FN3 domain-siRNA conjugates with enzyme replacement therapy

Abstract

The present disclosure relates to compositions in combination with enzyme replacement therapy, such as siRNA molecules and FN3 domains conjugated thereto, as well as methods of making and using these compositions. Also disclosed is a method of treating a glycogen storage disease comprising administering a composition comprising a siRNA molecule and an FN3 domain conjugated thereto, and an enzyme replacement therapy (ERT) for treating the glycogen storage disease.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 330,797, filed on April 14, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] Embodiments of the present invention relate to siRNA molecules that can be conjugated to a fibronectin type III domain (FN3) and methods of using the same in combination with enzyme replacement therapy. Background Art

[0004] Therapeutic nucleic acids include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense nucleic acids, antagomirs, antimirs, microRNA mimics, supermirs, U1 adapters and aptamers. In the case of siRNA or miRNA, these nucleic acids can be used to reduce the intracellular level of specific proteins by the process called RNA interference (RNAi). The therapeutic application of RNAi is very extensive, because siRNA and miRNA constructs can be synthesized with any nucleotide sequence for target protein. So far, siRNA constructs have all demonstrated the ability of specificity to reduce target protein in both in vitro and in vivo models. In addition, siRNA constructs are currently being evaluated in clinical research and have been approved for a variety of diseases.

[0005] However, two problems that siRNA constructs are currently facing are, first, their sensitivity to nuclease digestion in blood plasma, and secondly, when used as free siRNA or miRNA systemically, they obtain limited ability to enter the intracellular compartment, in which they can bind to RISC (RNA-induced silencing complex). Some delivery systems (such as lipid nanoparticles formed by cationic lipids and other lipid components (such as cholesterol and PEG lipids), carbohydrates (such as GalNac trimers)) have been used to promote the cellular uptake of oligonucleotides. However, these have not yet demonstrated successful siRNA effectively delivered to the expected target in the tissue except the liver.

[0006] There remains a need for compositions and methods for delivering siRNA to its intended cellular target. Furthermore, what is needed are FN3 domains that can specifically bind to CD71 with optimized properties for clinical use, and methods of using such molecules for enabling new therapeutic agents that can enter cells through receptor-mediated CD71 internalization. Embodiments of the present invention meet these needs and others. Summary of the invention

[0007] In some embodiments, methods of treating a glycogen storage disease in a subject in need thereof are provided. In some embodiments, the method comprises administering a composition comprising one or more FN3 domains linked to a siRNA molecule comprising a sense strand and an antisense strand as 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's disease or Forbes' disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen's disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdledisease (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 / Diabetic Nephropathy, Lafora Disease, Hypoxia and Adult Polyglucan 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 glycogen protein-1 (GYG1).

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

[0011] In some embodiments, a FN3 domain comprising the amino acid sequence of any FN3 domain provided herein is provided. 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 are provided herein, such as dsRNA or siRNA molecules. In some embodiments, oligonucleotides have sequences as provided herein, with or without modifications as 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 comprising one or more FN3 domains conjugated to siRNA molecules are provided.

[0015] In some embodiments, there is provided a compound having formula (X1) n -(X2) q -(X3) y -L-X4, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; and X4 is an oligonucleotide molecule, wherein n, q and y are each independently 0 or 1.

[0016] In some embodiments, there is provided a compound having the formula C-(X1) n -(X2) q -(X3) y -L-X4, wherein 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 molecule; L is a linker; and X4 is an oligonucleotide molecule, wherein n, q and y are each independently 0 or 1.

[0017] In some embodiments, there is provided a compound having formula (X1) n -(X2) q -(X3) y -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 extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer or an albumin binding domain (ABD), wherein n, q and y are each independently 0 or 1.

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

[0019] In some embodiments, there is provided a compound having the formula C-X4-L-(X1) n -(X2) q -(X3) y The invention provides a composition of the invention, wherein 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 molecule; L is a linker; and X4 is an oligonucleotide molecule, wherein n, q and y are each independently 0 or 1.

[0020] In some embodiments, there is provided a compound having the formula X4-L-(X1) 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 molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer or an albumin binding domain (ABD), wherein n, q and y are each independently 0 or 1.

[0021] In some embodiments, there is provided a compound having the formula C-(X1) n -(X2) q [L-X4]-(X3) y The invention provides a composition of the invention, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer or an albumin binding domain (ABD), wherein n, q and y are each independently 0 or 1.

[0022] In some embodiments, there is provided a compound having formula (X1) n -(X2) q [L-X4]-(X3) y -C, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer or an albumin binding domain (ABD), wherein n, q and y are each independently 0 or 1.

[0023] In some embodiments, pharmaceutical compositions comprising one or more compositions provided herein are provided.

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

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

[0026] In some embodiments, isolated polynucleotides encoding the FN3 domains described herein are provided.

[0027] In some embodiments, vectors comprising a polynucleotide described herein are provided.

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

[0029] In some embodiments, a method of producing a 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 CD71.

[0030] In some embodiments, a pharmaceutical composition comprising an FN3 domain that binds to CD71 linked to an oligonucleotide molecule and a pharmaceutically acceptable carrier is provided. In some embodiments, a kit comprising one or more FN3 domains with or without an oligonucleotide molecule is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a flow chart showing the characteristics of evaluating and considering siRNA screening.

[0032] Figure 2 is a diagram of an RNA sequencing experiment identifying changes in the transcriptome following transfection of cells with siRNA against HHH, where the arrow identifies a significant reduction in GYS1 transcripts.

[0033] Figure 3 Results of a target binding assay using over 6,000 receptors in a proteome array are presented, with data indicating that CD71 is the only binding target of the FN3 domain.

[0034] Figure 4A Shown is knockdown of GYS1 mRNA in mouse gastrocnemius muscle using 3 different FN3 domain-siRNA conjugates compared to vehicle alone. Figure 4BShown is knockdown of GYS1 protein in mouse gastrocnemius muscle using 3 different FN3 domain-siRNA conjugates compared to vehicle alone.

[0035] Figure 5 It was shown that GYS1 knockdown was highly specific to skeletal muscle using 3 different FN3 domain-siRNA conjugates compared to siRNA against a different target (AHA-1). DETAILED DESCRIPTION

[0036] 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.

[0037] As used herein, the term "type III fibronectin domain (FN3)" (FN3 domain) refers to a domain that occurs frequently in proteins including fibronectin, tenascin, 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-naturally synthesized 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, such as the 3rd FN3 domain of tenascin (TN3), or the 10th FN3 domain of fibronectin (FN10). As used throughout, "centyrin" refers to an FN3 domain. In addition, the FN3 domains described herein are not antibodies because they do not have a variable heavy chain (V H ) and / or light chain (V L ) structure.

[0038] As used herein, "autoimmune disease" refers to disease conditions and states in which an individual's immune response is directed against the individual's own components, resulting in an undesirable and often debilitating condition. "Autoimmune disease" as used herein is intended to further include autoimmune conditions, syndromes, and the like. Autoimmune diseases include, but are not limited to, Addison's disease, allergies, allergic rhinitis, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune atrophic gastritis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune parotitis, autoimmune uveitis, celiac disease, primary biliary cirrhosis, benign lymphocytic ileitis, COPD, colitis, coronary heart disease, Crohn's disease, diabetes (type I), depression, diabetes including type 1 diabetes and / or type 2 diabetes, epididymitis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease), hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD), immune response to recombinant drug products (e.g., 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, spondyloarthropathies, 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 a primary target and in some cases a secondary target. Such conditions include, but are not limited to, AIDS, atopic allergies, bronchial asthma, eczema, leprosy, schizophrenia, genetic depression, tissue and organ transplantation, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus's phenomenon, allergic reactions, and alcohol and drug addiction.

[0039] As used herein, "capture agent" refers to a substance that binds to a specific type of cell and is able to separate that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulation agents, molecules that bind to specific cell types, and the like.

[0040] As used herein, "sample" refers to a collection of similar fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues present in a subject. Exemplary samples are tissue biopsies; fine needle aspirates; surgically removed tissues; organ cultures; cell cultures and biological fluids, such as blood, serum and serosal fluids; plasma; lymph; urine; saliva; cystic fluid; tears; feces; sputum; mucosal secretions of secretory tissues and organs; vaginal secretions; fluids of the ascites, pleura, pericardium, peritoneum, abdomen and other body cavities; fluids collected by bronchial lavage; synovial fluid; liquid solutions in contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media and lavage fluids, etc.

[0041] "Substitution" or "substituted" or "mutation" or "mutant" refers to the alteration, deletion or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to produce a variant of the sequence.

[0042] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as substitutions, insertions, or deletions.

[0043] "Specifically binds" or "specific binding" refers to the FN3 domain being present at about 1×10 -6 M or less, for example, about 1×10 -7 M or smaller, about 1×10 -8 M or smaller, about 1×10 -9 M or smaller, about 1×10 -10 M or smaller, about 1×10 -11 M or smaller, about 1×10 -12 M or less or about 1×10 -13 M or less dissociation constant (K D ) binds to its target (such as CD71). Alternatively, "specific binding" refers to the ability of the FN3 domain to bind to its target (e.g., CD71) in a standard solution ELISA assay at least 5 times higher than a negative control. Figure 3The proteome array shown in demonstrates specific binding. In some embodiments, the negative control is an FN3 domain that does not bind to CD71. In some embodiments, the FN3 domain that specifically binds to CD71 may have cross-reactivity with other related antigens, for example, with the same predetermined antigen (homolog) from other species, such as Macaca Fascicularis (cynomolgousmonkey, cyno) or chimpanzee (Pantroglodytes) (chimpanzee).

[0044] A "library" refers to a collection of variants. A library can consist of polypeptide or polynucleotide variants.

[0045] "Stability" refers to the ability of a molecule to remain in a folded state under physiological conditions such that it retains at least one of its normal functional activities, e.g., binding to a predetermined antigen, such as CD71.

[0046] "CD71" refers to a human CD71 protein having an amino acid sequence of SEQ ID NO: 2 or 5. In some embodiments, SEQ ID NO: 2 is a full-length human CD71 protein. In some embodiments, SEQ ID NO: 5 is the extracellular domain of human CD71.

[0047] "Tencon" refers to a synthetic type III fibronectin (FN3) domain having the consensus sequence shown in SEQ ID NO: 1 (LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKV GEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEF TT) and described in U.S. Patent Publication No. 2010 / 0216708, which is hereby incorporated by reference in its entirety.

[0048] As used herein, "cancer cell" or "tumor cell" refers to cancerous cells, precancerous cells or transformed cells in vivo, in vitro and in tissue culture, which have spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can be caused by infection with transforming viruses and the incorporation of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is exemplified by, for example, morphological changes in vitro, in vivo and in vitro, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant tumors, tumor-specific marker levels, invasiveness, tumor growth or inhibition in suitable animal hosts (such as nude mice), etc. (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd Edition, 1994)).

[0049] "Dendritic cells" refer to a type of antigen presenting cell (APC) that plays an important role in the adaptive immune system. The main function of dendritic cells is to present antigens to T lymphocytes and secrete cytokines that can further directly or indirectly regulate immune responses. Dendritic cells have the ability to induce primary immune responses in inactive or resting naive T lymphocytes.

[0050] "Immune cells" refers to cells of the immune system classified as lymphocytes (T cells, B cells, and NK cells), neutrophils, or monocytes / macrophages. These are all types of white blood cells.

[0051] Myocytes are cells that make up various muscle tissues and are classified as skeletal muscle cells, smooth muscle cells, and cardiomyocytes. Skeletal muscle cells are multinuclear and striated. Smooth muscle cells are mononuclear rather than striated. Cardiomyocytes are mononuclear and striated and are only found in the heart. As used herein, "myocells" refer to skeletal muscle cells and smooth muscle cells, rather than cardiomyocytes. As used herein, "cardiomyocytes" refer only to cardiomyocytes.

[0052] "Vector" refers to a polynucleotide that can replicate within a biological system or can move between such systems. Vector polynucleotides typically contain elements, such as an origin of replication, a polyadenylation signal, or a selection marker, whose function is to promote the replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include cells, viruses, animals, plants, and reconstructed biological systems that utilize biological components that can replicate vectors. The polynucleotides comprising the vector may be DNA or RNA molecules or hybrids thereof.

[0053] "Expression vector" refers to a vector that can be used in a biological system or a recombinant biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0054] "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.

[0055] "Polypeptide" or "protein" means a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as "peptides."

[0056] "Effective" refers to the presence of a specific number of antigen-specific binding sites in a molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six antigen-specific binding sites in a molecule, respectively.

[0057] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably.

[0058] "Isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides, such as FN3 domains) that have been substantially separated and / or purified from other components of a system, such as those produced in recombinant cells, and proteins that have been subjected to at least one purification or isolation step. "Isolated FN3 domains" refers to FN3 domains that are substantially free of other cellular material and / or chemicals, and encompass FN3 domains isolated to a higher degree of purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0059] "Enzyme replacement therapy" or "ERT" refers to the use of a pharmaceutical composition comprising one or more enzymes administered to a subject. In some embodiments, the enzyme is administered to supplement or replace an enzyme that is missing or deficient in some aspect of the subject. Typically, the one or more enzymes involved in the treatment are administered to the subject by intravenous infusion of a pharmaceutical composition comprising one or more enzymes. As described herein, enzyme replacement therapy can be administered with a composition comprising an FN3 domain, which 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 comprising an FN3 domain linked to an additional therapeutic agent as provided herein.

[0060] In some embodiments, a composition comprising a polypeptide, such as a polypeptide comprising an FN3 domain, is provided, which FN3 domain can be linked to an oligonucleotide molecule. The oligonucleotide molecule can be, for example, an siRNA molecule, an antisense oligonucleotide, an mRNA, etc.

[0061] Therefore, in some embodiments, siRNA is a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand (passenger strand) and an antisense strand (guide strand). In some embodiments, the length of each strand of a dsRNA agent can be within the range of 12-40 nucleotides. For example, each strand can be 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length or 21-23 nucleotides in length.

[0062] In some embodiments, sense strand and antisense strand generally form double-stranded dsRNA.The duplex region of dsRNA agent can be 12-40 nucleotide pair length.For example, duplex region can be 14-40 nucleotide pair length, 17-30 nucleotide pair length, 25-35 nucleotide pair length, 27-35 nucleotide pair length, 17-23 nucleotide pair length, 17-21 nucleotide pair length, 17-19 nucleotide pair length, 19-25 nucleotide pair length, 19-23 nucleotide pair length, 19-21 nucleotide pair length, 21-25 nucleotide pair length or 21-23 nucleotide pair length.In another example, duplex region is selected from 15,16,17,18,19,20,21,22,23,24,25,26 and 27 nucleotide pair lengths.

[0063] In some embodiments, dsRNA comprises one or more overhang regions and / or blocking groups of dsRNA agent at 3' end or 5' end or both ends of chain. Overhang can be 1-10 nucleotide length, 1-6 nucleotide length, for example 2-6 nucleotide length, 1-5 nucleotide length, 2-5 nucleotide length, 1-4 nucleotide length, 2-4 nucleotide length, 1-3 nucleotide length, 2-3 nucleotide length or 1-2 nucleotide length. Overhang can be the result that one chain is longer than another chain, or the result that two chains of the same length are staggered. Overhang can form mismatch with target mRNA, or it can be complementary to the gene sequence targeted, or it can be another sequence. The first chain and the second chain can also be connected, for example, by forming other bases of hairpin or by other non-base joints.

[0064] In some embodiments, the nucleotides in the overhang region of the dsRNA agent can each independently be a modified or unmodified nucleotide, including but not limited to 2'-sugar modified, such as 2-F, 2'-methyl, 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl) and any combination thereof. For example, TT(UU) can be an overhang sequence at either end of either chain. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be another sequence.

[0065] 5' overhang or 3' overhang at the sense strand, antisense strand or two chains of dsRNA agent can be phosphorylated.In some embodiments, the overhang region contains two nucleotides with thiophosphate, dithiophosphate, phosphonate, phosphoramidate or methylsulfonyl phosphoramidate between two nucleotides, wherein two nucleotides can be identical or different.In one embodiment, the overhang is present in the 3' end of sense strand, antisense strand or two chains.In one embodiment, 3' overhang is present in the antisense strand.In one embodiment, 3' overhang is present in the sense strand.

[0066] The dsRNA agent may only include a single overhang, which can enhance the interference activity of the dsRNA without affecting its overall stability. For example, a single-stranded overhang is located at the 3' end of the sense strand, or at the 3' end of the antisense strand. The dsRNA may also have a flat end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Typically, the antisense strand of the dsRNA has a nucleotide overhang at the 3' end, and the 5' end is a flat end. Although not bound by theory, the asymmetric flat end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are conducive to loading the guide strand into RISC. For example, a single overhang includes at least two, three, four, five, six, seven, eight, nine or ten nucleotide lengths.

[0067] In some embodiments, the dsRNA agent may also have two blunt ends at both ends of the dsRNA duplex.

[0068] In some embodiments, each nucleotide in the sense and antisense strands of the dsRNA agent can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more changes in one or two non-linked phosphate oxygens and / or one or more linked phosphate oxygens; changes in the composition of ribose sugars, such as changes in the 2 hydroxyl groups on the ribose sugar; wholesale replacement of phosphate moieties with "dephosphorylation" linkers; modification or replacement of naturally occurring bases; and substitution or modification of the ribose-phosphate backbone.

[0069] In some embodiments, all or some bases in the 3' overhang or 5' overhang can be modified, for example, with modifications as described herein. Modifications can include, for example, using modifications known in the art to use modifications at the 2' position of ribose sugar, such as using deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications to replace the ribose sugar of the core base, and modifications in phosphate groups, such as phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate or mesyl phosphoramidate modifications. The overhang need not be homologous to the target sequence.

[0070] 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. The strands 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.

[0071] In some embodiments, at least two different modifications are generally present on the sense strand and the antisense strand. The two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides or other substances.

[0072] In one embodiment, the sense strand and the antisense strand each comprise two differently modified nucleotides selected from 2'-fluoro, 2'-O-methyl, or 2'-deoxy.

[0073] The dsRNA agent may also include at least one phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide bond. Phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate internucleotide bond modification may occur on any nucleotide of the sense strand or antisense strand, or may occur at any position of the strand. For example, internucleotide bond modification may occur on each nucleotide on the sense strand and / or antisense strand; each internucleotide bond modification may occur in an alternating pattern on the sense strand or antisense strand; or sense strand or antisense strand may include an internucleotide bond modification of an alternating pattern. The alternating pattern of internucleotide bond modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of internucleotide bond modification on the sense strand may be offset relative to the alternating pattern of internucleotide bond modification on the antisense strand.

[0074] In some embodiments, the dsRNA agent comprises a key modification between phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate nucleotides in the overhang region.For example, the overhang region comprises two nucleotides, and it has a key between phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate nucleotides between two nucleotides.It is also possible to carry out a key modification between nucleotides to connect the overhang nucleotides with the terminal pairing nucleotides in the duplex region.For example, at least 2,3,4 or all overhang nucleotides can be connected by a key between phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate nucleotides, and optionally, there can be a key between other phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, mesylphosphoramidate or methylphosphonate nucleotides, and it connects the overhang nucleotides to the paired nucleotides of the next-to-the-side overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, wherein two of the three nucleotides are overhang nucleotides and the third is a paired nucleotide immediately adjacent to the overhang nucleotide. In some embodiments, these terminal three nucleotides may be at the 3' end of the antisense strand.

[0075] In some embodiments, the dsRNA compositions are linked by modified bases or nucleoside analogs as described in U.S. Pat. No. 7,427,672, which is incorporated herein by reference. In some embodiments, the modified bases or nucleoside analogs are referred to as linkers or L in the formulas described herein.

[0076] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and its salts:

[0077]

[0078] wherein the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group optionally having a substituent, R1 and R2 are the same or different and each represents 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(R4)R5, wherein R4 and R5 are the same or different and each represents a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a thiol group, a thiol 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, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a functional molecular unit substituent, and m represents an integer from 0 to 2, and n represents an integer from 0 to 3. In some embodiments, m and n are zero.

[0079] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aromatic ring substituted with a lower alkyl group, a lower alkoxy group, a halogen group or a cyano group, or a silyl group.

[0080] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt 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.

[0081] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aromatic ring substituted with a lower alkyl group, a lower alkoxy group, a halogen group or a 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.

[0082] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein R2 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyldiphenylsilyl group, --P(OC2H4CN)(N(i-Pr)2), --P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphate group.

[0083] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein R3 is a hydrogen atom, a phenoxyacetyl group, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted with one to three 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).

[0084] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear transfer signal peptide.

[0085] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein the base is purine-9-yl, 2-oxopyrimidin-1-yl, or purine-9-yl or 2-oxopyrimidin-1-yl having a substituent selected from the following α group: α group: hydroxyl, hydroxyl protected with a protecting group for nucleic acid synthesis, alkoxy having 1 to 5 carbon atoms, thiol, thiol protected with a protecting group for nucleic acid synthesis, alkylthio having 1 to 5 carbon atoms, amino, amino protected with a protecting group for nucleic acid synthesis, amino substituted with an alkyl having 1 to 5 carbon atoms, alkyl having 1 to 5 carbon atoms, and halogen atoms.

[0086] In some embodiments, the modified base or nucleoside analog has a structure as shown in Chemical Formula I and a salt thereof, wherein the base is 6-aminopurine-9-yl (i.e., adenine), 6-aminopurine-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurine-9-yl, 2-amino-6-chloropurine-9-yl, 2-amino-6-chloropurine-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurine-9-yl, 2-amino-6-chloropurine-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (i.e., guanyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl , 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methyl oxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-thiol-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., thymidyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0087] In some embodiments, the modified base or nucleoside analog has a structure as shown in Formula I and a salt thereof, wherein m is 0 and n is 1.

[0088] In some embodiments, the modified base or nucleoside analog is a DNA oligonucleotide or RNA oligonucleotide analog containing one or more types of unit structures of nucleoside analogs having a structure as shown in Chemical Formula II, or a pharmacologically acceptable salt thereof, provided that the connection form between the corresponding nucleosides in the oligonucleotide analog is in addition to the phosphodiester bond [--OP(O2 - )O--] in addition to the same phosphodiester bond, it may contain one or two or more phosphorothioate bonds [--OP(O)(S - )O--], a phosphorodithioate bond [--O2PS2--], a phosphonate bond [--PO(OH)2--], a phosphoramidate bond [--O=P(OH)2--] or a methylsulfonylphosphoramidate bond [--OP(O)(N)(SO2)(CH3)O--], and if two or more of one or more types of these structures are included, the bases between these structures may be the same or different:

[0089]

[0090] wherein base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group optionally having a substituent, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group or a functional molecular unit substituent, and m represents an integer from 0 to 2, and n represents an integer from 0 to 3. In some embodiments, m and n are 0.

[0091] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by one to three aromatic groups, a methyl group substituted by one to three aromatic groups having an aromatic ring substituted by a lower alkyl group, a lower alkoxy group, a halogen group or a cyano group, or a silyl group.

[0092] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, 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.

[0093] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by one to three aromatic groups, a methyl group substituted by one to three aromatic groups having an aromatic ring substituted by a lower alkyl group, a lower alkoxy group, a halogen group or a 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.

[0094] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein R2 is a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a tert-butyldiphenylsilyl group, --P(OC2H4CN)(N(i-Pr)2), --P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenylphosphate group or a 4-chlorophenylphosphate group.

[0095] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein R3 is a hydrogen atom, a phenoxyacetyl group, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted with one to three 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).

[0096] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear transfer signal peptide.

[0097] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein the base is purine-9-yl, 2-oxopyrimidin-1-yl, or purine-9-yl or 2-oxopyrimidin-1-yl having a substituent selected from the following α group: α group: hydroxyl, hydroxyl protected with a protecting group for nucleic acid synthesis, alkoxy having 1 to 5 carbon atoms, thiol, thiol protected with a protecting group for nucleic acid synthesis, alkylthio having 1 to 5 carbon atoms, amino, amino protected with a protecting group for nucleic acid synthesis, amino substituted with an alkyl having 1 to 5 carbon atoms, alkyl having 1 to 5 carbon atoms, and halogen atoms.

[0098] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein the base is 6-aminopurine-9-yl (i.e., adenine), 6-aminopurine-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurine-9-yl, 2-amino-6-chloropurine-9-yl, 2-amino-6-chloropurine-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurine-9-yl, 2-amino-6-chloropurine-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guanyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9 -yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 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., uracilyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thymidyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0099] In some embodiments, the oligonucleotide analog or a pharmacologically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein m is 0 and n is 1.

[0100] In some embodiments, the compositions described herein also include polymers (polymer part C). In some cases, polymers are natural or synthetic polymers, which are composed of a cross-linked network of long chains of branched or unbranched monomers and / or two-dimensional or three-dimensional monomers. In some cases, polymers include polysaccharides, lignin, rubber or polyalkylene oxides (e.g., polyethylene glycol). In some cases, at least one polymer includes but is not limited to α-dihydroxy polyethylene glycol, ω-dihydroxy polyethylene glycol, biodegradable lactone-based polymers, such as polyacrylic acid, polylactic acid (PLA), poly (glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polybutylene glycol (PTG) or polyurethane, and mixtures thereof. As used herein, mixtures refer to the use of different polymers in the same compound and relate to block copolymers. In some cases, block copolymers are polymers in which at least one portion of a polymer is composed of a monomer of another polymer. In some cases, the polymer comprises polyalkylene oxide. In some cases, the polymer comprises PEG. In some cases, the polymer comprises polyethyleneimine (PEI) or hydroxyethyl starch (HES).

[0101] In some cases, C is a PEG moiety. In some cases, the PEG moiety is conjugated at the 5' end of the oligonucleotide molecule, and the binding 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, and the binding moiety is conjugated at the 5' end of the oligonucleotide molecule. In some cases, the PEG moiety is conjugated to the internal site of the oligonucleotide molecule. In some cases, the PEG moiety, the binding moiety or a combination thereof is conjugated to the internal site of the oligonucleotide molecule. In some cases, conjugation is direct conjugation. In some cases, conjugation is through natural connection.

[0102] In some embodiments, polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, polydisperse materials include the dispersed distribution of different molecular weights of materials, characterized in that average weight (weight average) size and dispersity. In some cases, monodisperse PEG includes molecules of a size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weight represents the mean value of the molecular weight of polyalkylene oxide (e.g., PEG) molecules.

[0103] 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, 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.

[0104] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000 In some cases, the molecular weight of C is about 900, 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 cases, the molecular weight of C is about 200 Da. In some cases, the molecular weight of C is about 300 Da. In some cases, the molecular weight of C is about 400 Da. In some cases, the molecular weight of C is about 500 Da. In some cases, the molecular weight of C is 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 800Da. In some cases, the molecular weight of C is about 900Da. In some cases, the molecular weight of C is about 1000Da. In some cases, the molecular weight of C is about 1100Da. In some cases, the molecular weight of C is about 1200Da. In some cases, the molecular weight of C is about 1300Da. In some cases, the molecular weight of C is about 1400Da. In some cases, the molecular weight of C is about 1450Da. In some cases, the molecular weight of C is about 1500Da. In some cases, the molecular weight of C is about 1600Da. In some cases, the molecular weight of C is about 1700Da. In some cases, the molecular weight of C is about 1800Da.In some cases, the molecular weight of C is about 1900Da. In some cases, the molecular weight of C is about 2000Da. In some cases, the molecular weight of C is about 2100Da. In some cases, the molecular weight of C is about 2200Da. In some cases, the molecular weight of C is about 2300Da. In some cases, the molecular weight of C is about 2400Da. In some cases, the molecular weight of C is about 2500Da. In some cases, the molecular weight of C is about 2600Da. In some cases, the molecular weight of C is about 2700Da. In some cases, the molecular weight of C is about 2800Da. In some cases, the molecular weight of C is about 2900Da. In some cases, the molecular weight of C is about 3000Da. In some cases, the molecular weight of C is about 3250Da. In some cases, the molecular weight of C is about 3350Da. In some cases, the molecular weight of C is about 3500Da. In some cases, the molecular weight of C is about 3750Da. In some cases, the molecular weight of C is about 4000Da. In some cases, the molecular weight of C is about 4250Da. In some cases, the molecular weight of C is about 4500Da. In some cases, the molecular weight of C is about 4600Da. In some cases, the molecular weight of C is about 4750Da. In some cases, the molecular weight of C is about 5000Da. In some cases, the molecular weight of C is about 5500Da. In some cases, the molecular weight of C is about 6000Da. In some cases, the molecular weight of C is about 6500Da. In some cases, the molecular weight of C is about 7000Da. In some cases, the molecular weight of C is about 7500Da. In some cases, the molecular weight of C is about 8000Da. In some cases, the molecular weight of C is about 10,000Da. In some cases, the molecular weight of C is about 12,000Da. In some cases, the molecular weight of C is about 20,000Da. 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.

[0105] In some embodiments, polyalkylene oxide (e.g., PEG) is a discrete PEG, wherein discrete PEG is a polymerized PEG comprising more than one repeating ethylene oxide unit. In some cases, discrete PEG (dPEG) comprises 2 to 60, 2 to 50 or 2 to 48 repeating ethylene oxide units. In some cases, dPEG comprises 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, dPEG comprises about 2 or more repeating ethylene oxide units. In some cases, dPEG comprises about 3 or more repeating ethylene oxide units. In some cases, dPEG comprises about 4 or more repeating ethylene oxide units. In some cases, dPEG comprises about 5 or more repeating ethylene oxide units. In some cases, dPEG comprises about 6 or more repeating ethylene oxide units. In some cases, dPEG comprises about 7 or more repeating ethylene oxide units. In some cases, dPEG comprises about 8 or more repeating ethylene oxide units. In some cases, dPEG comprises about 9 or more repeating ethylene oxide units. In some cases, dPEG comprises about 10 or more repeating ethylene oxide units. In some cases, dPEG comprises about 11 or more repeating ethylene oxide units. In some cases, dPEG comprises about 12 or more repeating ethylene oxide units. In some cases, dPEG comprises about 13 or more repeating ethylene oxide units. In some cases, dPEG comprises about 14 or more repeating ethylene oxide units. In some cases, dPEG comprises about 15 or more repeating ethylene oxide units. In some cases, dPEG comprises about 16 or more repeating ethylene oxide units. In some cases, dPEG comprises about 17 or more repeating ethylene oxide units. In some cases, dPEG comprises about 18 or more repeating ethylene oxide units. In some cases, dPEG comprises about 19 or more repeating ethylene oxide units. In some cases, dPEG comprises about 20 or more repeating ethylene oxide units. In some cases, dPEG comprises about 22 or more repeating ethylene oxide units. In some cases, dPEG comprises about 24 or more repeating ethylene oxide units. In some cases, dPEG comprises about 26 or more repeating ethylene oxide units. In some cases, dPEG comprises about 28 or more repeating ethylene oxide units. In some cases, dPEG comprises about 30 or more repeating ethylene oxide units. In some cases, dPEG comprises about 35 or more repeating ethylene oxide units. In some cases, dPEG comprises about 40 or more repeating ethylene oxide units.In some cases, dPEG comprises about 42 or more repeating ethylene oxide units. In some cases, dPEG comprises about 48 or more repeating ethylene oxide units. In some cases, dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized in a stepwise manner by pure (e.g., about 95%, 98%, 99% or 99.5%) starting materials in the form of a single molecular weight compound. In some cases, dPEG has a specific molecular weight, rather than an average molecular weight. In some cases, dPEG described herein is from QuantaBiodesign, the dPEG of LMD.

[0106] In some embodiments, C is an albumin binding domain. In certain aspects, the albumin binding domain specifically binds to serum albumin, such as human serum albumin (HSA), to extend the half-life of the domain or another therapeutic agent associated or connected to the albumin binding domain. In some embodiments, the human serum albumin binding domain comprises an initiator methionine (Met) connected to the N-terminus of the molecule. In some embodiments, the human serum albumin binding domain comprises a cysteine ​​(Cys) connected to the C-terminus or N-terminus of the domain. Adding an N-terminal Met and / or a C-terminal Cys can promote expression of another molecule and / or conjugation with another molecule, which can be another half-life extension molecule, such as PEG, Fc region, etc.

[0107] 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 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 or 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 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 comprises an amino acid sequence that is at least or 85%, 86%, 87%, 88%, 89%, 90%, 901%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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, provided that the protein has an amino acid sequence corresponding to SEQ ID NO: NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 at position 10. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid. In some embodiments, the isolated albumin binding domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions compared to the amino acid sequence of SEQ ID NO: 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 in or at the C-terminus of 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. Although the positions are listed in 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, other examples of albumin binding domains can be found in US Pat. No. 10,925,932, which is hereby incorporated by reference.

[0108] Table 1

[0109]

[0110]

[0111]

[0112] In some embodiments, the dsRNA agent comprises a mismatch with the target, within the duplex, or a combination thereof. Mismatches may occur in the overhang region or in the duplex region. Base pairs can be graded based on their tendency to promote dissociation or melt (e.g., based on the free energy of association or dissociation of a particular pairing, the simplest method is to examine the pairing on the basis of a single pairing, although the next adjacent or similar analysis may also be used). In promoting dissociation, A:U is superior to G:C; G:U is superior to G:C; and I:C is superior to G:C (I = inosine). Mismatches (e.g., non-canonical pairings or except non-canonical pairings (as described elsewhere herein)) are superior to canonical (A:T, A:U, G:C) pairings; and pairings including universal bases are superior to canonical pairings.

[0113] In some embodiments, dsRNA agents may include a phosphorus-containing group at the 5' end of the sense strand or antisense strand. The 5' end phosphorus-containing group may be a 5' end phosphate (5'-P), a 5' end phosphorothioate (5'-PS), a 5' end phosphorodithioate (5'-PS2), a 5' end vinyl phosphonate (5'-VP), a 5' end methyl phosphonate (MePhos), a 5' end methylsulfonyl phosphoramidate (5'MsPA) or a 5'-deoxy-5'-C-malonyl group. When the 5' end phosphorus-containing group is a 5' end vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer, such as trans-vinyl phosphate or cis-vinyl phosphate, or a mixture thereof. The representative structures of these modifications may be found in, for example, U.S. Patent No. 10,233,448, which is hereby incorporated by reference in its entirety.

[0114] In some embodiments, the nucleotide analog or synthetic nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl of the ribose moiety. In some cases, the modification comprises H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, straight-chain and branched C1-C 10 Chain length. In some cases, the alkyl moiety also includes modification. In some cases, modification includes azo, keto, aldehyde, carboxyl, nitro, nitroso, nitrile, heterocycle (for example, imidazole, hydrazine or hydroxylamino), isocyanate or cyanate group or sulfur-containing group (for example, sulfoxide, sulfone, sulfide and disulfide). In some cases, the alkyl moiety also includes other heteroatoms, such as O, S, N, Se, and each of these heteroatoms can be further replaced by alkyl as described above. In some cases, the carbon of the heterocyclic group is replaced by nitrogen, oxygen or sulfur. In some cases, heterocyclic substitution includes but is not limited to morpholino, imidazole and pyrrolidine.

[0115] In some cases, the modification at the 2' hydroxyl group is a 2'-O, -methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. Exemplary chemical structures of 2'-O-methyl modifications of adenosine molecules and 2'O-methoxyethyl modifications of uridine are shown below.

[0116]

[0117] In some cases, the modification at the 2' hydroxyl is a 2'-O-aminopropyl modification, wherein an extended amine group comprising a propyl linker binds the amine group to the 2' oxygen. In some cases, this modification neutralizes the total negative charge derived from the phosphate of the oligonucleotide molecule by introducing a positive charge from the amine group of each sugar, and thereby improves cellular uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is shown below.

[0118]

[0119] In some cases, the modification at the 2' hydroxyl is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which an oxygen molecule bound at the 2' carbon is linked to the 4' carbon via a methylene group, thereby forming a 2'-C, 4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of LNA is shown below. The representation shown on the left highlights the chemical linkage of the LNA monomer. The representation shown on the right highlights the locked 3'-endo (3E) conformation of the furanose ring of the LNA monomer.

[0120]

[0121] In some cases, the modification at the 2' hydroxyl comprises an ethylene nucleic acid (ENA), such as a 2'-4'-ethylene bridged nucleic acid, which locks the sugar conformation into a C3'-endo sugar fold conformation. ENAs are part of a class of bridged nucleic acids that also comprise LNAs, which are modified nucleic acids. Exemplary chemical structures of ENAs and bridged nucleic acids are shown below.

[0122]

[0123] In some embodiments, additional modifications at the 2' hydroxyl 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA).

[0124] In some embodiments, the nucleotide analogs comprise modified bases such as, but not limited to, 5-propynyl uridine, 5-propynyl cytidine, 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)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetyl cytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyl uridine, 5-methyloxyuridine, deazanucleotides (such as 7-deazaadenosine), 6-azo uridine, 6-azo Cytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine and 4-thiouridine and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl, any O- and N-alkylated purine and pyrimidine (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one), phenyl and modified phenyl (such as aminophenol or 2,4,6-trimethoxybenzene), modified cytosine as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those modified with respect to the sugar moiety, and nucleotides with sugars or their analogs that are not ribosyl. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose and other sugars, heterocycles or carbocycles. The term nucleotide also includes universal bases known in the art. As an example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole or nebularine.

[0125] In some embodiments, nucleotide analogs also include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, 1', 5'-anhydrohexitol nucleic acid (HNA) or a combination thereof.Morpholino or phosphorodiamidate morpholino oligomers (PMO) include synthetic molecules whose structures simulate natural nucleic acid structures by deviating from normal sugar and phosphate structures.In some cases, the five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, a nitrogen and an oxygen.In some cases, ribose monomers are connected by diaminophosphoryl groups rather than phosphate groups.In this case, the main chain changes and removes all positive and negative charges, so that the morpholino neutral molecules can pass through the cell membrane without the help of a cell delivery agent, such as those used by charged oligonucleotides.

[0126]

[0127] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar rings or phosphate bonds, and the bases are attached via oligosaccharide-like molecules and appropriately spaced, thus eliminating backbone charge.

[0128]

[0129] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some cases, the modified internucleotide bond includes but is not limited to thiophosphate, methylsulfonyl phosphoramidate, dithiophosphate, methylphosphonate, 5'-alkylenephosphonate, 5'-methylphosphonate, 3'-alkylenephosphonate, boron trifluoride, borophosphate and 3'-5' bond or 2'-5' bond selenophosphate, phosphotriester, thioalkylphosphotriester, hydrogen phosphonate bond, alkylphosphonate, alkyl phosphate, aryl phosphate, selenophosphate, diselenogenate, phosphinate, phosphoramidate, 3'-alkylphosphoramidate, aminoalkylphosphoramidate, thiophosphoramidate, piperazine phosphate (phosphoropiperazidate), anilinethio Phosphate (phosphoroanilothioate), aniline phosphate (phosphoroanilidate), ketone, sulfone, sulfonamide, carbonate, carbamate, methylene hydrazine, methylene dimethyl hydrazine, methylal, thiomethyl acetal, oxime, methylene imino, methylene imino, thioamide, with ribose acetyl key, aminoethylglycine, silyl or siloxane key, with or without hetero atom alkyl or cycloalkyl key, for example saturated or unsaturated and / or substituted and / or containing hetero atom 1 to 10 carbon, with morpholino structure, amide, polyamide, wherein base is directly or indirectly attached to the nitrogen hetero atom of backbone, and their combination.Phosphorothioate antisense oligonucleotide (PS ASO) is the antisense oligonucleotide comprising thiophosphate key.Methylsulfonyl phosphoramidate antisense oligonucleotide (MsPA ASO) is the antisense oligonucleotide comprising methylsulfonyl phosphoramidate key.

[0130] In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate, a methylsulfonylphosphoramidate, or a thiolphosphonate modification. In some cases, the modified nucleotides include, but are not limited to, 2'-fluoroN3-P5'-phosphoramidite.

[0131] In some cases, modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)).

[0132] In some embodiments, one or more modifications further optionally include modifications of ribose moieties, phosphate backbones and nucleosides, or modifications of nucleotide analogs at the 3' end or 5' end. For example, the 3' end optionally includes a 3' cationic group, or by flipping nucleosides with a 3'-3' key at the 3' end. In another alternative, the 3' end is optionally conjugated to an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In another alternative, the 3' end is optionally conjugated to an abasic site, such as a depurinic or pyrimidine site. In some cases, the 5' end is conjugated to an aminoalkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5' end is conjugated to an abasic site, such as a depurinic or pyrimidine site.

[0133] In some embodiments, the oligonucleotide molecule comprises one or more synthetic nucleotide analogs described herein. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more synthetic nucleotide analogs described herein. In some embodiments, the synthetic nucleotide 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-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or a combination 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 synthetic nucleotide analogs selected from 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-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite or a combination thereof. In some cases, the oligonucleotide molecule 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 thiolphosphonate nucleotides.

[0134] 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.

[0135] 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.

[0136] In some cases, the oligonucleotide molecule comprises at least one of: about 10% to about 80% modified, about 20% to about 80% modified, about 30% to about 80% modified, about 40% to about 80% modified, about 50% to about 80% modified, about 60% to about 80% modified, and about 70% to about 80% modified.

[0137] In some cases, the oligonucleotide molecule comprises at least one of: about 10% to about 70% modified, about 20% to about 70% modified, about 30% to about 70% modified, about 40% to about 70% modified, about 50% to about 70% modified, and about 60% to about 70% modified.

[0138] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 60% modified, about 20% to about 60% modified, about 30% to about 60% modified, about 40% to about 60% modified, and about 50% to about 60% modified.

[0139] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 50% modified, about 20% to about 50% modified, about 30% to about 50% modified, and about 40% to about 50% modified.

[0140] In some cases, the oligonucleotide molecule comprises at least one of: about 10% to about 40% modified, about 20% to about 40% modified, and about 30% to about 40% modified.

[0141] In some cases, the oligonucleotide molecule comprises at least one of: about 10% to about 30% modified and about 20% to about 30% modified.

[0142] In some cases, the oligonucleotide molecule comprises about 10% to about 20% modifications.

[0143] In some cases, the oligonucleotide molecule comprises about 15% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% modification.

[0144] In other cases, the oligonucleotide molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% modification.

[0145] 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.

[0146] 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.

[0147] In some cases, about 5 to about 100% of oligonucleotide molecules include synthetic nucleotide analogs as 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 oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 5% of oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 10% of oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 15% of oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 20% of oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 25% of oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 30% of oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 35% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 40% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 45% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 50% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 55% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 60% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 65% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 70% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 75% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 80% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 85% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 90% of oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 95% of the oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 96% of the oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 97% of the oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 98% of the oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 99% of the oligonucleotide molecules comprise synthetic nucleotide analogs as described herein. In some cases, about 100% of the oligonucleotide molecules comprise synthetic nucleotide analogs as described herein.In some embodiments, the 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-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof.

[0148] In some embodiments, the oligonucleotide molecule comprises about 1 to about 25 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 1 modification, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 2 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 3 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 4 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 5 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 6 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 7 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 8 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 9 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 10 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 11 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 12 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 13 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 14 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 15 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 16 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 17 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 18 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 19 modifications, wherein the modifications comprise synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 20 modifications, wherein the modifications comprise synthetic nucleotide analogs as described herein. In some embodiments, the oligonucleotide molecule comprises about 21 modifications, wherein the modifications comprise synthetic nucleotide analogs as described herein.In some embodiments, the oligonucleotide molecule comprises about 22 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 23 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 24 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 25 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 26 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 27 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 28 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 29 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 30 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 31 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 32 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 33 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 34 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 35 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 36 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 37 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 38 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 39 modifications, wherein the modification comprises a synthetic nucleotide analog as described herein. In some embodiments, the oligonucleotide molecule comprises about 40 modifications, wherein the modifications comprise synthetic nucleotide analogs described herein.

[0149] In some embodiments, the oligonucleotide molecule is assembled from two separate polynucleotides, wherein one polynucleotide comprises the sense strand of the oligonucleotide molecule, 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 is a polynucleotide linker or a non-nucleotide linker in some cases.

[0150] 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-methyl pyrimidine nucleotides, and the purine nucleotides in the sense strand comprise 2'-deoxy purine 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'-fluoro pyrimidine nucleotides, and wherein the purine nucleotides present in the sense strand comprise 2'-deoxy purine nucleotides.

[0151] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides when present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and the purine nucleotides when present in the antisense strand are 2'-O-methyl purine nucleotides.

[0152] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides when present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and wherein the purine nucleotides when present in the antisense strand comprise 2'-deoxy-purine nucleotides.

[0153] 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., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, etc.) of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, at least two, three, four, five, six or seven of the plurality of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides. In some embodiments, consecutive 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, consecutive 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 includes 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 includes 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 '5' end or at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at the 5' end of the polynucleotide '3' end. Also optionally, such at least two, at least three, at least four 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides.

[0154] 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.

[0155] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and one of the sense strand and the antisense strand has at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides at the 5' end, while the other strand has at least two consecutive 2'-O-methyl modified nucleotides at the 5' end. In some embodiments, when the strand has at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides at the 5' end, the strand also includes at least two, at least three consecutive 2'-O-methyl modified nucleotides at the 3' end of the at least two consecutive 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, one of the sense strand and the antisense strand has at least two, at least three, at least four, at least five, at least six, or at least seven consecutive 2'-O-methyl modified nucleotides, which are connected to 2'-deoxy-2'-fluoro modified nucleotides at its 5' end and / or 3' end. In some embodiments, one of the sense strand and the antisense strand has at least four, at least five nucleotides having alternating 2'-O-methyl modified nucleotides and 2'-deoxy-2'-fluoro modified nucleotides.

[0156] In some embodiments, the oligonucleotide molecule (eg, siRNA) has the formula as shown in Formula I:

[0157] N1N2N3N4N5N6N7N8N9N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 Sense strand (SS)

[0158] N 21 N 20 N 19 N 18 N 17 N 16 N 15 N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1 antisense strand (AS),

[0159] Wherein each nucleotide represented by N is independently A, U, C or G or a modified nucleotide base, such as those provided herein. The N1 nucleotide of the sense strand and the antisense strand represents the 5' end of the corresponding strand. For the sake of clarity, although Formula I utilizes N1, N2, N3, etc. in both the sense strand and the antisense strand, the nucleotide bases do not need to be the same, nor are they intended to be the same. The siRNA shown in Formula I will be complementary to the target sequence.

[0160] For example, in some embodiments, the sense strand comprises 2' O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone at N1 and N2; 12 and N 17 2'-Fluoro modified nucleotides at N4, N5, N6, N 10 、N 11 、N 13 、N 14 、N 15 、N 16 、N 18 and N 19 2'O-methyl modified nucleotides at

[0161] In some embodiments, the antisense strand comprises a vinylphosphonate moiety attached to N1; a 2' fluoro-modified nucleotide with a phosphorothioate (PS) modified backbone at N2; 10 、N 11 、N 12 、N 13 、N 15 、N 16 、N 17 、N 18 and N 19 2'O-methyl modified nucleotides at N 14 2' fluorine-modified nucleotides at N 20 and N 21 2' O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone.

[0162] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a terminal capping moiety at the 5' end, the 3' end, or both the 5' end and the 3' end of the sense strand. In other embodiments, the terminal capping moiety is an inverted deoxy abasic moiety.

[0163] 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.

[0164] In some embodiments, the 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 methylsulfonylphosphoramidate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally at the 3' end, the 5' end, or both the 3' end and the 5' end of the sense strand. and wherein 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 methylsulfonylphosphoramidate 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, the 5' end, or both the 3' end and the 5' end 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 strand and / or the antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or methylsulfonylphosphoramidate internucleotide linkages are present or absent in the same or different strands and / or a terminal cap molecule at the 3' end, the 5' end, or both the 3' end and the 5' end.

[0165] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises 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 methylsulfonylphosphoramidate 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' end, the 5' end, or both the 3' end and the 5' end of the sense strand. and wherein the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or methylsulfonylphosphoramidate 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, the 5' end, or both the 3' end and the 5' end 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 strand and / or the antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro nucleotides, 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 methylsulfonylphosphoramidate internucleotide linkages are present or absent in the same or different strands and / or a terminal cap molecule at the 3' end, the 5' end, or both the 3' end and the 5' end.

[0166] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense 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 methylsulfonylphosphoramidate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5 In some embodiments, the present invention provides a plurality of (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 at the 3' end, 5' end, or both the 3' end and 5' end of the sense strand and / or the antisense strand, and optionally a terminal cap molecule at the 3' end, 5' end, or both the 3' end and 5' end 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 methylsulfonylphosphoramidate 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, the 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more pyrimidine nucleotides of the sense strand and / or the antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or methylsulfonylphosphoramidate internucleotide linkages are present or absent in the same or different strands and / or a terminal cap molecule at the 3' end, the 5' end, or both the 3' end and the 5' end.

[0167] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or methylsulfonylphosphoramidate 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' end, 5' end, or 3' and 5' ends 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, phosphonate, phosphoramidate, or methylsulfonylphosphoramidate 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, the 5' end, or both the 3' end and the 5' end 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 strand and / or the antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro nucleotides, about 1 to about 5, e.g., about 1, 2, 3, 4, 5 or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate or methylsulfonylphosphoramidate internucleotide linkages are present or absent in the same or different strands and / or a terminal cap molecule at the 3' end, the 5' end, or both the 3' end and the 5' end.

[0168] In some embodiments, the oligonucleotide molecules described herein are chemically modified short interfering nucleic acid molecules, which have about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioates, phosphorodithioates, phosphonates, phosphoramidates or mesylphosphoramidate internucleotide bonds in each chain of the oligonucleotide molecules. In some embodiments, the oligonucleotide molecules include sense strands and antisense strands, and the antisense strand includes a phosphate backbone modification at the 3' end of the antisense strand. Alternatively or additionally, the oligonucleotide molecules include sense strands and antisense strands, and the sense strand includes a phosphate backbone modification at the 5' end of the antisense strand. In some cases, the phosphate backbone modification is a thiophosphate. In some cases, the phosphate backbone modification is a phosphorodithioate. In some cases, the phosphate backbone modification is a phosphonate. In some cases, the phosphate backbone modification is phosphoramidate. In some cases, the phosphate backbone modification is mesylphosphoramidate. In some embodiments, the sense strand or antisense strand has three continuous nucleosides coupled by two thiophosphate backbones. In some embodiments, the sense strand or antisense strand has three continuous nucleosides coupled by two dithiophosphate backbones. In some embodiments, the sense strand or antisense strand has three continuous nucleosides coupled by two phosphonate backbones. In some embodiments, the sense strand or antisense strand has three continuous nucleosides coupled by two phosphoramidate backbones. In some embodiments, the sense strand or antisense strand has three continuous nucleosides coupled by two mesylphosphoramidate backbones.

[0169] In another embodiment, oligonucleotide molecules as herein described include 2'-5' internucleotide bonds. In some cases, 2'-5' internucleotide bonds are located at 3' ends, 5' ends, or 3' ends and 5' ends of one or two sequence chains. In addition, 2'-5' internucleotide bonds are present in various other positions in one or two sequence chains, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (including each internucleotide bond of pyrimidine nucleotides) in one or two chains of oligonucleotide molecules include 2'-5' internucleotide bonds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (including each internucleotide bond of purine nucleotides) in one or two chains of oligonucleotide molecules include 2'-5' internucleotide bonds.

[0170] In some embodiments, the oligonucleotide molecule is a single-stranded molecule that mediates RNAi activity in a cell or in vitro reconstitution system, wherein the oligonucleotide molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the oligonucleotide molecule are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternatively a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the oligonucleotide molecule are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides). is a 2'-deoxy purine nucleotide or alternatively a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and a terminal cap modification, which is optionally present at the 3' end, the 5' end, or both the 3' end and the 5' end of the antisense sequence, the oligonucleotide molecule optionally further comprises from about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxy nucleotides at the 3' end of the oligonucleotide molecule, wherein the terminal nucleotide further comprises one or more (e.g., 1, 2, 3, or 4) phosphorothioate or methylsulfonyl phosphoramidate internucleotide linkages, and wherein the oligonucleotide molecule optionally further comprises a terminal phosphate group, such as a 5' terminal phosphate group.

[0171] In some cases, one or more synthetic nucleotide analogs described herein are resistant to nucleases such as, for example, ribonucleases (such as RNase H), deoxyribonucleases (such as DNase), or exonucleases (such as 5'-3' exonucleases and 3'-5' exonucleases) when compared to natural polynucleotide molecules and endonucleases. In some cases, one or more synthetic nucleotide analogs comprising 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON -Methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite or a combination thereof are resistant to nucleases such as, for example, ribonucleases (such as RNase H), deoxyribonucleases (such as DNA enzymes) 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, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, 2'-O-aminopropyl modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-deoxy modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistant).In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-ON-methylacetamido (2'-O-NMA) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, LNA modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, ENA modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, HNA modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, morpholino is nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, PNA modified oligonucleotide molecules are resistant to nucleases (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, methylphosphonate nucleotide modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, thiolphosphonate nucleotide modified oligonucleotide molecules are nuclease resistant (e.g., RNase H, DNA enzyme, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some cases, the oligonucleotide molecule comprising 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.

[0172] In some embodiments, one or more synthetic nucleotide analogs described herein have increased binding affinity for their mRNA targets relative to equivalent naturally occurring polynucleotide molecules. One or more of the synthetic nucleotide analogs comprising a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide or 2'-fluoro N3-P5'-phosphoramidite have increased binding affinity for their mRNA targets relative to the equivalent natural polynucleotide molecules. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by 2'-O-methyl have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by 2'-O-methoxyethyl (2'-O-MOE) have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by 2'-O-aminopropyl have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by 2'-O-aminopropyl have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by 2'-deoxy have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by 2'-deoxy-2'-fluorine have the binding affinity of increase to their mRNA targets. In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. In some cases, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules.In some cases, relative to equivalent natural polynucleotide molecules, 2'-ON-methylacetylamino (2'-O-NMA) modified oligonucleotide molecules have increased binding affinity to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, LNA modified oligonucleotide molecules have increased binding affinity to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, ENA modified oligonucleotide molecules have increased binding affinity to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, PNA modified oligonucleotide molecules have increased binding affinity to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, HNA modified oligonucleotide molecules have increased binding affinity to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, morpholino modified oligonucleotide molecules have increased binding affinity to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by methylphosphonate nucleotides have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by thiolphosphonate nucleotides have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules modified by thiolphosphonate nucleotides have the binding affinity of increase to their mRNA targets. In some cases, relative to equivalent natural polynucleotide molecules, the oligonucleotide molecules comprising 2'-fluoro N3-P5'-phosphoramidite have the binding affinity of increase to its mRNA targets. In some cases, the affinity of increase is illustrated by lower Kd, higher melting temperature (Tm) or their combination.

[0173] In some embodiments, oligonucleotide molecules as described herein are chirally pure (or stereo pure) polynucleic acid molecules, or polynucleic acid molecules comprising single enantiomers. In some cases, oligonucleotide molecules comprise L-nucleotides. In some cases, oligonucleotide molecules comprise D-nucleotides. In some cases, oligonucleotide molecule compositions comprise and are less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or its mirror image enantiomer less. In some cases, oligonucleotide molecule compositions comprise and are less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or its racemic mixture less.

[0174] In some embodiments, the oligonucleotide molecules described herein are further modified to include an aptamer conjugate portion. In some cases, the aptamer conjugate portion is a DNA aptamer conjugate portion. In some cases, the aptamer conjugate portion is an Alphamer, which includes an aptamer portion that recognizes a specific cell surface target and a portion that presents a specific epitope for attachment of a circulating antibody.

[0175] In other embodiments, oligonucleotide molecules as herein described are modified to increase its stability.In some embodiments, oligonucleotide molecules are RNA (for example, siRNA).In some cases, oligonucleotide molecules are modified by one or more of the above-mentioned modifications to increase its stability.In some cases, oligonucleotide molecules are at 2' hydroxyl positions, such as by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxidation, 2'-deoxidation-2'-fluorine, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modification or by locking or bridging ribose conformation (for example, LNA or ENA) modification. In some cases, oligonucleotide molecules are modified by 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, oligonucleotide molecules also include morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides and / or 2'-fluoro N3-P5'-phosphoramidites to increase their stability. In some cases, oligonucleotide molecules are chirally pure (or stereo-pure) oligonucleotide molecules. In some cases, chirally pure (or stereo-pure) oligonucleotide molecules are modified to increase their stability. It is obvious to those skilled in the art that RNA is suitably modified to increase the stability of delivery.

[0176] In some embodiments, the oligonucleotide molecules include 2' modifications. In some embodiments, the nucleotides of the oligonucleotide molecules at positions 3, 7, 8, 9, 12 and 17 from the 5' end of the sense strand are not modified with 2'O-methyl modifications. In some embodiments, the nucleotides of the oligonucleotide molecules at positions 3, 7, 8, 9, 12 and 17 from the 5' end of the sense strand are modified with 2' fluorine modifications. In some embodiments, the nucleotides of the oligonucleotide molecules at positions 2 and 14 from the 5' end of the antisense strand are not modified with 2'O-methyl modifications. In some embodiments, the nucleotides of the oligonucleotide molecules at positions 2 and 14 from the 5' end of the antisense strand are modified with 2' fluorine modifications. In some embodiments, any nucleotide may further include 5'-phosphorothioate group modifications. In some embodiments, the nucleotides of the oligonucleotide molecules at positions 1 and 2 from the 5' end of the sense strand are modified with 5'-phosphorothioate group modifications. 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 strand or the antisense strand of the oligonucleotide molecule may further comprise a vinylphosphonate modification. In some embodiments, the nucleotides of the oligonucleotide molecule at position 1 from the 5' end of the antisense strand are modified with a vinylphosphonate modification.

[0177] In some cases, the oligonucleotide molecule is a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. In some cases, the oligonucleotide molecule is assembled from two separate polynucleotides, wherein one strand is a sense strand and the other strand is an antisense strand, wherein the antisense strand and the sense strand are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to a nucleotide sequence in the other strand; e.g., wherein the antisense strand and the sense strand form a duplex or double-stranded structure, e.g., wherein the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. Alternatively, the oligonucleotide molecule is assembled from a single oligonucleotide, wherein the self-complementary sense and antisense regions of the oligonucleotide molecule are connected by a nucleic acid-based or non-nucleic acid-based linker.

[0178] In some cases, the oligonucleotide molecule is a polynucleotide having a duplex, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure, having a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. In other cases, the oligonucleotide molecule is a circular single-stranded polynucleotide having two or more loop structures and a stem comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed in vivo or in vitro to produce an active oligonucleotide molecule capable of mediating RNAi. In other cases, the oligonucleotide molecule further comprises a single-stranded polynucleotide having a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., when such an oligonucleotide molecule does not require the presence of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof within the oligonucleotide molecule), wherein the single-stranded polynucleotide further comprises a terminal phosphate group, such as a 5'-phosphate or a 5',3'-diphosphate.

[0179] In some cases, the asymmetric hairpin is a linear oligonucleotide molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region comprising fewer nucleotides than the antisense region, to such an extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with a loop. For example, the asymmetric hairpin oligonucleotide molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) long enough to mediate RNAi in a cell or in vitro system and a loop region comprising about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, the asymmetric hairpin oligonucleotide molecule also comprises a chemically modified 5'-terminal phosphate group. In other cases, the loop portion of the asymmetric hairpin oligonucleotide molecule comprises nucleotides, non-nucleotides, a linker molecule, or a conjugate molecule.

[0180] In some embodiments, an asymmetric duplex is an oligonucleotide molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises 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 comprises an antisense region of sufficient length to mediate RNAi in a cell or in vitro system (e.g., about 19 to about 22 nucleotides) and a sense region of about 3 to about 19 nucleotides that are complementary to the antisense region.

[0181] In some cases, universal base refers to a nucleotide base analog that forms a base pair with each natural DNA / RNA base, with little or no difference between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, pyrrolecarboxamide and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole known in the art.

[0182] In some embodiments, the dsRNA agent is 5' phosphorylated or includes a phosphoryl analog at the 5' initial end. 5'-Phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO2(O)P--O-5'); 5'-diphosphate ((HO)2(O)P--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 non-methylated) (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'-monothioate (phosphorothioate; (HO)2(S)P--O-5'); 5'-monodithioate (phosphorothioate; (HO)(HS)(S)P--O-5'); 5'-phosphorothioate ((H O)2(O)P--S-5'); dithiophosphates [--O2PS2--]; phosphonates [--PO(OH)2--]; phosphoramidates [--O=P(OH)2--]; methylsulfonylphosphoramidate (CH3)(SO2)(N)P(O)2--O-5'); any additional combination of oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2( (O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., such as RP(OH)(O)--O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R = alkylether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., such as RP(OH)(O)--O-5'-). In some embodiments, the modification can be placed in the antisense strand of the dsRNA agent.

[0183] 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 sequence in GYS1, wherein the first nucleotide of the target sequence starts at any nucleotide in the GYS1 mRNA transcript in the coding region or the 5' untranslated region or the 3' untranslated region (UTR). For example, the first nucleotide of the target sequence can be selected so that it starts at nucleic acid position (nal, the number starting from the 5' end of the full length of the GYS1 mRNA, for example, the first nucleotide at the 5' end is nal.1) 1, nal 2, nal 3, nal 4, nal 5, nal6, nal 7, nal 8, nal 9, nal 10, nal 11, nal 12, nal 13, nal 14, nal 15, nal 15, nal 16, nal 17, or any other nucleic acid position in the coding region or non-coding region (5' untranslated region or 3' untranslated region) of the GYS1 mRNA. nal 285-nal 335, nal 335-nal 345, nal 385-nal 395, nal 515-nal 525, nal 665-nal 675, nal 675-nal 685, nal 695-nal 705, nal 715-nal 725, nal 735-nal 745, nal 755-nal 765, nal 775-nal 785, nal 795-nal 805, nal 815-nal 825, nal 835-nal 845 715,nal 875-nal 885,nal 885-nal 895,nal 895-nal 905,nal 1035-nal1045,nal 1045-nal 1055,nal 1125-nal 1135,nal 1135-nal 1145,nal 1145-nal 1155,nal 1155-nal 1165,nal 1125-nal 1135,nal1155-nal 1165, nal 1225-nal 1235, nal1235-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-nal1245, 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,nal1615-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, nal2260 -2400, nal 2400 -2500, nal 2500 -2600, nal 2600 -2700, nal 2700 -2800, nal2800 -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.

[0184] In some embodiments, the antisense strand of the dsRNA agent is complementary to the target RNA 100% to hybridize with it and inhibit its expression by RNA interference. The target RNA can be any RNA expressed in a cell. In another embodiment, the cell is a tumor cell, a hepatocyte, a myocyte, 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 complementary to the target RNA by 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%. 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 the expression of GYS in the assay described herein at a concentration of no more than 200nm as described herein and does not reduce the expression of other RNAs by more than 50%.

[0185] siRNA can target any gene or RNA (eg, mRNA) transcript of interest.

[0186] Other modifications and patterns of modification can be found, for example, in U.S. Pat. No. 10,233,448, which is hereby incorporated by reference.

[0187] Other modifications and patterns of modification can be found, for example, in Anderson et al. (Nucleic Acids Research, 2021, 49(16), 9026-9041), which is hereby incorporated by reference.

[0188] Other modifications and patterns of modification can be found, for example, in International Patent Application Publication No. WO2021 / 030778, which is hereby incorporated by reference.

[0189] Other modifications and patterns of modification can be found, for example, in International Patent Application Publication No. WO2021 / 030763, which is hereby incorporated by reference.

[0190] In some embodiments, the siRNA is linked to a protein, such as a FN3 domain. The 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 attached to the sense strand, which is used to facilitate the connection of the sense strand to the FN3 domain.

[0191] In some embodiments, provided herein is a compound having formula (X1) n -(X2) q -(X3) y-L-X4, 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, and X4 is a nucleic acid molecule, such as but not limited to a siRNA molecule, wherein 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 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 a FN3 domain that binds to albumin. Examples of such FN3 domains include, but are not limited to, those described in U.S. Patent Application Publication No. 20170348397 and U.S. Patent No. 9,156,887, which are hereby incorporated by reference in their entirety. The FN3 domain may incorporate other subunits, for example, through 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, such as, but not limited to, any half-life extending moiety 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.

[0192] In some embodiments, provided herein are compositions having the formula (X1)-(X2)-L-(X4), wherein X1 is a first FN3 domain, X2 is a second FN3 domain, L is a linker, and X4 is a nucleic acid molecule. In some embodiments, X4 is an siRNA molecule. In some embodiments, X1 is an FN3 domain that binds to one of CD71. In some embodiments, X2 is an 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 comprise (e.g., is free of) a compound or protein that binds to ASGPR.

[0193] In some embodiments, provided herein is a compound having the formula C-(X1): n -(X2) q [L-X4]-(X3) y The invention provides a composition of the invention, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q and y are each independently 0 or 1.

[0194] In some embodiments, provided herein is a compound having formula (X1) n -(X2) q [L-X4]-(X3) y -C, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q and y are each independently 0 or 1.

[0195] In some embodiments, provided herein is a compound having the formula C-(X1): n -(X2) q [L-X4]L-(X3) y The invention provides a composition of the invention, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q and y are each independently 0 or 1.

[0196] In some embodiments, provided herein is a compound having formula (X1) n -(X2) q [L-X4]L-(X3) y -C, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q and y are each independently 0 or 1.

[0197] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula C-L1-X s And B1 has the formula X AS -L2-F1, where:

[0198] C is a polymer, such as PEG;

[0199] L1 and L2 are each independently a linker;

[0200] X SIt is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule;

[0201] X AS It is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule;

[0202] F1 is a polypeptide comprising at least one FN3 domain;

[0203] Where X S and X AS Double-stranded oligonucleotide molecules are formed to form a composition / complex.

[0204] In some embodiments, C can be a molecule that extends the half-life of a molecule. Examples of such parts are described herein. In some embodiments, C can also be Endoporter, INF-7, TAT, polyarginine, polylysine or an amphipathic peptide. These parts can be used to replace other half-life extension parts provided herein or to be used together with other half-life extension parts provided herein. In some embodiments, C can be a molecule that delivers a complex to a cell, an endosome or an ER; the molecule is selected from those peptides listed in Table 2 below.

[0205] Table 2

[0206]

[0207]

[0208] In some embodiments, a composition or complex having a formula A1-B1 is provided, wherein A1 has a formula X s And B1 has the formula X AS -L2-F1.

[0209] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula C-L1-X s And B1 has the formula X AS .

[0210] In some embodiments, the sense strand is a sense strand as provided herein.

[0211] In some embodiments, the antisense strand is an antisense strand as provided herein.

[0212] In some embodiments, the sense strand and the antisense strand 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.

[0213] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula F1-L1-X s And B1 has the formula X AS -L2-C, where:

[0214] F1 is a polypeptide comprising at least one FN3 domain;

[0215] L1 and L2 are each independently a linker;

[0216] C is a polymer, such as PEG;

[0217] X S It is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule;

[0218] X AS It is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule;

[0219] Where X S and X AS A double-stranded oligonucleotide molecule is formed to form a composition / complex. In certain embodiments, C is optional.

[0220] In some embodiments, a composition or complex having a formula A1-B1 is provided, wherein A1 has a formula X s And B1 has the formula X AS -L2-C.

[0221] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula F1-L1-X s And B1 has the formula X AS .

[0222] In some embodiments, C is a natural or synthetic polymer, which is composed of a cross-linked network of long chains of branched or unbranched monomers and / or two-dimensional or three-dimensional monomers. In some cases, polymer includes polysaccharide, lignin, rubber or polyalkylene oxide, which can be, for example, polyethylene glycol. In some cases, at least one polymer includes but is not limited to α-dihydroxy polyethylene glycol, ω-dihydroxy polyethylene glycol, biodegradable lactone-based polymers, such as polyacrylic acid, polylactic acid (PLA), poly (glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polybutylene glycol (PTG) or polyurethane, and their mixture. As used herein, mixture refers to using different polymers in the same compound and relates to block copolymers. In some cases, block copolymers are polymers in which at least one part of a polymer is composed of a monomer of another polymer. In some cases, polymer includes polyalkylene oxide. In some cases, polymer includes PEG. In some cases, the polymer comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0223] In some embodiments, polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, polydisperse materials include the dispersed distribution of different molecular weights of materials, characterized in that average weight (weight average) size and dispersity. In some cases, monodisperse PEG includes molecules of a size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weight represents the mean value of the molecular weight of polyalkylene oxide (e.g., PEG) molecules.

[0224] 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, 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.

[0225] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000 In some cases, the molecular weight of C is about 900, 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 cases, the molecular weight of C is about 200 Da. In some cases, the molecular weight of C is about 300 Da. In some cases, the molecular weight of C is about 400 Da. In some cases, the molecular weight of C is about 500 Da. In some cases, the molecular weight of C is 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 800Da. In some cases, the molecular weight of C is about 900Da. In some cases, the molecular weight of C is about 1000Da. In some cases, the molecular weight of C is about 1100Da. In some cases, the molecular weight of C is about 1200Da. In some cases, the molecular weight of C is about 1300Da. In some cases, the molecular weight of C is about 1400Da. In some cases, the molecular weight of C is about 1450Da. In some cases, the molecular weight of C is about 1500Da. In some cases, the molecular weight of C is about 1600Da. In some cases, the molecular weight of C is about 1700Da. In some cases, the molecular weight of C is about 1800Da.In some cases, the molecular weight of C is about 1900Da. In some cases, the molecular weight of C is about 2000Da. In some cases, the molecular weight of C is about 2100Da. In some cases, the molecular weight of C is about 2200Da. In some cases, the molecular weight of C is about 2300Da. In some cases, the molecular weight of C is about 2400Da. In some cases, the molecular weight of C is about 2500Da. In some cases, the molecular weight of C is about 2600Da. In some cases, the molecular weight of C is about 2700Da. In some cases, the molecular weight of C is about 2800Da. In some cases, the molecular weight of C is about 2900Da. In some cases, the molecular weight of C is about 3000Da. In some cases, the molecular weight of C is about 3250Da. In some cases, the molecular weight of C is about 3350Da. In some cases, the molecular weight of C is about 3500Da. In some cases, the molecular weight of C is about 3750Da. In some cases, the molecular weight of C is about 4000Da. In some cases, the molecular weight of C is about 4250Da. In some cases, the molecular weight of C is about 4500Da. In some cases, the molecular weight of C is about 4600Da. In some cases, the molecular weight of C is about 4750Da. In some cases, the molecular weight of C is about 5000Da. In some cases, the molecular weight of C is about 5500Da. In some cases, the molecular weight of C is about 6000Da. In some cases, the molecular weight of C is about 6500Da. In some cases, the molecular weight of C is about 7000Da. In some cases, the molecular weight of C is about 7500Da. In some cases, the molecular weight of C is about 8000Da. In some cases, the molecular weight of C is about 10,000Da. In some cases, the molecular weight of C is about 12,000Da. In some cases, the molecular weight of C is about 20,000Da. 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.

[0226] In some embodiments, polyalkylene oxide (e.g., PEG) is a discrete PEG, wherein discrete PEG is a polymerized PEG comprising more than one repeating ethylene oxide unit. In some cases, discrete PEG (dPEG) comprises 2 to 60, 2 to 50 or 2 to 48 repeating ethylene oxide units. In some cases, dPEG comprises 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, dPEG comprises about 2 or more repeating ethylene oxide units. In some cases, dPEG comprises about 3 or more repeating ethylene oxide units. In some cases, dPEG comprises about 4 or more repeating ethylene oxide units. In some cases, dPEG comprises about 5 or more repeating ethylene oxide units. In some cases, dPEG comprises about 6 or more repeating ethylene oxide units. In some cases, dPEG comprises about 7 or more repeating ethylene oxide units. In some cases, dPEG comprises about 8 or more repeating ethylene oxide units. In some cases, dPEG comprises about 9 or more repeating ethylene oxide units. In some cases, dPEG comprises about 10 or more repeating ethylene oxide units. In some cases, dPEG comprises about 11 or more repeating ethylene oxide units. In some cases, dPEG comprises about 12 or more repeating ethylene oxide units. In some cases, dPEG comprises about 13 or more repeating ethylene oxide units. In some cases, dPEG comprises about 14 or more repeating ethylene oxide units. In some cases, dPEG comprises about 15 or more repeating ethylene oxide units. In some cases, dPEG comprises about 16 or more repeating ethylene oxide units. In some cases, dPEG comprises about 17 or more repeating ethylene oxide units. In some cases, dPEG comprises about 18 or more repeating ethylene oxide units. In some cases, dPEG comprises about 19 or more repeating ethylene oxide units. In some cases, dPEG comprises about 20 or more repeating ethylene oxide units. In some cases, dPEG comprises about 22 or more repeating ethylene oxide units. In some cases, dPEG comprises about 24 or more repeating ethylene oxide units. In some cases, dPEG comprises about 26 or more repeating ethylene oxide units. In some cases, dPEG comprises about 28 or more repeating ethylene oxide units. In some cases, dPEG comprises about 30 or more repeating ethylene oxide units. In some cases, dPEG comprises about 35 or more repeating ethylene oxide units. In some cases, dPEG comprises about 40 or more repeating ethylene oxide units.In some cases, dPEG comprises about 42 or more repeating ethylene oxide units. In some cases, dPEG comprises about 48 or more repeating ethylene oxide units. In some cases, dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized in a stepwise manner by pure (e.g., about 95%, 98%, 99% or 99.5%) starting materials in the form of a single molecular weight compound. In some cases, dPEG has a specific molecular weight, rather than an average molecular weight. In some cases, dPEG described herein is from QuantaBiodesign, the dPEG of LMD.

[0227] In some embodiments, L1 is a polymer that can be used to link polymer C to sense strand X. S Or connect the polypeptide of F1 to the sense strand X S In some embodiments, L1 has the following formula:

[0228]

[0229] Where X S , X AS and F1 are as defined above.

[0230] In some embodiments, n=0-20. In some embodiments, R and R1 are independently methyl. In some embodiments, R and R1 are independently present or both are 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 enzyme-cleavable peptide, such as but not limited to Val-Cit, Val-Ala, etc.

[0231] In some embodiments, L2 is a polypeptide that can be used to link F1 to the antisense strand X AS Or connect polymer C to antisense strand X AS any connector.

[0232] In some embodiments, L2 has the following formula in the complex:

[0233]

[0234] Where X AS and F1 are as defined above.

[0235] In some embodiments, n=0-20. In some embodiments, R and R1 are independently methyl. In some embodiments, R and R1 are independently present or both are 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 enzyme-cleavable peptide, such as but not limited to Val-Cit, Val-Ala, etc.

[0236] In some embodiments, the linker is covalently attached to F1 through a cysteine ​​residue present on F1, which can be illustrated as follows:

[0237]

[0238] In some embodiments, A1-B1 has the formula:

[0239]

[0240] wherein C is a polymer such as PEG, Endoporter, INF-7, TAT, polyarginine, polylysine, an amphiphilic peptide, or a peptide as listed in Table 2 provided herein, 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; and F1 is a polypeptide comprising at least one FN3 domain, wherein X S and X AS Double-stranded siRNA molecules are formed. The sense strand and antisense strand are represented by the "N" symbol, wherein each nucleotide represented by N is independently A, U, C or G or a modified nucleobase, such as those provided herein. The N1 nucleotides of the sense strand and antisense strand represent the 5' end of the corresponding strand. For the sake of clarity, although Formula I utilizes N1, N2, N3, etc. in both the sense strand and the antisense strand, the nucleotide bases do not need to be the same, nor are they intended to be the same. The siRNA shown in Formula I will be complementary to the target sequence.

[0241] For example, in some embodiments, the sense strand comprises 2' O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone at N1 and N2; 12 and N 17 2'-Fluoro modified nucleotides at N4, N5, N6, N 10 、N 11 、N 13 、N 14 、N 15 、N 16 、N 18 and N 19 2'O-methyl modified nucleotides at

[0242] In some embodiments, the antisense strand comprises a vinylphosphonate moiety attached to N1; a 2' fluoro-modified nucleotide with a phosphorothioate (PS) modified backbone at N2; 10 、N 11 、N 12 、N 13 、N 15 、N 16 、N 17 、N 18 and N 19 2'O-methyl modified nucleotides at N 14 2' fluorine-modified nucleotides at N 20 and N 21 2' O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone.

[0243] In some embodiments, the compound has the formula:

[0244]

[0245] wherein F1 is a polypeptide comprising at least one FN3 domain and is conjugated to a linker L1, L1 being conjugated to X S Connection, where X S is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule, and X AS is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule; and wherein X S and X AS Double-stranded siRNA molecules are formed. The above linkers are non-limiting examples, and other types of linkers can be used.

[0246] In some embodiments, F1 comprises a compound having the formula (X1) 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 molecule; wherein n, q and y are each independently 0 or 1, provided 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.

[0247] In some embodiments, X1 is a CD71 FN3 binding domain, such as a domain provided herein. In some embodiments, X2 is a CD71 FN3 binding domain. In some embodiments, X1 and X2 are different CD71 FN3 binding domains. In some embodiments, the binding domains are the same. In some embodiments, X3 is a FN3 domain that binds to human serum albumin. In some embodiments, X3 is an Fc domain that does not have effector function that extends the half-life of the protein. In some embodiments, X1 is a first CD71 binding domain, X2 is a second CD71 binding domain, and X3 is a FN3 albumin binding domain. Examples of such polypeptides are provided herein and below. In some embodiments, provided herein are polypeptides having the formula C-(X1) n -(X2) q -(X3) y -L-X4, wherein C is a polymer such as PEG, Endoporter, INF-7, TAT, polyarginine, polylysine, an amphiphilic peptide, or a peptide provided in Table 2; X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q and y are each independently 0 or 1.

[0248] In some embodiments, provided herein is a compound having formula (X1) n -(X2) q -(X3) y -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 extension molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, wherein n, q and y are each independently 0 or 1.

[0249] In some embodiments, provided herein are compounds having the formula X4-L-(X1): n -(X2) q -(X3) y The invention provides a composition of the invention, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q and y are each independently 0 or 1.

[0250] In some embodiments, provided herein are compounds having the formula C-X4-L-(X1): n -(X2) q -(X3) yThe invention provides a composition of the invention, 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 molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q and y are each independently 0 or 1.

[0251] In some embodiments, provided herein are compounds having the formula X4-L-(X1): 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 molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, wherein n, q and y are each independently 0 or 1.

[0252] In some embodiments, the GYS1 siRNA pair can follow the following sequence: sense strand (5'-3')nsnsnnnnNfNfNfnnnnnnnnsnsa and antisense strand (5'-3')UfsNfsnnnNfnnnnnnnNfnNfnnnsusu, wherein (n) is 2'-O-Me (methyl), (nf) is 2'-F (fluoro), and (s) is a phosphorothioate backbone modification. Each nucleotide in the sense and antisense strands is modified independently or in combination at the ribose and nucleobase positions.

[0253] In some embodiments, the siRNA molecule comprises a sequence pair from Table 3A or Table 3B.

[0254] Table 3A: siRNA sense and antisense sequences

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] Abbreviations: (n / N = any nucleotide) mN = 2'-O-methyl residue, fN = 2'-F residue, * = phosphorothioate and (idT) = inverted Dt, (VP) 2'-O methyl vinylphosphonate uridine. Brackets indicate individual bases.

[0261] Table 3B

[0262]

[0263]

[0264]

[0265]

[0266] In some embodiments, the polynucleotides described above include polynucleotides that do not include 2'-O-methylvinylphosphonate uridine as the 5' nucleotide on the antisense strand of the siRNA.

[0267] 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 comprising a duplex. In some embodiments, the polynucleotide comprises a sense strand and an antisense strand. In some embodiments, comprising a sequence as shown in Table 3A or Table 3B. In some embodiments, comprising a sequence as shown in Table 3A or Table 3B, but without base modification. In some embodiments, the pharmaceutical composition comprises a siRNA pair as provided herein. In some embodiments, the siRNA pair is not conjugated to a FN3 domain.

[0268] In some embodiments, the oligonucleotide molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using methods known in the art. For example, naturally occurring nucleotides or various modified nucleotides are used to chemically synthesize oligonucleotide molecules that are designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the oligonucleotide molecule and the target nucleic acid. Alternatively, an expression vector is used to biologically produce the oligonucleotide molecules, wherein the oligonucleotide molecules have been subcloned into the expression vector in an antisense orientation (i.e., the RNA transcribed from the inserted oligonucleotide molecule will be the antisense orientation of the target polynucleic acid molecule of interest).

[0269] In some embodiments, the oligonucleotide molecules are synthesized by a tandem synthesis method in which the two strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide hybridization and allow purification of the separate fragments or strands of the duplex.

[0270] In some cases, oligonucleotide molecules are also assembled from two different nucleic acid strands or fragments, where one fragment includes the sense region of the molecule and the second fragment includes the antisense region of the molecule.

[0271] In some cases, although with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate or mesyl phosphoramidate, the bond between oligonucleotide molecule nucleotide is chemically modified, the bond improves stability. Excessive modification sometimes causes toxicity or activity reduction. Therefore, when designing nucleic acid molecules, the amount of bond between these nucleotides is minimized in some cases. In this case, the reduction of the concentration of these bonds reduces the toxicity of these molecules, improves effect and higher specificity.

[0272] As described herein, in some embodiments, the nucleic acid molecule can be modified to 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 or a modified 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 used to connect the dsRNA to the FN3 domain. The linker can be covalently attached to, for example, a cysteine ​​residue on a naturally occurring or substituted FN3 domain as described herein and, for example, in U.S. Patent No. 10,196,446, which is hereby incorporated by reference in its entirety. Non-limiting examples of such modified chains of dsRNA are shown in Table 4 below.

[0273] Table 4: Pairs with linkers and / or vinyl phosphonates

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] In some embodiments, the A to PPPP siRNA pairs provided above comprise a linker at the 3' end of the sense strand. In some embodiments, the A to PPPP siRNA pairs provided above comprise a vinylphosphonate at the 5' end of the sense strand.

[0281] Abbreviations: (n / N = any nucleotide) mN = 2'-O-methyl residue, fN = 2'-F residue, * = phosphorothioate, (idT) = inverted Dt, (VP) 2'-O methylvinylphosphonic acid uridine, BMPS = propylmaleimide,

[0282] The structure of the linker (L) is shown in Table 5 below.

[0283] Table 5: Representative examples of linkers (L)

[0284]

[0285]

[0286] Other linkers, such as linkers formed using click chemistry, amide coupling, reductive amination, oxime, enzyme coupling (such as transglutaminase), and sortage conjugation, may also be used. The linkers provided herein are exemplary in nature, and other linkers prepared using other such methods may also be used.

[0287] When linked to siRNA, the structure L-(X4) can be represented by the following formula:

[0288]

[0289] Although some siRNA sequences with some modified nucleobases are illustrated herein, sequences without such modifications are also provided herein. That is, the sequence may include the sequence shown in the table provided herein without any modification. In some embodiments, the unmodified siRNA sequence may also include a joint at the 5' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule may be modified to include 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 joint at the 3' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule may be modified to include vinyl phosphonate at the 3' end of the antisense strand of the dsRNA. The joint may be as provided herein.

[0290] In some embodiments, a FN3 protein comprising a polypeptide is provided, the polypeptide comprising a polypeptide that binds to CD71. In some embodiments, the polypeptide comprises an FN3 domain that binds to CD71. In some embodiments, a polypeptide comprising a sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599 or 708-710 is provided. In some embodiments, the polypeptide that binds to CD71 comprises a sequence of SEQ ID NO: 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 specifically binds to CD71.

[0291] In some embodiments, the CD71 binding FN3 domain is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon 27 sequence of SEQ ID NO: 4 (LPAPKNLVVSRVTEDSARLSWTAP DAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT), optionally with a substitution at residue position 11, 14, 17, 37, 46, 73, or 86 (corresponding to the residue numbering of SEQ ID NO: 4).

[0292] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0293] In some embodiments, the protein comprises a polypeptide comprising 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

[0294] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 X 13 VKG GX 14 X 15 SX 16 PLX 17 AX 18 FTT

[0295] Among them, X8, X9, X 17 and X 18 are each independently any amino acid except methionine or proline, and

[0296] X1 is selected from D, F, Y or H,

[0297] X2 is selected from Y, G, A or V,

[0298] X3 is selected from I, T, L, A or H,

[0299] X4 is selected from S, Y or P,

[0300] X5 is selected from Y, G, Q or R,

[0301] X6 is selected from G or P,

[0302] X7 is selected from A, Y, P, D or S,

[0303] X 10 Selected from W, N, S or E,

[0304] X 11 Selected from L, Y or G,

[0305] X 12 is selected from D, Q, H or V,

[0306] X 13 Select from G or S,

[0307] X 14 Selected from R, G, F, L or D,

[0308] X 15 is selected from W, S, P or L, and

[0309] X 16 Select from T, V, M or S.

[0310] In some embodiments:

[0311] X1 is selected from D, F, Y or H,

[0312] X2 is selected from G, A or V,

[0313] X3 is selected from T, L, A or H,

[0314] X4 is selected from Y or P,

[0315] X5 is selected from G, Q or R,

[0316] X6 is selected from G or P,

[0317] X7 is selected from Y, P, D or S,

[0318] X 10 Selected from W, N, S or E,

[0319] X 11 Selected from L, Y or G,

[0320] X 12 is selected from Q, H or V,

[0321] X 13 Select from G or S,

[0322] X 14 Selected from G, F, L or D,

[0323] X15 is selected from S, P or L, and

[0324] X 16 Select from V, M or S.

[0325] In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X 16 As shown in the sequence of SEQ ID NO: 288. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X3, X4, X5, X6, X7, X8, X9, X10, X11 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X 16 As shown in the sequence of SEQ ID NO: 289. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X3, X4, X5, X6, X7, X8, X9, X10, X11 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X 16 As shown in the sequence of SEQ ID NO: 290. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X3, X4, X5, X6, X7, X8, X9, X10, X11 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X 16 As shown in the sequence of SEQ ID NO:291.

[0326] In some embodiments, X8, X9, X 17 and X 18 are independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,

[0327] histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine or valine. 17 and X 18is independently not alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17 and X 18 In some embodiments, X8, X9, X 17and X 18 independently valine.

[0328] In some embodiments, the sequence is as shown in SEQ ID NO: 288, except that the sequences corresponding to X8, X9, X 17 and X 18 The position of the position may be any other amino acid residue as described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 Not S, and X 18 Not I.

[0329] In some embodiments, the sequence is as shown in SEQ ID NO: 289, except that the sequences corresponding to X8, X9, X 17 and X 18 The position of the position may be any other amino acid residue as described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 Not S, and X 18 Not I.

[0330] In some embodiments, the sequence is as shown in SEQ ID NO: 290, except that the sequences corresponding to X8, X9, X 17 and X 18 The position of the position may be any other amino acid residue as described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 Not S, and X 18 Not I.

[0331] In some embodiments, the sequence is as shown in SEQ ID NO: 291, except for the residues corresponding to X8, X9, X 17 and X 18 The position of the position may be any other amino acid residue as described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 Not S, and X 18 Not I.

[0332] In some embodiments, the protein comprises a polypeptide comprising an amino acid sequence that is at least 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: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.

[0333] Percent identity can be determined by aligning the two sequences using BlastP, available through the NCBI website, using default parameters.

[0334] In some embodiments, a type III fibronectin (FN3) domain that binds or specifically binds to a human CD71 protein (SEQ ID No: 2 or 5) is provided. As provided herein, the FN3 domain can bind to the CD71 protein. It is also provided that, even if not explicitly stated, these domains can also specifically bind to the CD71 protein. Thus, for example, an FN3 domain that binds to CD71 also includes an FN3 domain protein that specifically binds to CD71. These molecules can be used, for example, for therapeutic and diagnostic applications and imaging. In some embodiments

[0335] Provided herein are polynucleotides encoding the FN3 domains disclosed herein or complementary nucleic acids thereof, vectors, host cells, and methods of making and using the same.

[0336] In some embodiments, isolated FN3 domains that bind or specifically bind to CD71 are provided.

[0337] 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.

[0338] In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker, such as those provided herein. Exemplary linkers include, but are not limited to, (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)5AAA(SEQ ID NO:727) or (EAAAK) 1-5 (SEQ ID NO:728). In some embodiments, the linker comprises or is the following amino acid sequence: EAAAKEAAAKEA AAKEAAAK (SEQ ID NO:729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:730); APAPAPAPAP (SEQ ID NO:731); or EAAAK (SEQ ID NO:732).

[0339] In some embodiments, the FN3 domain may be less than about 1×10 -7 M, for example, less than about 1×10 -8 M, less than about 1×10 -9 M, less than about 1×10 -10 M, less than about 1×10 -11 M, less than about 1×10 -12 M or less than about 1×10 -13 The dissociation constant (K D ) binding to CD71 as measured by surface plasmon resonance or Kinexa method, as practiced by those skilled in the art. The affinity of a particular FN3 domain-antigen interaction measured may vary if measured under different conditions (e.g., osmotic pressure, pH). Therefore, affinity and other antigen binding parameters (e.g., K D , K on , K off ) are measured using standardized solutions of protein scaffold and antigen and a standardized buffer (such as the buffer described herein).

[0340] In some embodiments, the FN3 domain can bind CD71 at least 5-fold higher than the signal obtained for a negative control in a standard solution ELISA assay.

[0341] In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises an initiator methionine (Met) attached to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises a cysteine ​​(Cys) attached to the C-terminus of the FN3 domain. Addition of an N-terminal Met and / or a C-terminal Cys can facilitate expression and / or conjugation to extend half-life and provide other functions of the molecule.

[0342] The FN3 domain may also comprise a cysteine ​​substitution, such as those described in U.S. Patent No. 10,196,446, which is hereby incorporated by reference in its entirety. Briefly, in some embodiments, the polypeptides provided herein may comprise 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 an 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 equivalent positions in related FN3 domains.

[0343] The amino acid sequence of SEQ ID NO: 1 of U.S. Patent No. 10,196,446 is:

[0344] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVG EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFT T (specified here, SEQ ID NO:713)

[0345] 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.

[0346] Cysteine ​​substitutions at positions in a domain or protein include replacing an existing amino acid residue with a cysteine ​​residue. In some embodiments, instead of substitution, cysteine ​​is inserted into a sequence adjacent to the above position. Other examples of cysteine ​​modifications can be found in, for example, U.S. Patent Application Publication No. 20170362301, which is hereby incorporated by reference in its entirety. Sequence alignments can be performed using BlastP, using, for example, the default parameters on the NCBI website.

[0347] In some embodiments, a cysteine ​​residue is inserted at any position in a domain or protein.

[0348] In some embodiments, the FN3 domain that binds CD71 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 into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent into the cell. Cytotoxic agents can be used as therapeutic agents. 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 into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of an oligonucleotide into the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a hepatocyte. 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.

[0349] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0350] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 301. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 302. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 305. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 306. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 310. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 312. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 313. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 314. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 315. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 316. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 317. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 318. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 325. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 326. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 327. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 328. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 329. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 330. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 331. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 332. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 333. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 334. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 336. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 337. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 338. In some embodiments, the isolated FN3 domain that binds to 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 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 347. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 348. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 349. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 350. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 351. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 352. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 353. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 354. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 355. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 356. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 357. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 358. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 359. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 360. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 367. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 368. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 369. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 371. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 372. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 373. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 374. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 375. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 376. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 377. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 378. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 379. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 380. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 381. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 388. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 389. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 390. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 391. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 392. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 393. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 394. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 395. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 396. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 397. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 398. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 399. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 400. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 401. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 402. In some embodiments, the isolated FN3 domain that binds to 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 to 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 CD71 comprises the amino acid sequence of SEQ ID NO: 425. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 427. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 429.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 430. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 431. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 432. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 433. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 434. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 435. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 436. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 437. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 438. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 439. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 440. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 441. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 443. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 444. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 451. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 452. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 453. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 455. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 456. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 457. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 458. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 459. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 461. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 463. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 464. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 465. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 467. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 469. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 471.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 473. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 475. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 479. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 481. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 483. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 484. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 485. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 493. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 495. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 497. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 499. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 521. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 522. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 523. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 524. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 525. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 527. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 528. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 529. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 536. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 537. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 538. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 539. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 540. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 541. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 542. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 543. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 544. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 545. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 546. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 547. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 548. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 549. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 550. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 557. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 558. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 559. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 560. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 561. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 562. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 563. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 564. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 565. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 566. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 567. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 568. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 569. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 570. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 571. In some embodiments, the isolated FN3 domain that binds to 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 CD71 comprises the amino acid sequence of SEQ ID NO: 710.

[0351] In some embodiments, the isolated FN3 domain that binds CD71 comprises an initiator methionine (Met) attached to the N-terminus of the molecule.

[0352] In some embodiments, the isolated FN3 domain that binds 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 SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710. The percent identity can be determined by aligning the two sequences using BlastP, available through the NCBI website, using default parameters. The sequence of the FN3 domain that binds to CD71 can be found, for example, in Table 6 below.

[0353] Table 6: FN3 domain sequences that bind to CD71

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] 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.

[0368] Table 7

[0369]

[0370] 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, for example, (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)5AAA(SEQ ID NO: 727) or (EAAAK) 1-5 (SEQ ID NO:728). These are non-limiting examples, and other linkers may also be used. The number of GGGGS or GGGGA repeats may 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 EAAAKEAAAKEAAAKEAAAK (SEQ ID NO:729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:730); APAPAPAPAP (SEQ ID NO:731); or EAAAK (SEQ ID NO:732).

[0371] 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 the amino acid sequence of SEQ ID NO: 599. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of one of SEQ ID NOs: 592-599.

[0372] In some embodiments, the FN3 domain may be less than about 1×10 -7 M, for example, less than about 1×10 - 8 M, less than about 1×10 -9 M, less than about 1×10 -10 M, less than about 1×10 -11 M, less than about 1×10 -12 M or less than about 1×10 -13 The dissociation constant (K D ) binding to CD71 as measured by surface plasmon resonance or Kinexa method, as practiced by those skilled in the art. The affinity of a particular FN3 domain-antigen interaction measured may vary if measured under different conditions (e.g., osmotic pressure, pH). Therefore, affinity and other antigen binding parameters (e.g., K D , K on , K off ) are measured using standardized solutions of protein scaffold and antigen and a standardized buffer (such as the buffer described herein).

[0373] In some embodiments, the FN3 domain may bind to its target at least 5-fold higher than the signal obtained for a negative control in a standard solution ELISA assay.

[0374] In some embodiments, the FN3 domain that binds or specifically binds to its target protein comprises an initiator methionine (Met) attached 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) attached to the C-terminus of the FN3 domain. Addition of an N-terminal Met and / or a C-terminal Cys may facilitate expression and / or conjugation of a half-life extending molecule.

[0375] The FN3 domain may also comprise a cysteine ​​substitution, such as those described in U.S. Pat. No. 10,196,446, which is hereby incorporated by reference in its entirety. Briefly, in some embodiments, a polypeptide comprising a FN3 domain may have a FN3 domain having a residue substituted with cysteine, which residue may be referred to as a cysteine ​​engineered fibronectin type III (FN3) domain. In some embodiments, the FN3 domain may comprise 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 (LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAI NLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT; SEQ ID NO: 713) of U.S. Patent No. 10,196,446, which is hereby incorporated by reference in its entirety, and equivalent positions in related FN3 domains. A cysteine ​​substitution at a position in a domain or protein includes replacing an existing amino acid residue with a cysteine ​​residue. Other examples of cysteine ​​modifications can be found in, for example, U.S. Patent Application Publication No. 20170362301, which is hereby incorporated by reference in its entirety. Sequence alignments can be performed using BlastP, using, for example, the default parameters on the NCBI website.

[0376] In some embodiments, the FN3 domain bound 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 marker or therapeutic agent into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent into the cell. Cytotoxic agents can be used as therapeutic agents. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of any detectable marker, therapeutic agent and / or cytotoxic agent disclosed herein into the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a hepatocyte, a lung cell, a muscle cell, an immune cell, a dendritic cell, a CNS cell or a heart cell. In some embodiments, the therapeutic agent is an siRNA molecule as provided herein. The FN3 domain that binds to CD71 conjugated with a detectable marker can be used to evaluate the expression of CD71 on a sample (such as a tumor tissue) in vivo or in vitro. The FN3 domain that binds to CD71 conjugated with a detectable marker can be used to evaluate the expression of CD71 on a sample blood, immune cells, muscle cells or dendritic cells in vivo or in vitro.

[0377] As provided herein, a different FN3 domain attached to a siRNA molecule can also be conjugated or linked to another FN3 domain that binds to a different target. This would enable the molecule to be multispecific (e.g., bispecific, trispecific, etc.), such that it binds to a first target and another target, such as a target. In some embodiments, a first FN3 binding domain is linked to another FN3 domain that binds to an antigen expressed by a tumor cell (a tumor antigen).

[0378] 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 one serine, for example GGGGS. Other examples of linkers are provided herein and may also be used.

[0379] 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)5AAA(SEQ ID NO: 727) or (EAAAK) 1-5(SEQ ID NO:728). These are non-limiting examples, and other linkers may also be used. The number of GGGGS or GGGGA repeats may 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 one or more GGGGS repeats and one or more EAAAK repeats. In some embodiments, the linker comprises one or more GGGGS repeats and one or more "AP" repeats. In some embodiments, the linker comprises EAAAKEAAAKEAAAKEAAAK (SEQ ID NO:729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:730); APAPAPAPAP (SEQ ID NO:731); or EAAAK (SEQ ID NO:732).

[0380] Without being bound by any particular theory, in some embodiments, the FN3 domains linked to nucleic acid molecules can be used to target the delivery of therapeutic agents to cells (e.g., tumor cells) that express one or more binding partners of the FN3 domains and result in intracellular accumulation of the nucleic acid molecule therein. This can allow the siRNA molecule to properly interact with the cellular machinery to inhibit expression of the target gene, improve efficacy, and in some embodiments also avoid toxicity caused by non-targeted administration of the same siRNA molecule.

[0381] The FN3 domains described herein that bind to their specific target proteins can be produced as monomers, dimers or multimers, for example as a means of increasing the valency of target molecule binding and thus its affinity, or to produce bispecific or multispecific scaffolds that simultaneously bind to two or more different target molecules. Dimers and multimers can be produced by linking monospecific, bispecific or multispecific protein scaffolds, for example by including amino acid linkers, such as linkers 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)5AAA(SEQ ID NO: 727) or (EAAAK) 1-5(SEQ ID NO:728). In some embodiments, the linker comprises or is the following amino acid sequence: EAAAKEAAAKE AAAKEAAAK (SEQ ID NO:729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:730); APAPAPAPAP (SEQ ID NO:731); or EAAAK (SEQ ID NO:732).

[0382] Dimers and multimers can be connected to each other in the N-direction to the C-direction. The use of naturally occurring and synthetic peptide linkers to connect polypeptides to newly connected 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 connect the domains provided herein and in the formula provided above.

[0383] Half-life extension

[0384] In some embodiments, the FN3 domain can also incorporate other subunits, for example, through 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 binds to albumin, albumin variants, albumin binding proteins and / or domains and fragments and analogs thereof. Extending the half-life of the entire molecule.

[0385] 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. 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 or 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 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 comprises an amino acid sequence that is at least or 85%, 86%, 87%, 88%, 89%, 90%, 901%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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, provided that the protein has an amino acid sequence corresponding to SEQ ID NO: NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 at position 10. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid. In some embodiments, the isolated albumin binding domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions compared to the amino acid sequence of SEQ ID NO: 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 in or at the C-terminus of 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. Although the positions are listed in 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, other examples of albumin binding domains can be found in US Pat. No. 10,925,932, which is hereby incorporated by reference.

[0386] All or a portion of an antibody constant region can be attached to an FN3 domain to confer antibody-like properties, particularly those associated with an Fc region, such as Fc effector functions, such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor; BCR), and can be further modified by modifying residues in the Fc responsible for these activities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

[0387] Other moieties may be incorporated into the FN3 domains, such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of varying chain lengths, e.g., laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, suberic acid, tetradecanedioic acid, octadecanedioic acid, docosandioic acid, etc., polylysine, octane, carbohydrates (glucan, cellulose, oligosaccharides or polysaccharides) to obtain desired properties. These moieties may be fused directly to the protein scaffold coding sequence and may be produced by standard cloning and expression techniques. Alternatively, these moieties may be attached to the recombinantly produced molecules disclosed herein using well-known chemical coupling methods.

[0388] A PEG moiety can be added to the FN3 domain, for example, by incorporating a cysteine ​​residue into the C-terminus of the molecule, or engineering a cysteine ​​into a residue position facing away from the binding face of the molecule and attaching the PEG group to the cysteine ​​using well known methods.

[0389] The functionality of FN3 domains incorporated into other moieties can be compared by several well-known assays. For example, properties altered by incorporation of an Fc domain and / or Fc domain variant can be determined in an Fc receptor binding assay using a soluble form of the receptor, such as FcγRI, FcγRII, FcγRIII, or FcRn receptor, or using well-known cell-based assays measuring, for example, ADCC or CDC, or evaluating the pharmacokinetic properties of the molecules disclosed herein in an in vivo model.

[0390] The compositions provided herein can be prepared by preparing a FN3 protein and a nucleic acid molecule and linking them together. Techniques for linking proteins to nucleic acid molecules are known, and any method can be used. For example, in some embodiments, a nucleic acid molecule is modified with a linker (e.g., such as a linker provided herein), and then the protein is mixed with the nucleic acid molecule comprising the linker to form a composition. For example, in some embodiments, a FN3 domain is conjugated to a siRNA cysteine ​​using thiol-maleimide chemistry. In some embodiments, a cysteine-containing FN3 domain can be reduced with a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) in, for example, phosphate-buffered saline (or any other suitable buffer) to produce a free thiol. Then, in some embodiments, the FN3 domain containing the free thiol is mixed with a maleimide-linked modified siRNA duplex and incubated under conditions that form a linked complex. In some embodiments, the mixture is incubated at RT for 0-5 hours or about 1, 2, 3, 4, or 5 hours. The reaction can be quenched, for example, with N-ethylmaleimide. In some embodiments, affinity chromatography and ion exchange can be used to purify the conjugate. Other methods can also be used, and this is just one non-limiting embodiment.

[0391] Methods of 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.

[0392] In some embodiments, the FN3 domain that specifically binds to CD71 comprises an amino acid sequence of SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599 or 708-710, wherein a histidine tag has been attached to the N-terminus or C-terminus of the polypeptide for ease of purification. In some embodiments, the histidine tag comprises six histidine residues. In other embodiments, the His tag is connected to the FN3 domain by at least one glycine residue or about 2 to about 4 glycine residues. Thus, after purification of the FN3 domain and cleavage of the His tag from the polypeptide, one or more glycines may be left at the N-terminus or C-terminus. In some embodiments, if the His tag is removed from the N-terminus, all glycines are removed. In some embodiments, if the His tag is removed from the C-terminus, one or more glycines are retained.

[0393] In some embodiments, the FN3 domain that specifically binds CD71 comprises the amino acid sequence of SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, wherein the N-terminal methionine is retained after purification of the FN3 domain.

[0394] Reagent test kit

[0395] In some embodiments, kits comprising the compositions described herein are provided.

[0396] The kit can be used for therapeutic purposes and as a diagnostic kit.

[0397] In some embodiments, the kit comprises a FN3 domain conjugated to a nucleic acid molecule.

[0398] In some embodiments, a kit for treating glycogen storage disease is provided. In some embodiments, the kit comprises a first container comprising a pharmaceutical composition containing one or more FN3 domains linked to an siRNA comprising a sense strand and an antisense strand; and a second container comprising a pharmaceutical composition comprising an enzyme replacement therapy (ERT) for treating glycogen storage disease. In some embodiments, the one or more FN3 domains comprise an FN3 domain that binds to CD71. In some embodiments, the siRNA targets Gys1. In some embodiments, the one or more FN3 domains comprise 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), Corey or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Anderson disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Taluy disease (GSD7, muscle phosphofructokinase

[0399] (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucan body disease. In some embodiments, 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 glycogen protein-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 comprises a pharmaceutical composition comprising a CD71-binding FN3 domain linked to a siRNA targeting Gys1, and the second container comprises a pharmaceutical composition comprising an enzyme comprising GAA.

[0400] Use of conjugated FN3 domains

[0401] The compositions provided herein can be used to diagnose, monitor, regulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of a human disease or a particular condition in a cell, tissue, organ, fluid, or generally in a host.

[0402] In some embodiments, the method selectively reduces GYS1 mRNA and protein in skeletal muscle. In certain embodiments, GYS1 mRNA and protein is not reduced in liver and / or kidney.

[0403] In some embodiments, the reduction in GYS1 mRNA and protein persists for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or greater than 5 weeks following administration of a conjugate described herein.

[0404] In some embodiments, the FN3 domain can facilitate delivery into CD71-positive tissues (e.g., skeletal muscle, smooth muscle) to treat muscle diseases.

[0405] In some embodiments, the FN3 domain can facilitate delivery to activated lymphocytes, dendritic cells, or other immune cells to treat immune diseases.

[0406] In some embodiments, the polypeptide bound to CD71 is directed to immune cells. In some embodiments, the polypeptide bound to CD71 is directed to dendritic cells. In some embodiments, a method for treating an autoimmune disease in a subject in need is provided. In some embodiments, these methods include administering to a 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 NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein that is connected 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, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, and immune thrombocytopenic purpura.

[0407] In some embodiments, methods of treating a subject with Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency) are provided, the methods comprising administering to the subject a composition provided herein. In some embodiments, these methods comprise 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 No: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein that is linked or conjugated to a therapeutic agent.

[0408] In some embodiments, a method for treating a glycogen storage disease in a subject in need thereof is provided, the method comprising administering a composition provided herein. In some embodiments, the glycogen storage disease is selected from the group consisting of Corey's disease 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, Anderson's disease (GSD4, glycogen debranching enzyme (GBE1) deficiency), Taluy's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency) and adult polyglucan body disease. In some embodiments, the glycogen storage disease is selected from the group consisting of: glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke's disease), Hers' disease (GSD6, liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (Fanconi-Bickel syndrome), and glycogen synthase (GYS2) deficiency (GSD0). syndrome) (GSD11, glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), β-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0409] In some embodiments, a composition as provided herein or any composition is provided for use in the preparation of a pharmaceutical composition or drug 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, breast clear cell carcinoma, endometrial clear cell carcinoma, ovarian clear cell carcinoma, uterine clear cell carcinoma, hepatocellular carcinoma, pancreatic cancer, prostate cancer, soft tissue cancer, Ewing's sarcoma, and non-small cell lung cancer.

[0410] In some embodiments, CD71 cells are cells involved in CNS diseases, inflammatory / immune diseases (such as MS and brain infectious diseases). In some embodiments, the polypeptide bound to CD71 is directed to the central nervous system. In some embodiments, a method for treating a neurological condition and / or a brain tumor in a subject in need is provided. In some embodiments, these methods include administering to the subject a polypeptide or pharmaceutical composition bound to CD71. In some embodiments, the polypeptide is an FN3 domain bound 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 connected or conjugated to a therapeutic agent. In some embodiments, brain tumors are selected from the group consisting of: non-malignant brain tumors, benign brain tumors, 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 disease, Pompe disease, polyglucan body disease in adults, stroke, spinal cord injury, ataxia, Bell's Palsy, cerebral aneurysm, epilepsy, seizures, Guillain-Barré syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, migraine, encephalitis, sepsis, and myasthenia gravis.

[0411] In some embodiments, a method of treating a subject with cancer is provided, the method comprising administering a composition provided herein to the subject. 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 No: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein that is linked or conjugated to a therapeutic agent.

[0412] In some embodiments, the subject has a solid tumor.

[0413] In some embodiments, the solid tumor is melanoma.

[0414] 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.

[0415] In some embodiments, the solid tumor is renal cell carcinoma (RCC).

[0416] In some embodiments, the solid tumor is mesothelioma.

[0417] In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).

[0418] In some embodiments, the solid tumor is colorectal cancer.

[0419] In some embodiments, the solid tumor is prostate cancer. In some embodiments, the solid tumor is castration-resistant prostate cancer.

[0420] In some embodiments, the solid tumor is stomach cancer.

[0421] In some embodiments, the solid tumor is ovarian cancer.

[0422] In some embodiments, the solid tumor is gastric cancer.

[0423] In some embodiments, the solid tumor is liver cancer.

[0424] In some embodiments, the solid tumor is pancreatic cancer.

[0425] In some embodiments, the solid tumor is thyroid cancer.

[0426] In some embodiments, the solid tumor is head and neck squamous cell carcinoma.

[0427] In some embodiments, the solid tumor is esophageal cancer or gastrointestinal cancer.

[0428] In some embodiments, the solid tumor is breast cancer.

[0429] In some embodiments, the solid tumor is fallopian tube cancer.

[0430] In some embodiments, the solid tumor is brain cancer.

[0431] In some embodiments, the solid tumor is urethral cancer.

[0432] In some embodiments, the solid tumor is a genitourinary cancer.

[0433] In some embodiments, the solid tumor is endometriosis.

[0434] In some embodiments, the solid tumor is cervical cancer.

[0435] In some embodiments, the solid tumor is a metastatic lesion of cancer.

[0436] In some embodiments, the subject has a hematological malignancy.

[0437] In some embodiments, the hematological malignancy is a lymphoma, myeloma, or leukemia. In some embodiments, the hematological malignancy is a 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.

[0438] In some embodiments, the hematological malignancy is myelodysplastic syndrome.

[0439] 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).

[0440] In some embodiments, the hematological malignancy is multiple myeloma (MM).

[0441] In some embodiments, the hematological malignancy is a plasmacytoma.

[0442] In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the soft tissue cancer is Ewing sarcoma.

[0443] In some embodiments, methods of treating cancer in a subject in need are provided. In some embodiments, these methods include administering any composition provided herein to the subject. In some embodiments, uses of the compositions provided herein in the preparation of pharmaceutical compositions or drugs for treating cancer are provided. In some embodiments, the compositions can be used to treat cancer.

[0444] In some embodiments, methods are provided for treating Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency) in a subject in need thereof. In some embodiments, the methods comprise administering to the subject any composition provided herein. In some embodiments, uses of the compositions provided herein for preparing a pharmaceutical composition or medicament for treating Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency) are provided. In some embodiments, the compositions can be used to treat Pompe disease (GSD2, acid alpha-glucosidase (GAA) deficiency).

[0445] In some embodiments, methods of treating glycogen storage disease in a subject in need thereof are provided. In some embodiments, these methods comprise administering to a subject any composition provided herein. In some embodiments, uses of the compositions provided herein in the preparation of pharmaceutical compositions or medicaments for treating glycogen storage disease are provided. In some embodiments, the compositions can be used to treat glycogen storage disease.

[0446] In some embodiments, a method for treating a glycogen storage disease in a subject in need thereof is provided, the method comprising administering a composition provided herein. In some embodiments, the glycogen storage disease is selected from the group consisting of Corey's disease 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, Anderson's disease (GSD4, glycogen debranching enzyme (GBE1) deficiency), Taluy's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency) and adult polyglucan body disease. In some embodiments, the glycogen storage disease is selected from the group consisting of: glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke's disease), Hers' disease (GSD6, liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (Fanconi-Bickel syndrome), and glycogen synthase (GYS2) deficiency (GSD0). syndrome) (GSD11, glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), β-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0447] In some embodiments, the polypeptide bound to CD71 is directed to the central nervous system. In some embodiments, a method for treating a neurological condition and / or a brain tumor in a subject in need is provided. In some embodiments, these methods include administering to a subject a polypeptide or pharmaceutical composition bound to CD71. In some embodiments, the polypeptide is an FN3 domain bound 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 connected or conjugated to a therapeutic agent. In some embodiments, the brain tumor is selected from the group consisting of: non-malignant brain tumors, benign brain tumors, 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 disease, Pompe disease, adult polyglucan body disease, stroke, spinal cord injury, ataxia, Bell's palsy, cerebral aneurysm, epilepsy, seizure, Guillain-Barré syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, migraine, encephalitis, sepsis, and myasthenia gravis. In some embodiments, a method of treating a neurological condition and / or a brain tumor in a subject, the method comprising administering to the subject an FN3 domain that binds to CD71, and the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide (such as siRNA, ASO, etc.), an FN3 domain that binds another target, etc.).

[0448] In some embodiments, methods for treating Pompe disease in a subject in need are provided. In some embodiments, these methods include administering to a 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 No: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein that is connected or conjugated to a therapeutic agent. In some embodiments, a method for treating Pompe disease in a subject, the method comprising administering to the subject an FN3 domain that binds to CD71, and the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide (such as siRNA, ASO, etc.), an FN3 domain that binds to another target, etc.).

[0449] In some embodiments, the polypeptide bound to CD71 is directed to immune cells. In some embodiments, the polypeptide bound to CD71 is directed to dendritic cells. In some embodiments, a method for treating an autoimmune disease in a subject in need is provided. In some embodiments, these methods include administering to the subject a polypeptide or pharmaceutical composition bound to CD71. In some embodiments, the polypeptide is an FN3 domain bound 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 connected 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, 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 comprises administering to the subject an FN3 domain that binds CD71, and the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide (such as siRNA, ASO, etc.), an FN3 domain that binds another target, etc.).

[0450] In some embodiments, methods of reducing the expression of a target gene in a cell are provided. In some embodiments, these methods include delivering a composition or pharmaceutical composition provided herein to a cell. 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, as the evidence provided herein demonstrates that siRNA molecules can be effectively delivered when conjugated to a FN3 domain. In some embodiments, siRNA targeting GYS1 is attached to a FN3 domain. In some embodiments, the FN3 polypeptide (domain) is a polypeptide that binds to CD71. In some embodiments, the FN3 polypeptide is as provided herein or as provided in PCT Application No. PCT / US20 / 55509, U.S. Application No. 17 / 070,337, PCT Application No. PCT / US20 / 55470, or U.S. Application No. 17 / 070,020, each of which is hereby incorporated by reference in its entirety. In some embodiments, the siRNA is not conjugated to a FN3 domain.

[0451] In some embodiments, methods for reducing the expression of a target gene in a cell are provided. In some embodiments, these methods include delivering a composition or pharmaceutical composition provided herein to a cell. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the method for reducing the expression of a target gene results in a reduction in target gene expression of about 99%, 90%-99%, 50%-90%, or 10%-50%.

[0452] In some embodiments, a method of reducing the expression of GYS1 is provided. In some embodiments, the reduced expression is the expression (amount) of GYS1 mRNA. In some embodiments, the method of reducing the expression of GYS1 results in a reduction in GYS1 expression of about 99%, 90%-99%, 50%-90% or 10%-50%. In some embodiments, the reduced expression is the expression (amount) of GYS1 protein. In some embodiments, the reduced protein is glycogen. In some embodiments, the reduction of glycogen occurs in muscle cells. In some embodiments, the reduction of glycogen occurs in cardiac cells. In some embodiments, the method includes delivering a siRNA molecule targeting GYS1 as provided herein to a cell. In some embodiments, the siRNA is conjugated to a FN3 domain. In some embodiments, the FN3 domain is a 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 identical. In some embodiments, the two FN3 domains are different, i.e., bind to different regions or amino acid residues of CD71, i.e., different epitopes. In some embodiments, the method comprises administering to a subject (patient) a GYS1 siRNA molecule, such as those provided herein. In some embodiments, the GYS1 siRNA administered to the subject is conjugated or linked to a FN3 domain. In some embodiments, the FN3 domain is a 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 identical. In some embodiments, the two FN3 domains are different, i.e., bind to different regions or amino acid residues of CD71, i.e., different epitopes. In some embodiments, the CD71 binding domain is a polypeptide provided herein.

[0453] In some embodiments, the siRNA is conjugated to a cyclic peptide of Formula III:

[0454]

[0455] or a protonated form thereof, wherein: R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; at least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4, R5, R6, R7 are independently H or an amino acid side chain; at least one of R4, R5, R6, R7 is 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-di The side chain of aminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, b-homoarginine, 3-(l-piperidinyl)alanine; AAsc is an amino acid side chain; and q is 1, 2, 3 or 4; wherein the cyclic peptide of formula @@ is not FfΦRrRrE as disclosed in International Patent Publication No. WO2022 / 213118, which is hereby incorporated by reference in its entirety. "FfΦRrRrE" refers to a structure in which "F" is L-Phe; "f" is D-Phe; "Φ" is L-2-naphthylalanine; "R" is L-arginine; and "r" is D-arginine. "FfΦRrRrE" 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.

[0456] In some embodiments, a method for delivering siRNA molecules to cells in a subject is provided. In some embodiments, these methods include administering to a subject a pharmaceutical composition comprising a composition as 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 expressing the protein. In some embodiments, the protein is expressed on the cell surface. In some embodiments, the cell is a tumor cell, a hepatocyte, an immune cell, a dendritic cell, a cardiac cell, a myocyte, a CNS cell, or a cell within the blood-brain barrier. In some embodiments, siRNA downregulates the expression of a target gene in a cell. In some embodiments, the target gene is GYS1.

[0457] In some embodiments, provided herein are compositions comprising a compound of Formula IV:

[0458]

[0459] or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein: Y 2 and Y 3 Each is C, or Y 2 and Y 3 One of them is N and Y 2 and Y 3 The other one is C; X 1 and X 2 Each independently is H, C 1-6 Alkyl or C 1-6 Alkoxy; X 3 and X 4 Each is independently H, halo, C1-6 alkyl, C1-6 alkoxy or 5-20 membered heteroaryl, wherein X 3 and X 4 C 1-6 The alkyl group is optionally substituted with one or more halo groups; X 5 It is H, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-10 Cycloalkyl; (1) L 1 does not exist; and Q 1 Selected from (i) to (iv): (i) phenyl, wherein Q 1 The phenyl group is replaced by one or more halogenated groups, C 1-6 Alkyl, C 2-6 Alkenyl, -NH2, -NH-C(O)-(C 1-6 alkyl), -NH-C(O)-(3-15 membered heterocyclic group), C 3- The C1-6 alkyl group is optionally substituted with one or more halo groups, -NH-C(O)-NH(C1-6 alkyl), -NH-C(O)-C 1-6 Alkyl or -NH-C(O)-C 1-6 substituted by alkoxy, C3-10 cycloalkyl is optionally substituted by one or more halo or C1-6 alkyl, and 5-20 membered heteroaryl is optionally substituted by one or more C1-6 alkyl, (ii) 3-15 membered heterocyclyl, wherein Q 1 The 3-15 membered heterocyclic group is optionally substituted with one or more oxo groups or C 1-6 Alkyl substituted, (iii) 5-20 membered heteroaryl, wherein Q 1 The 5-20 membered heteroaryl is optionally substituted with one or more halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Alkoxy, -NH2 or C 3-10wherein the C1-6 alkyl is optionally substituted by one or more halo groups, and the C3-10 cycloalkyl is optionally substituted by one or more halo groups or C1-6 alkyl groups, and (iv) C 3-10 Cycloalkyl; or (2) L 1 is -CH2-; and Q 1 It is C 3-10 Cycloalkyl; L 2 is -C(O)- or -S(O)2-; R 1 is H or C1-6 alkyl; R k is H, halo, -OH, -NH2 or -NH-C(O)C 1-6 Alkyl; R m is H, -OH or C 1-6 Alkyl; R n It is H, C 1-6 Alkyl or C 3-10 Cycloalkyl, or R n Together with the carbon atom to which it is attached, it forms a C3-5 cycloalkyl group; or R k With R m or R n and the atoms to which they are attached together form a cyclopropyl group; and R 2 Selected from (i) to (vii): (i) C1-6 alkyl, wherein R 2 The C1-6 alkyl group is optionally replaced by one or more R a Substitution, where R a It is (a) -OH, (b) cyano, (c) C 2-6 Alkynyl, (d)C 6-20 Aryl, where R a C 6-20 The aryl group is optionally substituted by one or more halo groups, cyano groups, C1-6 alkoxy groups or -NH-C(O)-C1-6 alkyl groups; (e) a 3-15 membered heterocyclic group, wherein R a The 3-15 membered heterocyclic group is optionally substituted by one or more R c Substitution, where R c is a halo group, an oxo group, a C1-6 alkyl group, a C1-6 alkoxy group, a -C(O)-C1-6 alkyl group or a -C(O)-C1-6 alkoxy group, wherein R c C 1-6 The alkyl group is optionally substituted with one or more halo or C2-6 alkynyl groups, and R c The -C(O)-C1-6alkoxy group is optionally substituted by one or more halo groups, (f)-N(R c )(R d ), where -N(R c )(R d ) c and Rd are independently H, C1-6 alkyl, -C(O)-C 1-6 Alkyl, -C(O)-C 1-6 Alkoxy, -C(O)-NH2, -C(O)-NH(C 1-6 alkyl), -C(O)-N(C 1-6 alkyl)2, -C(O)-(3-15 membered heterocyclyl), -CH2-C(O)-NH2, 3-15 membered heterocyclyl or 5-20 membered heteroaryl, wherein R c or R d C 1-6 The alkyl group is optionally substituted with one or more -C(O)-NH2, R c or R d O-C(O)-C 1-6 The alkyl group is optionally substituted with one or more halo groups, R c or R d The 3-15 membered heterocyclyl and 5-20 membered heteroaryl are independently optionally substituted by one or more C1-6 alkyl groups, R c or R d -C(O)-(3-15 membered heterocyclyl) is optionally substituted by one or more halo, -C(O)-C1-6 alkoxy or C1-6 alkyl, wherein C1-6 alkyl is optionally substituted by one or more halo, C1-6 alkoxy or C3-10 cycloalkyl, and R c or R d -C(O)-N(C 1-6 Alkyl)2C 1-6 The alkyl groups are each independently optionally substituted with one or more halo groups or C6-20 aryl groups, (g) -OR e , where R e It is C 1-6 Alkyl, C 6-20 Aryl, -C(O)-(3-15 membered heterocyclic group), -C(O)-N-(C 1-6 alkyl) 2 or 5-20 membered heteroaryl, wherein R e C1-6 alkyl / optionally substituted by one or more C1-6 alkoxy, wherein the C1-6 alkoxy is optionally substituted by one or more C2-6 alkynyl, R e C 6-20 The aryl group is optionally substituted with one or more C 1-6 Alkyl substituted, and R e The -C(O)-(3-15 membered heterocyclyl) is optionally substituted by one or more C1-6 alkyl, C1-6 alkoxy or -C(O)-C1-6 alkoxy, wherein the C1-6 alkyl is optionally substituted by one or more halo, C 1-6 Alkoxy or C 3-10Cycloalkyl substitution, (h)-C(O)-R e , where R e is -NH2, -OH or a 3-15 membered heterocyclic group, or (i) -S(O)2-R f , where R f It is C 1-6 alkyl or 3-15 membered heterocyclic group, provided that when R 2 When is an unsubstituted methyl group, then (1) Q 1 is a 5-20 membered heteroaryl group, wherein Q 1 The 5-20 membered heteroaryl is optionally substituted with one or more halo, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, -NH2, C3- 10 cycloalkyl or -OH, and wherein Q 1 is not unsubstituted pyridyl, or (2) Q 1 is phenyl, where Q 1 The phenyl group is substituted by: (i) at least one C 3-6 Alkyl, at least one C 3-6 The alkyl group is optionally substituted with one or more halo groups, or (ii) at least one C 3-10 Cycloalkyl, wherein at least one C 3-10 The cycloalkyl group is optionally substituted with one or more halo or C 1-6 or (iii) at least one 5-20 membered heteroaryl, wherein at least one 5-20 membered heteroaryl is optionally substituted with one or more C1-6 alkyl groups, (ii) C 3-10 Cycloalkyl, where R 2 C 3-10 The cycloalkyl group is optionally substituted with one or more R q Substitution, where R q is a 5-20 membered heteroaryl or a C6-20 aryl, wherein R q The C6-20 aryl group is optionally substituted by one or more C1-6 alkoxy groups, (iii) a 3-15 membered heterocyclic group, wherein R 2 The 3-15 membered heterocyclic group is optionally substituted with one or more halo, oxo, C 1-6 Alkyl, -C(O)-C 1-6 alkyl or 5-20 membered heteroaryl, (iv) 5-20 membered heteroaryl or -(C 1-4 alkyl)(5-20 membered heteroaryl), wherein C 1-4 The alkyl group is optionally substituted with one or more -OH, halo, -NH2, -NH(C 1-6 Alkyl), -N(C 1- 6 alkyl)2, and wherein the 5-20 membered heteroaryl is optionally substituted by one or more R s Substitution, where R sis a halogenated group, C 1-6 Alkyl, C 1-6 Alkoxy, -NH2, -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, wherein R s C 1-6 The alkyl group is optionally substituted with one or more halo, C 1-6 Alkoxy, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NH-C(O)C 1-6 Alkyl or -NH-C(O)-C 1-6 Alkoxy substituted, and R s The 3-15 membered heterocyclic group is optionally substituted by one or more halo or -C(O)-C 1-6 Alkoxy substituted, (v)-N(R g )(R h ), where R g and R h independently H or C 1-6 Alkyl, (vi)-C(O)-R j , where R j It 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, where R 2 C 6-20 The aryl group is optionally substituted with one or more 5-20 membered heteroaryl groups or -OR p Substitution, where R p is a 3-15 membered heterocyclic group, wherein R p The 3-15 membered heterocyclyl is optionally substituted with one or more -C(O)-C1-6 alkyl groups as described in International Patent Publication No. WO2022 / 198196, which is hereby incorporated by reference in its entirety.

[0460] In some embodiments, provided herein are compositions that can be used for diagnosis, monitoring, regulation, treatment, alleviation, help prevent the incidence of human diseases or specific symptom in cells, tissues, organs, fluids or common hosts or reduce their symptoms, and also show the characteristics that can pass through the blood-brain barrier. The blood-brain barrier (BBB) ​​prevents most macromolecules (e.g., DNA, RNA and polypeptides) and many small molecules from entering the brain. The BBB is mainly composed of specialized endothelial cells with highly restrictive tight connections, and therefore, the BBB controls small substances and large substances from entering the central nervous system from the blood. This structure makes the treatment and management of patients with neurological diseases and illnesses (e.g., brain cancer) difficult, because many therapeutic agents cannot be delivered through the BBB with the desired efficiency. Other situations related to BBB destruction include: stroke, diabetes, epileptic seizures, hypertensive encephalopathy, acquired immunodeficiency syndrome, traumatic brain injury, multiple sclerosis, Lafora disease, Pompe disease, adult polyglucan body disease, Parkinson's disease (PD) and Alzheimer's disease. This ability is particularly useful for treating brain cancer, including, for example: astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma and congenital tumors; or spinal cord cancer, such as neurofibroma, meningioma, glioma and sarcoma. This can also be used to treat Pompe disease and / or glycogen storage disease. In certain embodiments, the compositions provided herein can be used to deliver therapeutic agents or cytotoxic agents, such as through the blood-brain barrier. In certain embodiments, the compositions provided herein can be used to deliver therapeutic agents or cytotoxic agents, such as into muscle.

[0461] In some embodiments, a method for treating a glycogen storage disease in a subject in need thereof is provided, the method comprising administering a composition provided herein. In some embodiments, the glycogen storage disease is selected from the group consisting of Corey's disease 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, Anderson's disease (GSD4, glycogen debranching enzyme (GBE1) deficiency), Taluy's disease (GSD7, muscle phosphofructokinase (PFKM) deficiency) and adult polyglucan body disease. In some embodiments, the glycogen storage disease is selected from the group consisting of: glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke's disease), Hers' disease (GSD6, liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (Fanconi-Bickel syndrome), and glycogen synthase (GYS2) deficiency (GSD0). syndrome) (GSD11, glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), β-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0462] In some embodiments, the compositions or pharmaceutical compositions provided herein can be used to treat muscle diseases, such as muscular dystrophy, DMD, etc.

[0463] In some embodiments, the compositions or pharmaceutical compositions provided herein can be administered alone or in combination with other therapeutic agents, i.e., administered simultaneously or sequentially. In some embodiments, other or additional therapeutic agents are other anti-tumor agents or therapeutic agents. Different tumor types and tumor stages may require the use of various auxiliary compounds that can be used to treat cancer. For example, the compositions provided herein can be used in combination with various chemotherapeutic agents, such as paclitaxel, tyrosine kinase inhibitors, folinic acid, fluorouracil, irinotecan, phosphatase inhibitors, MEK inhibitors, etc. The composition can also be used in combination with drugs (such as anti-PD-1 or anti-CTLA-4, etc.) that regulate the immune response to tumors. Additional treatment can be an agent that regulates the immune system, such as an antibody targeting PD-1 or PD-L1.

[0464] In some embodiments, the compositions or pharmaceutical compositions provided herein can be administered in combination with enzyme replacement therapy (ERT). In some embodiments, the ERT is an ERT designed to treat glycogen storage diseases. In some embodiments, the glycogen storage disease is selected from the group consisting of: Pompe Disease (GSD2, glucosidase alpha acid (GAA) deficiency), Cori's disease or Forbes' disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Andersen's disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdledisease (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 / Diabetic Nephropathy, Lafora Disease, Hypoxia and Adult Polyglucan Body Disease. In some embodiments, the glycogen storage disease is selected from the group consisting of: glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1, von Gierke's disease), Hers' disease (GSD6, liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (Fanconi-Bickel syndrome), and glycogen synthase (GYS2) deficiency (GSD0). syndrome) (GSD11, glucose transporter (GLUT2) deficiency, aldolase A deficiency (GSD12), β-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0465] 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), glycogen protein-1 (GYG1), or any combination thereof. In some embodiments, ERT comprises administration of glucosidase alpha acid (GAA). In some embodiments, ERT comprises administration of glycogen debranching enzyme (AGL). In some embodiments, ERT includes administration of glycogen branching enzyme (BGE1). In some embodiments, ERT includes administration of muscle glycogen phosphorylase (PYGM). In some embodiments, ERT includes administration of muscle phosphofructokinase (PFKM). In some embodiments, ERT includes administration of aldolase A (ALDOA). In some embodiments, ERT includes administration of malin. In some embodiments, ERT includes administration of laforin. In some embodiments, ERT includes administration of glycogen synthase (GYS2). In some embodiments, ERT includes administration of glucose-6-phosphatase (G6PC / SLC37A4). In some embodiments, ERT includes administration of phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1). In some embodiments, ERT includes administration of phosphoglycerate mutase (PGAM2). In some embodiments, ERT includes administration of muscle lactate dehydrogenase (LDHA). In some embodiments, ERT includes administration of glucose transporter (GLUT2). In some embodiments, ERT includes administration of β-enolase (ENO3). In some embodiments, ERT comprises administration of glycogenin-1 (GYG1).

[0466] In some embodiments, methods of treating glycogen storage disease in a subject in need thereof are provided. In some embodiments, these methods comprise administering any composition provided herein and ERT to a subject. In some embodiments, use of the compositions provided herein and ERT in the preparation of a pharmaceutical composition or medicament for treating glycogen storage disease is provided. In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject prior to administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0467] In some embodiments, methods for treating Pompe disease in a subject in need are provided. In some embodiments, these methods include administering any composition provided herein and an ERT comprising glucosidase alpha acid (GAA) to a subject. In some embodiments, the compositions provided herein and the ERT comprising glucosidase alpha acid (GAA) are provided for use in the preparation of a pharmaceutical composition or drug for treating Pompe disease. In some embodiments, the composition is a composition comprising an FN3 domain, which may be connected to another 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 a subject before administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0468] In some embodiments, methods of treating Corey's disease or Forbes' disease (GSD3) in a subject in need thereof are provided. In some embodiments, these methods comprise administering to a subject any composition provided herein and an ERT comprising a glycogen debranching enzyme (AGL). In some embodiments, the use of a composition provided herein and an ERT comprising a glycogen debranching enzyme (AGL) in the preparation of a pharmaceutical composition or medicament for treating Corey's disease or Forbes' disease (GSD3) is provided. In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject prior to administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0469] In some embodiments, methods for treating Anderson's disease (GSD4) in a subject in need thereof are provided. In some embodiments, these methods include administering to a subject any composition provided herein and an ERT comprising a glycogen branching enzyme (GBE1). In some embodiments, the compositions provided herein and the ERT comprising a glycogen branching enzyme (GBE1) are used in the preparation of a pharmaceutical composition or a medicament for treating Anderson's disease (GSD4). In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject before administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0470] In some embodiments, methods for treating McArdle's disease (GSD5) in a subject in need thereof are provided. In some embodiments, these methods include administering to a subject any composition provided herein and an ERT comprising glycogen phosphorylase (PYGM). In some embodiments, the use of a composition provided herein and an ERT comprising glycogen phosphorylase (PYGM) in the preparation of a pharmaceutical composition or a drug for treating McArdle's disease (GSD5) is provided. In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject before administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0471] In some embodiments, methods of treating Talui disease (GSD7) in a subject in need thereof are provided. In some embodiments, the methods comprise administering to a subject any composition provided herein and an ERT comprising myofructokinase (PFKM). In some embodiments, the compositions provided herein and the ERT comprising myofructokinase (PFKM) are used in the preparation of a pharmaceutical composition or medicament for treating Talui disease (GSD7). In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject prior to administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0472] In some embodiments, methods of treating Aldolase A Deficiency (GSD12) in a subject in need thereof are provided. In some embodiments, the methods comprise administering to a subject any composition provided herein and an ERT comprising Aldolase A (ALDOA). In some embodiments, the compositions provided herein and the ERT comprising Aldolase A (ALDOA) ​​are used in the preparation of a pharmaceutical composition or a medicament for treating Aldolase A Deficiency (GSD12). In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject prior to administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0473] In some embodiments, methods for treating Lafora disease in a subject in need thereof are provided. In some embodiments, these methods include administering to a subject any composition provided herein and an ERT comprising malin, laforin, or both. In some embodiments, the use of a composition provided herein and an ERT comprising malin, laforin, or both in the preparation of a pharmaceutical composition or drug for treating Lafora disease is provided. In some embodiments, the composition is a composition comprising an FN3 domain, which may 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 a subject before administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administration of ERT.

[0474] In some embodiments, a method for treating an adult polyglucan body disease in a subject in need is provided. In some embodiments, these methods include administering to a subject any composition provided herein and an ERT comprising a glycogen branching enzyme (BGE1). In some embodiments, a composition provided herein and an ERT comprising a glycogen branching enzyme (BGE1) are provided for use in the preparation of a pharmaceutical composition or a drug for treating an adult polyglucan body disease. In some embodiments, the composition is a composition comprising an FN3 domain. In some embodiments, the composition is administered to a subject before administering ERT. In some embodiments, the composition is administered to a subject during administration of ERT. In some embodiments, the composition is administered to a subject after administering ERT.

[0475] "Treat" or "treatment" refers to therapeutic treatment and preventive measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as the development or spread of cancer. In some embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, alleviation of disease extent, stabilization of disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of disease state, and detectable or undetectable relief (partial or complete). "Treatment" can also refer to prolonged survival compared to the expected survival without treatment. Those in need of treatment include those already suffering from a condition or disorder and those prone to suffering from a condition or disorder or those of a condition or disorder to be prevented.

[0476] "Therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the necessary dosage and time period. The therapeutically effective amount of the compositions provided herein may vary according to factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective amount are improved health of the patient, reduction or shrinkage of tumor size, prevention or slowing of tumor growth, and / or that cancer cells do not metastasize to other locations in the body.

[0477] Administration / Pharmaceutical Composition

[0478] In some embodiments, a pharmaceutical composition of a composition provided herein and a pharmaceutically acceptable carrier is provided. For therapeutic use, the composition can be prepared as a pharmaceutical composition containing an effective amount of a domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient or vehicle used together with an active compound. Such vehicles can be liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally do not contain insoluble particles. It can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, stabilizers, thickeners, lubricants and colorants, etc. The concentration of the molecules disclosed herein in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5%, typically at least about 1% to as much as 15 or 20% by weight, and will be selected primarily based on the desired dose, fluid volume, viscosity, etc., in accordance with the particular mode 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, 21st edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, see especially pp. 958-989.

[0479] The mode of administration of the therapeutic use of the compositions disclosed herein can be any suitable route for delivering the agent to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal), using tablets, capsules, solutions, powders, gels, granular forms of preparations; and in syringes, implants, osmotic pumps, cartridges, micropumps; or other means known to those skilled in the art. Site-directed administration can be achieved, for example, by intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal or transdermal delivery.

[0480] The pharmaceutical composition can be provided as a kit comprising a container, the container comprising a pharmaceutical composition as described herein. The pharmaceutical composition can be provided, for example, in the form of a single-dose or multi-dose injectable solution, or as a sterile powder to be reconstituted before injection. Alternatively, such a kit may include a dry powder disperser, a liquid aerosol generator or a nebulizer for administering the pharmaceutical composition. Such a kit may also include written information about the indications and usage of the pharmaceutical composition.

[0481] In addition, the following implementation schemes are provided:

[0482] 1. A method of treating a glycogen storage disease in a subject in need thereof, the method comprising administering:

[0483] Compositions containing one or more FN3 domains linked to a siRNA molecule (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, such as provided herein; and

[0484] Enzyme replacement therapy (ERT).

[0485] 2. A method as described in embodiment 1, wherein the glycogen storage disease is selected from the group consisting of: Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Corey disease or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Anderson disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Talloy disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes / diabetic nephropathy, Lafora disease, hypoxia and adult polyglucan body disease.

[0486] 3. A method as described in 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 glycogen protein-1 (GYG1).

[0487] 4. The method of any one of embodiments 1 to 3, wherein a composition comprising a FN3 domain linked to a siRNA is administered to the subject prior to administration of the ERT.

[0488] 5. The method of any one of embodiments 1 to 3, wherein a composition comprising a FN3 domain linked to a siRNA is administered to the subject during administration of the ERT.

[0489] 6. The method of any one of embodiments 1 to 3, wherein a composition comprising a FN3 domain linked to a siRNA is administered to the subject after administration of the ERT.

[0490] 7. A method of treating Pompe disease in a subject in need thereof, the method comprising administering:

[0491] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0492] ERT containing glucosidase alpha acid (GAA).

[0493] 8. A method of treating Corey's disease (also known as Forbes' disease) in a subject in need thereof, the method comprising administering:

[0494] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0495] ERT containing glycogen debranching enzyme (AGL).

[0496] 9. A method of treating Anderson's disease (GSD4) in a subject in need thereof, the method comprising administering:

[0497] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0498] ERT containing glycogen branching enzyme (GBE1).

[0499] 10. A method of treating McArdle's disease (GSD5) in a subject in need thereof, the method comprising administering:

[0500] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0501] ERT containing glycogen phosphorylase (PYGM).

[0502] 11. A method of treating Tarui's disease (GSD7) in a subject in need thereof, the method comprising administering:

[0503] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0504] ERT containing phosphofructokinase (PFKM).

[0505] 12. A method of treating Aldolase A Deficiency (GSD12) in a subject in need thereof, the method comprising administering:

[0506] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0507] ERT containing aldolase A (ALDOA).

[0508] 13. A method of treating Lafora disease in a subject in need thereof, the method comprising administering:

[0509] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0510] ERTs containing malin, laforin, or both.

[0511] 14. A method of treating an adult polyglucan body disorder in a subject in need thereof, the method comprising administering:

[0512] A composition containing an FN3 domain linked to a siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) comprising a sense strand and an antisense strand, as provided herein; and

[0513] ERT containing glycogen branching enzyme (BGE1).

[0514] 15. The method of any one of embodiments 7 to 14, further comprising at least one additional ERT.

[0515] 16. The method of any one of embodiments 7 to 15, wherein a composition comprising a FN3 domain linked to a siRNA is administered to the subject prior to administration of the ERT.

[0516] 17. The method of any one of embodiments 7 to 15, wherein a composition comprising a FN3 domain linked to a siRNA is administered to the subject during administration of the ERT.

[0517] 18. The method of any one of embodiments 7 to 15, wherein a composition comprising a FN3 domain linked to siRNA is administered to the subject after administration of the ERT.

[0518] 19. The method of any one of embodiments 1 to 18, wherein the one or more FN3 domains comprises an FN3 domain that binds to CD71.

[0519] 20. The method of any one of embodiments 1 to 19, wherein the siRNA (or other oligonucleotide, such as an antisense oligonucleotide or as otherwise provided herein) molecule is an siRNA that reduces the expression of GYS1.

[0520] 21. The method of any one of embodiments 1 to 20, wherein the siRNA does not contain any modified nucleobases.

[0521] 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.

[0522] 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.

[0523] 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.

[0524] 25. The method of embodiment 24, wherein the vinylphosphonate modification is attached to the 5' end or the 3' end of the sense strand or the antisense strand.

[0525] 26. The method of any one of embodiments 1 to 25, wherein the sense strand comprises SEQ ID NO: 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, 631, 632, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645 32, 634, 636, 638, 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 a sequence as shown in Table 3A, Table 3B or Table 4.

[0526] 27. The method of any one of embodiments 1 to 26, wherein the antisense strand comprises SEQ ID NO: 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, 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 a sequence as shown in Table 3A, Table 3B or Table 4.

[0527] 28. A method as described in any of the preceding embodiments, wherein the siRNA molecule comprises 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, KK, LL, MM, NN, OO, PP, QQ, RR, SS, TT, UU, VV, WW, XX, YY, ZZ, AAA, BBB, CCC, DDD, EEE, F FF, GGG, HHH, III, JJJ, KKK, LLL, MMM, 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 an siRNA pair as shown in Table 3A, Table 3B, or Table 4.

[0528] 29. The method of any one of the preceding embodiments, wherein the sense strand comprises 19 nucleotides.

[0529] 30. The method of any one of the preceding embodiments, wherein the antisense strand comprises 21 nucleotides.

[0530] 31. The method of any one of embodiments 1 to 30, wherein the composition comprises the siRNA pairs provided in Table 4 having linkers and / or vinylphosphonate modifications shown in Table 5.

[0531] 32. The method of any one of the preceding embodiments, wherein the siRNA molecule has the formula as shown in Formula I:

[0532] N1N2N3N4N5N6N7N8N9N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 Sense strand (SS)

[0533] N 21 N 20 N 19 N 18 N 17 N 16 N 15 N 14 N 13 N12 N 11 N 10 N9N8N7N6N5N4N3N2N1 Antisense strand (AS)

[0534] wherein each nucleotide represented by N is independently A, U, C or G or a modified nucleotide base such as those provided herein.

[0535] 33. A method as described in embodiment 32, wherein the sense strand comprises 2'O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone at N1 and N2; 12 and N 17 2'-Fluoro modified nucleotides at N4, N5, N6, N 10 、N 11 、N 13 、N 14 、N 15 、N 16 、N 18 and N 19 2'O-methyl modified nucleotides at

[0536] 34. A method as described in embodiment 32, wherein the antisense strand comprises a vinyl phosphonate moiety attached to N1; a 2' fluoro-modified nucleotide with a phosphorothioate (PS) modified backbone at N2; 10 、N 11 、N 12 、N 13 、N 15 、N 16 、N 17 、N 18 and N 19 2'O-methyl modified nucleotides at N 14 2' fluorine-modified nucleotides at N 20 and N 21 2' O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone.

[0537] 35. The method of embodiment 32, wherein the vinylphosphonate moiety is attached to N1 of the antisense strand.

[0538] 36. The method of any one of the preceding embodiments, wherein the siRNA molecule has the formula as shown in Formula I:

[0539]

[0540] wherein F1 is a polypeptide comprising at least one FN3 domain and is conjugated to a linker L1, L1 being conjugated to X S Connection, where X S is the 5' to 3' oligonucleotide sense strand of the double-stranded siRNA molecule, and X AS is the 3' to 5' oligonucleotide antisense strand of the double-stranded siRNA molecule; and wherein X S and X AS Double-stranded siRNA molecules are formed.

[0541] 37. The method of embodiment 36, wherein F1 comprises a compound having the formula (X1) n -(X2) q -(X3) y The polypeptide of the invention, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; wherein n, q and y are each independently 0 or 1, provided that at least one of n, q and y is 1.

[0542] 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.

[0543] 39. The method of embodiment 38, wherein the cysteine ​​is at a position as described herein.

[0544] 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 the amino acid sequence based on SEQ ID NO: 713.

[0545] 41. The method of embodiment 40, wherein the cysteine ​​is located at a position corresponding to residue 6, 53 or 88.

[0546] 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.

[0547] 43. The method of any one of the preceding embodiments, wherein the one or more FN3 domains comprise at least two FN3 domains connected by a peptide linker.

[0548] 44. The method of any one of the preceding embodiments, wherein the composition comprises a first FN3 domain and a second FN3 domain.

[0549] 45. The method of embodiment 44, wherein the first FN3 domain and the second FN3 domain bind to different proteins.

[0550] 46. ​​The composition of embodiment 44, wherein the first FN3 domain and the second FN3 domain bind to the same protein.

[0551] 47. The method of any one of embodiments 44 to 46, wherein the first FN3 domain binds to CD71.

[0552] 48. The method of any one of embodiments 44 to 46, wherein the second FN3 domain binds to a different target that does not bind CD71.

[0553] 49. The method of 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.

[0554] 50. The method of any one of embodiments 43 to 49, further comprising a third FN3 domain.

[0555] 51. The method of embodiment 50, wherein the third FN3 domain is an FN3 domain that binds to CD71 or albumin.

[0556] 52. The method of embodiment 51, wherein the CD71-binding FN3 domain has an amino acid sequence as provided herein, including but not limited to SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599 or 708-710,...

Claims

1. A method of treating a glycogen storage disease in a subject in need thereof, the method comprising administering: A composition comprising one or more FN3 domains linked to a siRNA comprising a sense strand and an antisense strand, wherein the siRNA targets Gys1; and Enzyme replacement therapy (ERT) for the treatment of said glycogen storage diseases.

2. The method of claim 1, wherein the glycogen storage disease is selected from the group consisting of: Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Corey or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Anderson disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Talloy disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes / diabetic nephropathy, Lafora disease, hypoxia and adult polyglucan body disease.

3. The method of 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), 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 glycogen protein-1 (GYG1).

4. The method of any one of claims 1 to 3, wherein the one or more FN3 domains comprises an FN3 domain that binds to CD71. 5 . The method according to claim 1 , wherein the siRNA targeting Gys1 is an siRNA that reduces the expression of GYS1. The method of claim 4 , wherein the siRNA targeting Gys1 is an siRNA that reduces the expression of GYS1.

7. The method of claim 1, wherein: The glycogen storage disease is Pompe disease; The FN3 domain is a polypeptide that binds to CD71; and The ERT involves the administration of glucosidase alpha acid (GAA).

8. The method of claim 1, wherein: The glycogen storage disease is Lafora's disease; The FN3 domain is a polypeptide that binds to CD71; and The ERT comprises administration of malin, laforin, or both.

9. The method of claim 1, wherein the siRNA is covalently linked to a FN3 domain via a chemical linker that is covalently attached to the sense strand or the antisense strand of the siRNA.

10. The method of claim 1, wherein the siRNA molecule comprises an siRNA pair comprising OOOO, 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, KK, 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, MMM, NNN, OOO, PPP, QQQ, RRR, SSS, TTT, UUU, VVV, WWW, XXX, YYY, ZZZ, AAAA, BBBB, CCCC, DDDD, EEEE, FFFF, GGGG, HHHH, IIII, JJJ, KKKK, LLLL, MMMM, NNNN, PPPP or the sense strand and antisense strand as shown in Table 3A, Table 3B or Table 4.

11. The method of claim 1, wherein the siRNA linked to the FN3 domain is conjugated to a cysteine ​​residue of the FN3 domain.

12. The method of claim 11, 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 the amino acid sequence based on SEQ ID NO:

713.

13. The method of claim 12, wherein the cysteine ​​is located at a position corresponding to residue 6, 53 or 88 of the amino acid sequence of SEQ ID NO:

713.

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

15. The method of claim 1, wherein the FN3 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

16. The method of claim 1, wherein the FN3 domain that binds CD71 has amino acids of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

17. A pharmaceutical composition for treating a glycogen storage disease, comprising: a composition comprising one or more FN3 domains linked to a siRNA comprising a sense strand and an antisense strand; and an enzyme replacement therapy (ERT) for treating the glycogen storage disease.

18. A kit for treating a glycogen storage disease, the kit comprising a first container comprising a pharmaceutical composition comprising one or more FN3 domains linked to a siRNA comprising a sense strand and an antisense strand; and a second container comprising a pharmaceutical composition comprising an enzyme replacement therapy (ERT) for treating the glycogen storage disease.

19. The kit of claim 18, wherein the one or more FN3 domains comprises an FN3 domain that binds to CD71.

20. The kit of claim 18, wherein the siRNA targets Gys1.

21. The kit of claim 18, wherein the one or more FN3 domains comprises an FN3 domain that binds to CD71.

22. The kit of claim 18, wherein the glycogen storage disease is selected from the group consisting of: Pompe disease (GSD2, glucosidase alpha acid (GAA) deficiency), Corey or Forbes disease (GSD3, glycogen debranching enzyme (AGL) deficiency), Anderson disease (GSD4, glycogen branching enzyme (GBE1) deficiency), McArdle disease (GSD5, muscle glycogen phosphorylase (PYGM) deficiency), Talloy disease (GSD7, muscle phosphofructokinase (PFKM) deficiency), aldolase A deficiency (GSD12, aldolase A (ALDOA) ​​deficiency), type II diabetes / diabetic nephropathy, Lafora disease, hypoxia, and adult polyglucan body disease.

23. The kit of claim 18, 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), 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 glycogen protein-1 (GYG1), or a combination thereof.

24. The kit of claim 18, wherein the ERT comprises GAA, malin, laforin, or a combination thereof.

25. The kit of claim 18, wherein the first container comprises a pharmaceutical composition comprising a CD71-binding FN3 domain linked to a siRNA targeting Gys1, and the second container comprises a pharmaceutical composition comprising an enzyme of GAA.

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