersen's disease (GSD4, glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke disease), Haas disease (GSD6, hepatic glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency, syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11), glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0326] In some embodiments, the polypeptide that binds CD71 is directed to the central nervous system. In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject in need of treatment is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent. In some embodiments, the brain tumor is selected from the group consisting of non-malignant, benign, and malignant brain tumors. In some embodiments, the neurological condition is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Lafora's disease, Pompe's disease, adult polyglucosan body disease, stroke, spinal cord injury, ataxia, Bell's palsy, cerebral aneurysm, epilepsy, seizures, Guillain-Barre syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, migraine headache, encephalitis, sepsis, and myasthenia gravis. In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject, the method comprising administering to the subject an FN3 domain that binds CD71, the FN3 domain being conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide such as an siRNA, an ASO, an FN3 domain that binds to another target, etc.).

[0327] In some embodiments, a method of treating Pompe disease in a subject in need thereof is provided. 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 a FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent. In some embodiments, a method of treating Pompe disease in a subject is provided, the method comprising administering to the subject a FN3 domain that binds to CD71, the FN3 domain being conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide such as an siRNA, an ASO, an FN3 domain that binds to another target, etc.).

[0328] In some embodiments, the polypeptide that binds CD71 is directed to an immune cell. In some embodiments, the polypeptide that binds CD71 is directed to a dendritic cell. In some embodiments, a method of treating an autoimmune disease in a subject in need of treatment is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes mellitus, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, and immune thrombocytopenic purpura. In some embodiments, a method of 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 such as an siRNA, an ASO, an FN3 domain that binds to another target, etc.).

[0329] In some embodiments, a method for reducing expression of a target gene in a cell is provided. In some embodiments, the method includes delivering to a cell using a composition or pharmaceutical composition provided herein. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the target gene is GYS1. However, the target gene can be any target gene, since evidence provided herein demonstrates that siRNA molecules can be delivered efficiently when conjugated to an FN3 domain. In some embodiments, the siRNA targeting GYS1 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 provided herein or provided in PCT Application No. PCT / US20 / 55509, US Application No. 17 / 070,337, PCT Application No. PCT / US20 / 55470, or US 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.

[0330] In some embodiments, a method of reducing expression of a target gene in a cell is provided. In some embodiments, the method includes delivering to a cell with a composition or pharmaceutical composition provided herein. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the method of reducing expression of a target gene results in about 99%, 90-99%, 50-90%, or 10-50% reduction in expression of the target gene.

[0331] In some embodiments, a method of reducing expression of GYS1 is provided. In some embodiments, the reduced expression is expression (amount) of GYS1 mRNA. In some embodiments, the method of reducing expression of GYS1 results in about 99%, 90-99%, 50-90%, or 10-50% reduction in expression of GYS1. In some embodiments, the reduced expression is expression (amount) of GYS1 protein. In some embodiments, the reduced protein is glycogen. In some embodiments, the reduction in glycogen occurs in muscle cells. In some embodiments, the reduction in glycogen occurs in cardiac cells. In some embodiments, the method includes delivering to a cell with an siRNA molecule provided herein that targets GYS1. 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., bind to different epitopes. In some embodiments, the method comprises administering to a subject (patient) a GYS1 siRNA molecule, such as those provided herein. In some embodiments, the GYS1 siRNA 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., bind to different epitopes.In some embodiments, the CD71 binding domain is a polypeptide provided herein.

[0332] In some embodiments, the siRNA comprises a cyclic peptide of formula III: [ka] or a protonated form thereof, wherein R 1 , R 2 , and R 3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; R 1 , R 2 , and R 3 at least one of R is an aromatic or heteroaromatic side chain of an amino acid; 4 , R 5 , R 6 , R 7 are independently H or an amino acid side chain, R 4 , R 5 , R 6 , R 7is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, b-homoarginine, 3-(1-piperidinyl)alanine, AAsc is an amino acid side chain, and q is 1, 2, 3, or 4, and the cyclic peptide of formula @@ is not FfΦ RrRrE, as disclosed in International Patent Publication No. WO2022 / 213118, which is incorporated herein by reference in its entirety. "FfΦ RrRrE" refers to the structure where "F" is L-Phe, "f" is D-Phe, "Φ" is L-2-naphthylalanine, "R" is L-arginine, and "r" is D-arginine. "FfΦ RrRrE" is also described in Soudah et al. ("AntimiR-155 Cyclic Peptide-PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity", ACS Omega, 2019, 4(9):13954-13961), which is hereby incorporated by reference in its entirety.

[0333] In some embodiments, a method of delivering siRNA molecules to cells in a subject is provided. In some embodiments, the method comprises 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 dendritic cell, a cardiac cell, a muscle cell, a cell of the CNS, or a cell in the blood-brain barrier. In some embodiments, the siRNA downregulates the expression of a target gene in the cell. In some embodiments, the target gene is GYS1.

[0334] In some embodiments, a compound of formula IV, as described in International Patent Publication No. WO2022 / 198196, which is incorporated by reference in its entirety, is provided. [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein Y 2 and Y 3 are each C or Y 2 and Y 3 One of them is N and the other is Y 2 and Y 3 The other is C and X 1 and X 2 However, each independently, H, C 1-6 Alkyl, or C 1-6 Alkoxy, X 3 and X 4 are each independently H, halo, C alkyl, C alkoxy, or 5-20 membered heteroaryl, 3 and X 4 C 1-6 The alkyl is optionally substituted with one or more halo; and X 5 But, H, C 1-6 Alkyl, C1-6 Alkoxy, or C 3-10 cycloalkyl, any one of (1) L 1 does not exist, and Q 1 (i) to (iv): (i) phenyl (wherein, Q 1 The phenyl may be one or more halo, C 1-6 Alkyl, C 2-6 Alkenyl, -NH 2 , -NH-C(O)-(C 1-6 alkyl), -NH-C(O)-(3-15 membered heterocyclyl), C 3- 10 cycloalkyl, or 5-20 membered heteroaryl, wherein the C1-6 alkyl is selected from one or more of halo, -NH-C(O)-NH(C1-6 alkyl), -NH-C(O)-C 1-6 Alkyl, or -NH-C(O)-C 1-6 alkoxy optionally substituted, C3-10 cycloalkyl optionally substituted with one or more halo or C1-6 alkyl, and 5-20 membered heteroaryl optionally substituted with one or more C1-6 alkyl); (ii) 3-15 membered heterocyclyl, where Q 1 3-15 membered heterocyclyl is one or more oxo, or C 1-6 (iii) 5- to 20-membered heteroaryl, 1 5-20 membered heteroaryl is one or more halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Alkoxy, -NH 2 , or C 3-10 cycloalkyl, wherein C alkyl is optionally substituted with one or more halo, and C cycloalkyl is optionally substituted with one or more halo or C alkyl; and (iv) C 3-10 cycloalkyl; or (2) L 1 is -CH2-, and Q 1 But, C 3-10 is cycloalkyl, and L 2 -C(O)- or -S(O) 2 - and R1 is H or C alkyl, R k H, halo, -OH, -NH 2 or -NH-C(O)C 1-6 is alkyl, R m is H, -OH, or C 1-6 is alkyl, R n But, H, C 1-6 Alkyl or C 3-10 cycloalkyl or R n together with the carbon atom to which it is attached form a C3-5 cycloalkyl, or R k But R m Or R n together with the atom to which they are attached form a cyclopropyl group, R 2 (i) to (vii): (i) C1-6 alkyl (wherein, R 2 C1-6 alkyl is one or more of R a wherein R a (a) -OH, (b) cyano, (c) C 2-6 Alkynyl, (d) C 6-20 Aryl (wherein R a C 6-20 aryl is optionally substituted with one or more halo, cyano, C1-6 alkoxy, or -NH-C(O)-C1-6 alkyl; (e) 3- to 15-membered heterocyclyl, where R a 3 to 15-membered heterocyclyl is one or more of R c wherein R c is halo, oxo, C alkyl, C alkoxy, -C(O)-C alkyl, or -C(O)-C alkoxy, where R c C 1-6 The alkyl is optionally substituted with one or more halo or C alkynyl; R c -C(O)-C1-6 is optionally substituted with one or more halo; (f) -N(R c )(R d )(wherein -N(R c )(R d )Rc and R d are each independently H, C1-6 alkyl, -C(O)-C 1-6 Alkyl, -C(O)-C 1-6 Alkoxy, -C(O)-NH 2 , -C(O)-NH(C 1-6 alkyl), -C(O)-N(C 1-6 Alkyl) 2 , -C(O)-(3 to 15 membered heterocyclyl), -CH 2 -C(O)-NH 2 , 3- to 15-membered heterocyclyl, or 5- to 20-membered heteroaryl, wherein R c or R d C 1-6 Alkyl is optionally substituted with one or more -C(O)-NH2, R c or R d -C(O)-C 1-6 Alkyl is optionally substituted with one or more halo, R c or R d The 3- to 15-membered heterocyclyl and the 5- to 20-membered heteroaryl are independently optionally substituted with one or more C alkyl groups; R c or R d -C(O)-(3- to 15-membered heterocyclyl) is optionally substituted with one or more halo, -C(O)-C1-6 alkoxy, or C1-6 alkyl, and the C1-6 alkyl is optionally substituted with one or more halo, C1-6 alkoxy, or C3-1 0 Cycloalkyl and C 1-6 Optionally substituted with alkyl, R c or R d -C(O)-N(C 1-6 Alkyl) 2 are each independently optionally substituted with one or more halo or C aryl; e (In the formula, R e But, C 1-6 Alkyl, C 6-20 Aryl, -C(O)-(3-15 membered heterocyclyl), -C(O)-N-(C 1-6 Alkyl) 2 or 5-20 membered heteroaryl, Re wherein the C1-6 alkyl is optionally substituted with one or more C1-6 alkoxy, and the C1-6 alkoxy is optionally substituted with one or more C2-6 alkynyl; R e C 6-20 Aryl is one or more C 1-6 Optionally substituted with alkyl, R e -C(O)-(3 to 15 membered heterocyclyl) is optionally substituted with one or more C1-6 alkyl, C1-6 alkoxy, or -C(O)-C1-6 alkoxy, and the C1-6 alkyl is optionally substituted with one or more halo, C 1-6 Alkoxy or C 3-10 (h)-C(O)-R optionally substituted with cycloalkyl e (In the formula, R e is -NH2, -OH, or 3- to 15-membered heterocyclyl), or (i) -S(O) 2 -R f (In the formula, R f But, C 1-6 alkyl or 3- to 15-membered heterocyclyl, with the proviso that R 2 is unsubstituted methyl, then either (1)Q 1 is a 5- to 20-membered heteroaryl, 1 The 5-20 membered heteroaryl is selected from the group consisting of one or more halo, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, -NH2, C3- 10 Optionally substituted with cycloalkyl or -OH, Q 1 is not an unsubstituted pyridyl, or (2)Q 1 is phenyl, and Q 1 The phenyl has (i) at least one C 3-6 Alkyl, having at least one C 3-6 The alkyl is optionally substituted with one or more halo. 3-6 alkyl, or (ii) at least one C 3-10 Cycloalkyl, having at least one C 3-10 Cycloalkyl may be one or more halo or C 1-6 At least one C optionally substituted with alkyl3-10 cycloalkyl, or (iii) at least one 5-20 membered heteroaryl, where at least one 5-20 membered heteroaryl is substituted with at least one 5-20 membered heteroaryl optionally substituted with one or more C alkyl; 3-10 Cycloalkyl (R 2 C 3-10 Cycloalkyl is one or more R q Optionally substituted with R q is a 5-20 membered heteroaryl or C6-20 aryl; R q wherein R is a 3- to 15-membered heterocyclyl, and R is a 3- to 15-membered heterocyclyl. 2 3 to 15-membered heterocyclyl is substituted with one or more halo, oxo, C 1-6 Alkyl, -C(O)-C 1-6 (iv) 5-20 membered heteroaryl or -(C 1-4 alkyl)(5-20 membered heteroaryl) (wherein C 1-4 Alkyl may contain one or more of -OH, halo, -NH 2 , -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, and the 5-20 membered heteroaryl is optionally substituted with one or more R s Optionally substituted with R s But, Halo, C 1-6 Alkyl, C 1-6 Alkoxy, -NH 2 , -NH(C 1-6 alkyl), -N(C 1-6 Alkyl) 2 , -NH-C(O)-C 1- 6 alkyl, C6-20 aryl, C3-10 cycloalkyl, 3-15 membered heterocyclyl, 5-20 membered heteroaryl, or -C(O)-C1-6 alkoxy; R s C 1-6 Alkyl may be one or more halo, C 1-6 Alkoxy, -NH 2 , -NH(C1-6 alkyl), -N(C 1-6 Alkyl) 2 , -NH-C(O)C 1-6 Alkyl, or -NH-C(O)-C 1-6 Optionally substituted with alkoxy, R s The 3- to 15-membered heterocyclyl is selected from the group consisting of one or more halo or -C(O)-C 1-6 (v) -N(R g )(R h )(wherein, R g and R h are independently H or C 1-6 (vi) -C(O)-R j (In the formula, R j is C 3-10 Cycloalkyl, -NH(C 1-6 alkyl), -N(C 1-6 Alkyl) 2 or -NH(5-20 membered heteroaryl), and (vii) C 6-20 Aryl (wherein R 2 C 6-20 Aryl is one or more 5-20 membered heteroaryl or -OR p Optionally substituted with R p is 3-15 membered heterocyclyl, R p wherein the 3- to 15-membered heterocyclyl is optionally substituted with one or more -C(O)-C1-6 alkyl.

[0335] In some embodiments, the compositions provided herein can be used to diagnose, monitor, regulate, treat, alleviate, prevent the occurrence of, or help alleviate symptoms of, human diseases or specific pathologies in cells, tissues, organs, fluids, or generally in a host, and also exhibit properties that can cross the blood-brain barrier. The blood-brain barrier (BBB) ​​prevents most macromolecules (e.g., DNA, RNA, and polypeptides) as well as many small molecules from entering the brain. The BBB is primarily composed of specialized endothelial cells with highly restrictive tight junctions, so the passage of large and small substances from the blood to the central nervous system is controlled by the BBB. This structure makes it difficult to treat and manage patients with neurological diseases and disorders (e.g., brain tumors) because many therapeutic agents cannot be delivered across the BBB with the desired efficiency. Additional conditions involving disruption of the BBB include stroke, diabetes, seizures, hypertensive encephalopathy, acquired immune deficiency syndrome, traumatic brain injury, multiple sclerosis, Lafora's disease, Pompe's disease, adult polyglucosan body disease, Parkinson's disease (PD), and Alzheimer's disease. This capability is particularly useful for treating brain tumors, including, for example, astrocytoma, medulloblastoma, glioma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors, or spinal cancers, for example, neurofibroma, meningioma, glioma, and sarcoma. This is further useful in treating Pompe's disease and / or glycogen storage diseases. In certain embodiments, the compositions provided herein can be used, for example, to deliver therapeutic or cytotoxic agents across the blood-brain barrier. In certain embodiments, the compositions provided herein can be used, for example, to deliver therapeutic or cytotoxic agents into muscle.

[0336] In some embodiments, methods are provided for treating a glycogen storage disorder in a subject in need thereof, comprising administering a composition provided herein. In some embodiments, the glycogen storage disorder is selected from the group consisting of Cori's or Forbes' disease (GSD3, glycogen debranching enzyme (AGL) deficiency), McArdle's disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), type II diabetes / diabetic nephropathy, aldolase A deficiency (GSD12), Lafora's disease, hypoxia, Andersen's disease (GSD4, glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke disease), Haas disease (GSD6, hepatic glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency, syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11), glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0337] In some embodiments, the compositions or pharmaceutical compositions provided herein can be used to treat muscle diseases such as muscular dystrophy, DMD.

[0338] 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. In some embodiments, the other or additional therapeutic agents are other anti-tumor agents or therapeutic agents. Different tumor types and stages of tumors may require the use of various adjunct compounds useful in the treatment of cancer. For example, the compositions provided herein can be used in combination with various chemotherapeutic agents such as, inter alia, taxol, tyrosine kinase inhibitors, leucovorin, fluorouracil, irinotecan, phosphatase inhibitors, MEK inhibitors, etc. The compositions can also be used in combination with, inter alia, drugs that modulate the immune response against tumors such as anti-PD-1 or anti-CTLA-4. An additional treatment can be an agent that modulates the immune system, such as an antibody that targets PD-1 or PD-L1.

[0339] In some embodiments, the compositions or pharmaceutical compositions provided herein may be administered in combination with enzyme replacement therapy (ERT). In some embodiments, the ERT is an ERT designed to treat a glycogen storage disease. In some embodiments, the glycogen storage disease is selected from the group consisting of Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Cori disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Tarui disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke disease), Haas disease (GSD6, hepatic glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency, syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11), glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0340] Thus, in some embodiments, ERT comprises administration of one or more enzymes or gene replacement products selected from the group consisting of glucosidase alpha acid (GAA), glycogen debranching enzyme (AGL), glycogen branching enzyme (BGE1), muscle glycogen phosphorylase (PYGM), muscle phosphofructokinase (PFKM), aldolase A (ALDOA), malin, laforin, glycogen synthase (GYS2), glucose-6-phosphatase (G6PC / SLC37A4), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1), phosphoglycerate mutase (PGAM2), muscle lactate dehydrogenase (LDHA), glucose transporter (GLUT2), β-enolase (ENO3), glycogenin-1 (GYG1), or any combination thereof. In some embodiments, ERT comprises administration of glucosidase alpha acid (GAA). In some embodiments, the ERT comprises administering glycogen debranching enzyme (AGL). In some embodiments, the ERT comprises administering glycogen branching enzyme (BGE1). In some embodiments, the ERT comprises administering muscle glycogen phosphorylase (PYGM). In some embodiments, the ERT comprises administering muscle phosphofructokinase (PFKM). In some embodiments, the ERT comprises administering aldolase A (ALDOA). In some embodiments, the ERT comprises administering malin. In some embodiments, the ERT comprises administering laforin. In some embodiments, the ERT comprises administering glycogen synthase (GYS2). In some embodiments, the ERT comprises administering glucose-6-phosphatase (G6PC / SLC37A4). In some embodiments, the ERT comprises administering phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1). In some embodiments, the ERT comprises administering phosphoglycerate mutase (PGAM2). In some embodiments, the ERT comprises administering muscle lactate dehydrogenase (LDHA). In some embodiments, the ERT comprises administering glucose transporter (GLUT2).In some embodiments, the ERT comprises administering β-enolase (ENO3). In some embodiments, the ERT comprises administering glycogenin-1 (GYG1).

[0341] In some embodiments, a method of treating a glycogen storage disease in a subject in need of such treatment is provided. In some embodiments, the method includes administering to the subject any of the compositions provided herein and ERT. In some embodiments, the use of the compositions provided herein and ERT is provided in the preparation of a pharmaceutical composition or medicament for treating a glycogen storage disease. In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to an additional therapeutic agent, such as, but not limited to, an oligonucleotide (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of ERT. In some embodiments, the composition is administered to the subject during administration of ERT. In some embodiments, the composition is administered to the subject after administration of ERT.

[0342] In some embodiments, a method of treating Pompe disease in a subject in need of such treatment is provided. In some embodiments, the method includes administering to the subject any of the compositions provided herein and an ERT comprising glucosidase alpha acid (GAA). In some embodiments, the use of the compositions provided herein and an ERT comprising glucosidase alpha acid (GAA) is provided in the preparation of a pharmaceutical composition or medicament for treating Pompe disease. In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to an additional therapeutic agent, such as, but not limited to, an oligonucleotide (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of an ERT. In some embodiments, the composition is administered to the subject during administration of an ERT. In some embodiments, the composition is administered to the subject after administration of an ERT.

[0343] In some embodiments, a method of treating Cori's disease or Forbes' disease (GSD3) in a subject in need of such treatment is provided. In some embodiments, the method comprises administering to the subject an ERT comprising any of the compositions provided herein and glycogen debranching enzyme (AGL). In some embodiments, the use of the compositions provided herein and an ERT comprising glycogen debranching enzyme (AGL) is provided in the preparation of a pharmaceutical composition or medicament for treating Cori's disease or Forbes' disease (GSD3). In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to an additional therapeutic agent, such as, but not limited to, an oligonucleotide (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of an ERT. In some embodiments, the composition is administered to the subject during administration of an ERT. In some embodiments, the composition is administered to the subject after administration of an ERT.

[0344] In some embodiments, a method of treating Andersen's disease (GSD4) in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject an ERT comprising any of the compositions provided herein and glycogen branching enzyme (GBE1). In some embodiments, the use of an ERT comprising the compositions provided herein and glycogen branching enzyme (GBE1) is provided in the preparation of a pharmaceutical composition or agent for treating Andersen's disease (GSD4). In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to additional therapeutic agents such as, but not limited to, oligonucleotides (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of the ERT. In some embodiments, the composition is administered to the subject during administration of the ERT. In some embodiments, the composition is administered to the subject after administration of the ERT.

[0345] In some embodiments, a method of treating McArdle disease (GSD5) in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject an ERT comprising any of the compositions provided herein and muscle glycogen phosphorylase (PYGM). In some embodiments, the use of an ERT comprising the compositions provided herein and muscle glycogen phosphorylase (PYGM) is provided in the preparation of a pharmaceutical composition or agent for treating McArdle disease (GSD5). In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to additional therapeutic agents such as, but not limited to, oligonucleotides (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of the ERT. In some embodiments, the composition is administered to the subject during administration of the ERT. In some embodiments, the composition is administered to the subject after administration of the ERT.

[0346] In some embodiments, a method of treating Tarui's Disease (GSD7) in a subject in need of such treatment is provided. In some embodiments, the method comprises administering to the subject an ERT comprising any of the compositions provided herein and muscle phosphofructokinase (PFKM). In some embodiments, the use of the compositions provided herein and an ERT comprising muscle phosphofructokinase (PFKM) is provided in the preparation of a pharmaceutical composition or medicament for treating Tarui's Disease (GSD7). In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to an additional therapeutic agent, such as, but not limited to, an oligonucleotide (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of an ERT. In some embodiments, the composition is administered to the subject during administration of an ERT. In some embodiments, the composition is administered to the subject after administration of an ERT.

[0347] In some embodiments, a method of treating aldolase A deficiency (GSD12) in a subject in need of such treatment is provided. In some embodiments, the method includes administering to the subject any of the compositions provided herein and an ERT comprising aldolase A (ALDOA). In some embodiments, the use of the compositions provided herein and an ERT comprising aldolase A (ALDOA) ​​is provided in the preparation of a pharmaceutical composition or medicament for treating aldolase A deficiency (GSD12). In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to an additional therapeutic agent, such as, but not limited to, an oligonucleotide (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of an ERT. In some embodiments, the composition is administered to the subject during administration of an ERT. In some embodiments, the composition is administered to the subject after administration of an ERT.

[0348] In some embodiments, a method of treating Lafora's disease in a subject in need of such treatment is provided. In some embodiments, the method comprises administering to the subject any of the compositions provided herein and an ERT comprising malin, laforin, or both. In some embodiments, the use of the compositions provided herein and an ERT comprising malin, laforin, or both is provided in the preparation of a pharmaceutical composition or medicament for treating Lafora's disease. In some embodiments, the composition is a composition comprising an FN3 domain that can be linked to an additional therapeutic agent, such as, but not limited to, an oligonucleotide (e.g., siRNA, antisense, mRNA, miRNA, cDNA, etc.). In some embodiments, the composition is administered to the subject prior to administration of an ERT. In some embodiments, the composition is administered to the subject during administration of an ERT. In some embodiments, the composition is administered to the subject after administration of an ERT.

[0349] In some embodiments, a method of treating adult polyglucosan body disease in a subject in need of such treatment is provided. In some embodiments, the method comprises administering to the subject any of the compositions provided herein and an ERT comprising glycogen branching enzyme (BGE1). In some embodiments, the use of the compositions provided herein and an ERT comprising glycogen branching enzyme (BGE1) is provided in the preparation of a pharmaceutical composition or medicament for treating adult polyglucosan body disease. In some embodiments, the composition is a composition comprising an FN3 domain. In some embodiments, the composition is administered to the subject prior to administration of an ERT. In some embodiments, the composition is administered to the subject during administration of an ERT. In some embodiments, the composition is administered to the subject after administration of an ERT.

[0350] "Treating" or "treatment" refers to therapeutic treatment and preventative measures, the purpose of which is to prevent or slow down (alleviate) undesirable physiological changes or disorders, 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, reduction in the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, or those in need of preventing the condition or disorder.

[0351] "Therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amount of the compositions provided herein may vary depending on factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective amount are improvement in the patient's health, reduction or shrinkage of the size of the tumor, cessation or slowing of tumor growth, and / or absence of metastasis of cancer cells to other locations in the body.

[0352] Administration / Pharmaceutical Compositions

[0353] In some embodiments, pharmaceutical compositions of the compositions provided herein and pharma- ceutically acceptable carriers are provided. For therapeutic use, the compositions can be prepared as pharmaceutical compositions containing an effective amount of the domain or molecule as an active ingredient in a pharma- ceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle in which the 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 can contain pharma- ceutically 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 may vary widely, i.e., less than about 0.5% by weight, usually at least about 1% by weight up to 15% or 20% by weight, and is selected primarily based on the required dose, fluid volumes, viscosities, etc., according to the particular method of administration selected. Suitable vehicles and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see in particular pp. 958-989.

[0354] Methods of administration for therapeutic use of the compositions disclosed herein can be any suitable route that delivers an agent to a host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using formulations in tablets, capsules, solutions, powders, gels, particles, as is well known in the art, including syringes, implanted devices, osmotic pumps, cartridges, micropumps, or other means understood by those of skill in the art. Site-specific administration can be achieved, for example, by intra-articular, intrabronchial, intraabdominal, intracapsular, intracartilage, intracavitary, intracelial, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

[0355] The pharmaceutical composition can be provided as a kit comprising a container containing the pharmaceutical composition described herein.The pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted before injection.Alternatively, such a kit can include a dry powder disperser, liquid aerosol generator, or nebulizer for administration of the pharmaceutical composition.Such a kit can further include written information regarding the indications and use of the pharmaceutical composition.

[0356] Additionally, the following embodiments are also provided.

[0357] 1. A method of treating a glycogen storage disorder in a subject in need thereof, comprising: A composition comprising one or more FN3 domains linked to an siRNA molecule (or other oligonucleotides, such as antisense oligonucleotides, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and enzyme replacement therapy (ERT).

[0358] 2. The method of embodiment 1, wherein the glycogen storage disease is selected from the group consisting of Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Cori disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Tarui disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes mellitus / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucosan body disease.

[0359] 3. The method of embodiment 1 or 2, wherein the ERT comprises one or more enzymes selected from the group consisting of glucosidase alpha acid (GAA), glycogen debranching enzyme (AGL), glycogen branching enzyme (BGE1), muscle glycogen phosphorylase (PYGM), muscle phosphofructokinase (PFKM), aldolase A (ALDOA), malin, laforin, glycogen synthase (GYS2), glucose-6-phosphatase (G6PC / SLC37A4), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1), phosphoglycerate mutase (PGAM2), muscle lactate dehydrogenase (LDHA), glucose transporter (GLUT2), β-enolase (ENO3), and glycogenin-1 (GYG1).

[0360] 4. The method of any one of embodiments 1 to 3, wherein the composition comprising an FN3 domain linked to an siRNA is administered to the subject prior to administration of the ERT.

[0361] 5. The method of any one of embodiments 1 to 3, wherein the composition comprising an FN3 domain linked to an siRNA is administered to the subject during administration of the ERT.

[0362] 6. The method of any one of embodiments 1 to 3, wherein the composition comprising an FN3 domain linked to an siRNA is administered to the subject after administration of the ERT.

[0363] 7. A method of treating Pompe disease in a subject in need thereof, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising glucosidase alpha acid (GAA).

[0364] 8. A method for treating Coli disease (also known as Forbes' disease) in a subject in need thereof, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising glycogen debranching enzyme (AGL).

[0365] 9. A method of treating Andersen's disease (GSD4) in a subject in need of such treatment, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising glycogen branching enzyme (GBE1).

[0366] 10. A method of treating McArdle disease (GSD5) in a subject in need thereof, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising glycogen phosphorylase (PYGM).

[0367] 11. A method of treating Tarui's disease (GSD7) in a subject in need thereof, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising muscle phosphofructokinase (PFKM).

[0368] 12. A method of treating aldolase A deficiency (GSD12) in a subject in need of such treatment, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising aldolase A (ALDOA).

[0369] 13. A method of treating Lafora's disease in a subject in need thereof, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising malin, laforin, or both.

[0370] 14. A method of treating adult polyglucosan body disease in a subject in need thereof, comprising: A composition comprising an FN3 domain linked to an siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) comprising a sense strand and an antisense strand as provided herein; and an ERT comprising glycogen branching enzyme (BGE1).

[0371] 15. The method of any one of embodiments 7-14, further comprising at least one additional ERT.

[0372] 16. The method of any one of embodiments 7 to 15, wherein the composition comprising an FN3 domain linked to an siRNA is administered to the subject prior to administration of the ERT.

[0373] 17. The method of any one of embodiments 7 to 15, wherein the composition comprising an FN3 domain linked to an siRNA is administered to the subject during administration of the ERT.

[0374] 18. The method of any one of embodiments 7 to 15, wherein the composition comprising an FN3 domain linked to an siRNA is administered to the subject after administration of the ERT.

[0375] 19. The method of any one of embodiments 1-18, wherein the one or more FN3 domains comprises an FN3 domain that binds to CD71.

[0376] 20. The method of any one of embodiments 1 to 19, wherein the siRNA (or other oligonucleotide, such as an antisense oligonucleotide, or otherwise provided herein) molecule is an siRNA that reduces the expression of GYS1.

[0377] 21. The method of any one of embodiments 1 to 20, wherein the siRNA does not contain any modified nucleobases.

[0378] 22. The method of any one of embodiments 1 to 21, wherein the siRNA further comprises a linker covalently attached to the sense strand or the antisense strand of the siRNA.

[0379] 23. The method of embodiment 22, wherein the linker is attached to the 5' end or the 3' end of the sense strand or the antisense strand.

[0380] 24. The method of any one of embodiments 1 to 23, wherein the siRNA further comprises a vinylphosphonate modification on the sense strand or the antisense strand.

[0381] 25. The method of embodiment 24, wherein the vinyl phosphonate modification is attached to the 5'-end or the 3'-end of the sense strand or the antisense strand.

[0382] 26. The sense strand is selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 63 8, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 801-860, 921-980, or as set forth in Table 3A, Table 3B, or Table 4.

[0383] 27. The antisense strand is selected from the group consisting of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639 27. The method of any one of embodiments 1-26, comprising a sequence of: 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 690, 691, 693, 695, 697, 699, 701, 703, 705, 707, 861-920, 981-1042, or as set forth in Table 3A, Table 3B, or Table 4.

[0384] 28. The siRNA molecule is A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, AA, BB, CC, DD, EE, FF, GG, HH, II, JJ, K K, LL, MM, NN, OO, PP, QQ, RR, SS, TT, UU, VV, WW, XX, YY, ZZ, AAA, BBB, CCC, DDD, EEE, FFF, GGG, HHH, III, JJJ, KKK, LLL, MM The method of any one of the preceding embodiments, comprising the siRNA pair of: M, NNN, OOO, PPP, QQQ, RRR, SSS, TTT, UUU, VVV, WWW, XXX, YYY, ZZZ, AAAA, BBBB, CCCC, DDDD, EEEE, FFFF, GGGG, HHHH, IIII, JJJJ, KKKK, LLLL, MMMM, NNNN, OOOO, PPPP, or as described in Table 3A, Table 3B, or Table 4.

[0385] 29. The method of any one of the preceding embodiments, wherein the sense strand comprises 19 nucleotides.

[0386] 30. The method of any one of the preceding embodiments, wherein the antisense strand comprises 21 nucleotides.

[0387] 31. The method of any one of embodiments 1 to 30, wherein the composition comprises the siRNA pair provided in Table 4, together with a linker and / or vinyl phosphonate modification, as described in Table 5.

[0388] 32. The siRNA molecule has the formula exemplified in Formula I, [ka] The method of any one of the preceding embodiments, wherein each nucleotide represented by N is independently A, U, C, or G, or a modified nucleotide base such as those provided herein.

[0389] 33. 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, as well as N 4 , N 5 , N 6 , N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 , and N 19 33. The method of embodiment 32, wherein the nucleotide sequence comprises 2'O-methyl modified nucleotides.

[0390] 34. The antisense strand is N 1 A vinyl phosphonate moiety bound to N 2 2' fluoro-modified nucleotide with phosphorothioate (PS) modified backbone at N 3 , N4 , 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 to 2’O-methyl modified nucleotides, N 14 to 2’-fluoro modified nucleotides, and N 20 and N 21 to 2’O-methyl modified nucleotides having a phosphorothioate (PS) modified backbone as described in embodiment 32.

[0391] 35. The N of the antisense strand 1 is a vinylphosphonate moiety attached thereto, as described in embodiment 32.

[0392] 36. The siRNA molecule has the formula exemplified by formula I,

Chemical formula

[0393] 37. F 1 is (X 1 ) n -(X 2 )q -(X 3 ) y wherein X 1 is the first FN3 domain, and X 2 is the second FN3 domain, and X 3 is a third FN3 domain or a half-life extension, and n, q, and y are each independently 0 or 1, with the proviso that at least one of n, q, and y is 1.

[0394] 38. The method of any one of the preceding embodiments, wherein the FN3 domain is conjugated to the siRNA molecule via a cysteine ​​on the FN3 domain.

[0395] 39. The method of embodiment 38, wherein the cysteines are at the positions described herein.

[0396] 40. The method of embodiment 38 or 39, wherein the cysteine ​​in the FN3 domain is at a position corresponding to residue 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 the FN3 domain comprising an amino acid sequence based on SEQ ID NO: 713.

[0397] 41. The method of embodiment 40, wherein the cysteine ​​is located at a position corresponding to residue 6, 53, or 88.

[0398] 42. The method of any one of the preceding embodiments, wherein the FN3 domain has a sequence selected from the group consisting of SEQ ID NOs: 509, 708, and 710.

[0399] 43. The method of any one of the preceding embodiments, wherein the one or more FN3 domains comprises at least two FN3 domains linked by a peptide linker.

[0400] 44. The method according to any one of the preceding embodiments, wherein the composition comprises a first FN3 domain and a second FN3 domain.

[0401] 45. The method according to embodiment 44, wherein the first FN3 domain and the second FN3 domain bind to different proteins.

[0402] 46. The composition according to embodiment 44, wherein the first FN3 domain and the second FN3 domain bind to the same protein.

[0403] 47. The method according to any one of embodiments 44 to 46, wherein the first FN3 domain binds to CD71.

[0404] 48. The method according to any one of embodiments 44 to 46, wherein the second FN3 domain binds to a different target that does not bind to CD71.

[0405] 49. The method according to any one of the preceding embodiments, wherein the FN3 domain comprises a sequence that is at least 87% identical to, or identical to, the sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0406] 50. The method according to any one of embodiments 43 to 49, further comprising a third FN3 domain.

[0407] 51. The method according to embodiment 50, wherein the third FN3 domain is an FN3 domain that binds to CD71 or albumin.

[0408] 52. The method according to embodiment 51, wherein the FN3 domain that binds to CD71 has an amino acid sequence provided herein, including but not limited to SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710, or a binding fragment thereof.

[0409] 53. The method of embodiment 51, wherein the FN3 that binds to albumin has an amino acid sequence provided herein, including but not limited to, SEQ ID NOs: 101-119, or a binding fragment thereof.

[0410] 54. The composition comprising an FN3 domain linked to an siRNA, 1 ) n -(X 2 ) q -(X 3 ) y -LX 4 , C-(X 1 ) n -(X 2 ) q -LX 4 -(X 3 ) y , (X 1 ) n -(X 2 ) q -LX 4 -(X 3 ) y -C, C-(X 1 ) n -(X 2 ) q -LX 4 -L-(X 3 ) y , or (X 1 ) n -(X 2 ) q -LX 4 -L-(X 3 ) y -C, During the ceremony, X 1 is the first FN3 domain, X 2 is the second FN3 domain, X 3 is a third FN3 domain or a half-life extender; L is a linker, X 4 is a nucleic acid molecule (e.g., one or more strands of the siRNA), C is a polymer such as PEG, an albumin binding protein, 2. The method of embodiment 1, wherein n, q, and y are each independently 0 or 1.

[0411] 5...

Claims

1. A combination for treating glycogen storage disorders, (a) An FN3 domain that binds to CD71 linked to a siRNA targeting Gys1, wherein the siRNA includes a sense strand and an antisense strand, (b) A composition comprising enzyme replacement therapy (ERT), The aforementioned combination, including the above.

2. The combination according to claim 1, wherein the glycogen storage disorder is selected from the group consisting of Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Koli's disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen's disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle's disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Tarui's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes mellitus / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucosan body disease.

3. The combination according to claim 1, wherein the ERT comprises one or more enzymes selected from the group consisting of glucosidase alpha acid (GAA), glycogen debranching enzyme (AGL), glycogen branching enzyme (BGE1), muscle glycogen phosphorylase (PYGM), muscle phosphofructokinase (PFKM), aldolase A (ALDOA), marin, laforin, glycogen synthase (GYS2), glucose-6-phosphatase (G6PC / SLC37A4), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1), phosphoglycerate mutase (PGAM2), muscle lactate dehydrogenase (LDHA), glucose transporter (GLUT2), β-ennolase (ENO3), and glycogennin-1 (GYG1).

4. The combination according to any one of claims 1 to 3, wherein the siRNA targeting Gys1 is a siRNA that reduces the expression of GYS1.

5. The aforementioned glycogen storage disorder is Pompe disease. The FN3 domain is a polypeptide that binds to CD71. The aforementioned ERT includes administering glucosidase alpha acid (GAA); or The aforementioned glycogen storage disorder is Lafora disease. The FN3 domain is a polypeptide that binds to CD71. The aforementioned ERT includes administering marin, laforin, or both. The combination described in claim 1.

6. The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 704, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 705; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 614, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 615; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 632, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 633; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 10, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 11; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 12, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 13; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 14, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 15; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 16, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 17; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 18, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 19; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 20, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 21; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 22, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 23; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 24, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 25; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 26, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 27; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 28, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 29; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 30, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 31; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 32, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 33; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 34, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 35; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 36, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 37; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 38, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 39; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 40, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 41; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 42, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 43; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 44, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 45; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 46, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 47; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 48, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 49; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 50, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 51; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 52, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 53; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 54, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 55; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 56, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 57; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 58, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 59; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 60, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 61; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 62, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 63; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 64, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 65; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 66, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 67; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 68, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 69; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 600, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 601; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 602, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 603; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 604, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 605; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 606, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 607; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 608, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 609; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 610, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 611; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 612, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 613; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 616, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 617; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 618, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 619; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 620, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 621; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 622, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 623; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 624, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 625; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 626, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 627; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 628, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 629; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 630, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 631; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 634, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 635; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 636, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 637; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 638, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 639; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 640, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 641; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 642, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 643; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 644, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 645; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 646, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 647; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 648, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 649; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 650, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 651; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 652, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 653; The sense strand comprises the nucleic acid base sequence of SEQ ID NO: 654, and the antisense strand comprises the nucleic acid base sequence of SEQ ID NO: 655; or The sense strand contains the nucleic acid base sequence of SEQ ID NO: 656, and the antisense strand contains the nucleic acid base sequence of SEQ ID NO:

657. The combination described in claim 1.

7. The combination according to claim 1, wherein the siRNA is covalently bound to the FN3 domain via a chemical linker covalently bonded to the sense strand or antisense strand of the siRNA, and the siRNA linked to the FN3 domain is conjugated to a cysteine ​​residue of the FN3 domain.

8. The combination according to claim 7, wherein the cysteine ​​within the FN3 domain is located at a position corresponding to 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, or 93 of the FN3 domain, which includes the amino acid sequence based on Sequence ID No.

713.

9. The combination according to claim 1, wherein the FN3 domain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 509, 708, and 710.

10. The aforementioned FN3 domain, An amino acid sequence that is at least 95% identical to the amino acid sequences of SEQ ID NOs. 273, 288-291, 301-310, 312-572, 592-599, or 708-710; or Amino acid sequences of sequence numbers 273, 288-291, 301-310, 312-572, 592-599, or 708-710 The combination according to claim 1, including the following:

11. A kit for treating glycogen storage disorders, comprising: a first container containing a pharmaceutical composition comprising an FN3 domain that binds to CD71 linked to a Gys1-targeting siRNA, wherein the siRNA comprises a sense strand and an antisense strand; and a second container containing a pharmaceutical composition comprising enzyme replacement therapy (ERT) for treating the glycogen storage disorder.

12. The kit according to claim 11, wherein the glycogen storage disorder is selected from the group consisting of Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Koli's disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen's disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle's disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Tarui's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes mellitus / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucosan body disease.

13. The kit according to claim 11, wherein the ERT comprises glucosidase alpha acid (GAA), glycogen debranching enzyme (AGL), glycogen branching enzyme (BGE1), muscle glycogen phosphorylase (PYGM), muscle phosphofructokinase (PFKM), aldolase A (ALDOA), marin, laforin, glycogen synthase (GYS2), glucose-6-phosphatase (G6PC / SLC37A4), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1), phosphoglycerate mutase (PGAM2), muscle lactate dehydrogenase (LDHA), glucose transporter (GLUT2), β-ennolase (ENO3), and glycogennin-1 (GYG1), or a combination thereof.

14. The kit according to claim 11, wherein the first container contains a pharmaceutical composition comprising an FN3 domain bound to CD71 linked to a Gys1-targeting siRNA, and the second container contains a pharmaceutical composition comprising the GAA enzyme.

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