FN3 domain-siRNA conjugates and uses thereof

Abstract

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

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 174,776, filed April 14, 2021, U.S. Provisional Patent Application No. 63 / 203,776, filed July 30, 2021, and U.S. Provisional Patent Application No. 63 / 324,437, filed March 28, 2022, which are incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 17, 2022, is designated 145965_002612_SL.txt and is 427,548 bytes in size.

[0003] Field The present embodiments relate to siRNA molecules that are conjugated to a fibronectin type III domain (FN3), and methods of making and using the molecules. [Background technology]

[0004] Therapeutic nucleic acids include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide, ribozyme, plasmid, immunostimulatory nucleic acid, antisense, antagomir, antimir, microRNA mimic, supermir, U1 adaptor, and aptamer. In the case of siRNA or miRNA, these nucleic acids can downregulate the intracellular level of specific proteins through a process called RNA interference (RNAi). Since siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against target proteins, the therapeutic application of RNAi is extremely broad. At present, siRNA constructs have shown the ability to specifically downregulate target proteins in both in vitro and in vivo models. In addition, siRNA constructs have been evaluated in clinical studies and approved for various diseases at present.

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

[0006] There is still a need for compositions and methods for delivering siRNA to its intended cellular target.What is more, what is needed is a method of using such molecules for novel therapeutic agents that specifically bind to CD71, and that optimizes the properties of FN3 domain for clinical use, and that allows access to the inside of cells via receptor-mediated CD71 internalization.This embodiment meets these and other needs. [Means for solving the problem]

[0007] In some embodiments, a siRNA conjugated to an FN3 domain protein that binds CD71 is provided.

[0008] In some embodiments, a FN3 domain is provided that comprises any of the amino acid sequences of the FN3 domains presented herein. In some embodiments, the FN3 domain binds to CD71. In some embodiments, the FN3 domain specifically binds to CD71.

[0009] 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.

[0010] In some embodiments herein, oligonucleotides are provided, such as dsRNA molecules or siRNA molecules.In some embodiments, oligonucleotides have the sequence presented herein, with or without the modification presented herein.In some embodiments, oligonucleotides are provided by compositions, such as pharmaceutical compositions.In some embodiments, oligonucleotides are conjugated to polypeptides.

[0011] In some embodiments, compositions are provided that include one or more FN3 domains conjugated to a siRNA molecule.

[0012] In some embodiments, (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; X4 is an oligonucleotide molecule; and n, q, and y are each independently 0 or 1.

[0013] In some embodiments, 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; X4 is an oligonucleotide molecule; and n, q, and y are each independently 0 or 1.

[0014] In some embodiments, (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; C is a polymer or an albumin binding domain (ABD); and n, q, and y are each independently 0 or 1.

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

[0016] In some embodiments, C-X-L-(X1) n -(X2) q -(X3) y 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; X4 is an oligonucleotide molecule; and n, q, and y are each independently 0 or 1.

[0017] In some embodiments, X-L-(X) n -(X2) q -(X3) y 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; C is a polymer or an albumin binding domain (ABD); and n, q, and y are each independently 0 or 1.

[0018] In some embodiments, C-(X1) n -(X2) q [L-X4]-(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; C is a polymer or an albumin binding domain (ABD); and n, q, and y are each independently 0 or 1.

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

[0020] In some embodiments, pharmaceutical compositions are provided that include one or more of the compositions presented herein.

[0021] In some embodiments, provided is a method of treating Pompe disease (GSD2; acid alpha-glucosidase (GAA) deficiency) in a subject in need thereof, comprising administering a composition presented herein.

[0022] In some embodiments, a method of treating glycogen storage disease in a subject in need thereof is provided, comprising administering a composition as 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, Andersen's disease (GSD4; glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7; muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1; von Gierke disease), Haas disease (GSD6; hepatic glycogen phosphorylase (PYGL) deficiency or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11; glucose transporter (GLUT2) deficiency), aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0023] In some embodiments, provided are methods of treating cancer in a subject in need thereof, comprising administering to the subject a composition presented herein.

[0024] In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject in need thereof is provided, comprising administering to the subject a composition as provided herein. In some embodiments, the neurological condition is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Lafora's disease, Pompe's disease, adult polyglucosan body disease, stroke, spinal cord injury, ataxia, Bell's palsy, cerebral aneurysm, epilepsy, seizures, Guillain-Barre syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, migraine, encephalitis, sepsis, and myasthenia gravis.

[0025] In some embodiments, a method for treating autoimmune disease in a subject in need thereof is provided, comprising administering to the subject the composition presented herein.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.

[0026] In some embodiments, the present composition is used in the preparation of a pharmaceutical composition or medicament for treating cancer.In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia, myelodysplastic syndrome, gastric cancer, clear cell renal cell carcinoma, clear cell breast cancer, clear cell endometrial cancer, clear cell ovarian cancer, clear cell uterine cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, soft tissue cancer, Ewing's sarcoma, and non-small cell lung cancer.

[0027] In some embodiments, a method of reducing expression of a target gene in a cell is provided, comprising contacting the cell with a composition provided herein. In some embodiments, the cell is a tumor cell, a liver cell, a muscle cell, an immune cell, a dendritic cell, a cardiac cell, or a CNS cell.

[0028] In some embodiments, a method is provided for selectively reducing GYS1 mRNA and GYS1 protein in skeletal muscle. In certain embodiments, GYS1 mRNA and GYS1 protein are not reduced in liver and / or kidney.

[0029] In some embodiments, an isolated polynucleotide encoding an FN3 domain described herein is provided.

[0030] In some embodiments, a vector is provided that comprises a polynucleotide described herein.

[0031] In some embodiments, a host cell is provided that comprises a vector described herein.

[0032] In some embodiments, a method for producing FN3 domain is provided.In some embodiments, the method comprises culturing a host cell comprising a vector that encodes or expresses FN3 domain.In some embodiments, the method further comprises purifying FN3 domain.In some embodiments, the FN3 domain binds to CD71.

[0033] In some embodiments, a pharmaceutical composition is provided that includes a CD71-binding FN3 domain linked to an oligonucleotide molecule and a pharma- ceutically acceptable carrier. In some embodiments, a kit is provided that includes one or more of the FN3 domains, with or without an oligonucleotide molecule. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows a flow chart depicting the properties evaluated and considered for siRNA screening. [Diagram 2]FIG. 13 is a graph of an RNA sequencing experiment identifying changes in the transcriptome after cells were transfected with the siRNA pair HHH, where the arrow identifies a significant decrease in GYS1 transcript. [Diagram 3] FIG. 1 presents the results of a target binding assay using over 6,000 receptors in a proteomic array, where the data confirms that CD71 is the exclusive binding target of the FN3 domain. [Figure 4] Figure 4A is a graph supporting the knockdown of GYS1 mRNA in mouse gastrocnemius muscle using three different FN3 domain-siRNA conjugates compared to vehicle alone. Figure 4B is a graph supporting the knockdown of GYS1 protein in mouse gastrocnemius muscle using three different FN3 domain-siRNA conjugates compared to vehicle alone. [Diagram 5] FIG. 10 confirms that GYS1 knockdown is highly specific to skeletal muscle using three different FN3 domain-siRNA conjugates and compared with siRNA against a different target (AHA-1). [Figure 6] FIG. 2 shows an example of a Histrap chromatogram for purification of a conjugate (tagged protein). [Figure 7] FIG. 1 shows an example of a HIC chromatogram for purification of a conjugate (untagged protein). [Figure 8] FIG. 2 shows an example of an ion exchange chromatogram for purification of a conjugate (tagged / untagged protein). [Figure 9] FIG. 1 shows an example of analytical SEC for centirin-oligonucleotide conjugates. [Figure 10] FIG. 1 shows an example of an SDS PAGE gel for the conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] 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 so forth.

[0036] "Fibronectin type III (FN3) domain" (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, 89:8990-8994, 1992; Meinke et al., J Bacteriol, 175:1910-1918, 1993; Watanabe et al., J Biol Chem, 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin-C, the 15 different FN3 domains present in human fibronectin (FN), and the non-natural synthetic FN3 domains described, for example, in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the third FN3 domain of tenascin (TN3), or the tenth FN3 domain of fibronectin (FN10). As used throughout this specification, "sentilin" also refers to an FN3 domain. Additionally, the FN3 domains described herein include variable weight (V H ) chain and / or variable light (V L ) It is not an antibody because it does not have a chain structure.

[0037] As used herein, "autoimmune disease" refers to a disease condition and state in which an individual's immune response is directed against the individual's own components, resulting in an undesirable and often debilitating condition. As used herein, "autoimmune disease" is intended to further include autoimmune conditions, autoimmune syndromes, and the like. Autoimmune diseases include Addison's disease, allergies, allergic rhinitis, ankylosing spondylitis, asthma, atherosclerosis, autoimmune ear disease, autoimmune eye disease, autoimmune atrophic gastritis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune parotitis, autoimmune uveitis, celiac disease, primary biliary cirrhosis, benign lymphocytic vasculitis, COPD, colitis, coronary heart disease, Crohn's disease, diabetes mellitus (type I), depression, diabetes mellitus including type 1 diabetes and / or type 2 diabetes, epididymitis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, and inflammatory bowel disease (IBD). , immune responses to recombinant pharmaceuticals, including, but not limited to, Factor VII in hemophilia, juvenile idiopathic arthritis, systemic lupus erythematosus, lupus nephritis, male infertility, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, oncology diseases, osteoarthritis, pain, primary myxedema, pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, sympathetic ophthalmia, T-cell lymphoma, T-cell acute lymphoblastic leukemia, testicular angiocentric T-cell lymphoma, thyroiditis, transplant rejection, ulcerative colitis, autoimmune uveitis, and vasculitis. Autoimmune diseases include, but are not limited to, conditions in which the affected tissue is the primary target and, in some cases, the secondary target. Such conditions include, but are not limited to, AIDS, atopic allergies, bronchial asthma, eczema, leprosy, schizophrenia, hereditary depression, tissue / organ transplantation, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus phenomenon, anaphylaxis, and alcohol / drug intoxication.

[0038] The term "capture agent" refers to a substance that binds to a particular type of cell and allows for the separation of that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulation reagents, molecules that bind to specific cell types, etc.

[0039] A "sample" refers to a collection of fluids, cells, or tissues present within a subject, as well as similar fluids, cells, or tissues isolated from a subject. Exemplary samples are tissue biopsies, fine needle aspirates, surgically resected tissues, organ cultures, cell cultures, and fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucous secretions of secretory tissues / organs, vaginal secretions, ascites, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, fluids that have come into contact with a subject or biological source, such as cell / organ culture medium containing cell conditioned medium or organ conditioned medium, and lavage fluids.

[0040] "Substituting" or "substituted" or "mutating" or "mutated" refers to the alteration, deletion, or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of the sequence.

[0041] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, eg, a substitution, insertion, or deletion.

[0042] "Specifically binds to" or "specifically binds to" means that the FN3 domain is more than about 1×10 -6 M or less, e.g., about 1×10 -7 M or less, approximately 1 x 10 -8 M or less, approximately 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less, approximately 1 x 10 -11 M or less, approximately 1 x 10 -12M or less, or about 1 x 10 -13 M or a dissociation constant (K D ) to its target, such as CD71. Alternatively, "specific binding to" refers to the ability of an FN3 domain to bind to its target (e.g., CD71) at least 5-fold stronger than a negative control in a standard solution ELISA assay. Specific binding is also confirmed using a proteomic array, as described herein and shown in FIG. 3. In some embodiments, the negative control is an FN3 domain that does not bind to CD71. In some embodiments, an FN3 domain that specifically binds to CD71 may have cross-reactivity to other related antigens, e.g., the same given antigen from other species (homologues), such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee).

[0043] A "library" refers to a collection of variants. A library is composed of polypeptide variants or polynucleotide variants.

[0044] "Stability" refers to the ability of a molecule to remain folded under physiological conditions such that it retains at least one of its normal functional activities, e.g., binding to a given antigen, such as CD71.

[0045] "CD71" refers to the human CD71 protein having the amino acid sequence of SEQ ID NO: 2 or 5. In some embodiments, SEQ ID NO: 2 is the full-length human CD71 protein. In some embodiments, SEQ ID NO: 5 is the extracellular domain of human CD71.

[0046] "Tencon" refers to SEQ ID NO:1 LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT and described in U.S. Patent Publication No. 2010 / 0216708.

[0047] "Cancer cells" or "tumor cells" refer to cancerous, precancerous, or transformed cells, in vivo, ex vivo, and in tissue culture, that have spontaneous or induced phenotypic changes that do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and the incorporation of new genomic nucleic acid, or the incorporation of exogenous nucleic acid, but transformation can also occur spontaneously or following exposure to carcinogens, which may mutate endogenous genes. Transformation / cancer is exemplified by, for example, morphological changes, cellular immortalization, abnormalities in growth control, lesion formation, proliferation, malignancy, tumor-specific marker levels, invasiveness, tumor growth or inhibition, in suitable animal hosts, such as nude mice, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed., 1994)).

[0048] "Dendritic cells" refers 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 modulate immune responses, directly or indirectly. Dendritic cells have the ability to induce primary immune responses in inactive or resting naive T lymphocytes.

[0049] "Immune cells" refers to cells of the immune system, classified as lymphocytes (T cells, B cells, and NK cells), neutrophils, or monocytes / macrophages, which are all types of white blood cells.

[0050] "Vector" refers to a polynucleotide that can replicate in a biological system or can be transferred between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers that function to facilitate replication or maintenance of these polynucleotides in biological systems. Examples of such biological systems can include cells, viruses, animals, plants, and reconstituted biological systems that utilize biological components that can replicate vectors. The polynucleotides that comprise vectors can be DNA, RNA molecules, or hybrids thereof.

[0051] An "expression vector" refers to a vector utilized within a biological system, or within a biological system reconstituted to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0052] "Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical reaction. cDNA is a typical example of a polynucleotide.

[0053] "Polypeptide" or "protein" refers to a molecule containing at least two amino acid residues linked by peptide bonds to form a polypeptide. Small polypeptides, fewer than about 50 amino acids, are referred to as "peptides."

[0054] "Valent" refers to the presence of a certain number of binding sites specific for an antigen within a molecule. Thus, "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six, respectively, binding sites specific for an antigen within a molecule.

[0055] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except as noted, the terms "patient" and "subject" are used interchangeably.

[0056] "Isolated" refers to a molecule (such as a synthetic polynucleotide or polypeptide, such as an FN3 domain) that has been substantially separated and / or purified from other components of the system in which it is produced, such as a recombinant cell, as well as a homogenous population of a protein that has been subjected to at least one purification or isolation step. An "isolated FN3 domain" refers to an FN3 domain that is substantially free of other cellular material and / or chemicals, and includes FN3 domains that have been isolated to a high degree of purity, such as up to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0057] In some embodiments, compositions are provided that include a polypeptide, such as a polypeptide that includes an FN3 domain, linked to an oligonucleotide molecule. The oligonucleotide molecule can be, for example, a siRNA molecule.

[0058] Thus, in some embodiments, the siRNA is a double-stranded RNAi (dsRNA) agent capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand (passenger strand) and an antisense strand (guide strand). In some embodiments, each strand of the dsRNA agent can range from 12-40 nucleotides in length. 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.

[0059] In some embodiments, the sense strand and the antisense strand typically form a double-stranded dsRNA. The double-stranded region of the dsRNA agent can be 12-40 nucleotide pairs in length. For example, the double-stranded region can be 14-40 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotide pairs in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.

[0060] In some embodiments, the dsRNA comprises one or more overhanging regions and / or capping groups of the dsRNA agent at the 3' or 5' end of the strand or both. The overhang can be 1-10 nucleotides long, 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA, be complementary to the gene sequence being targeted, or be some other sequence. The first and second strands can also be joined by additional bases, e.g., to form a hairpin, or by other abasic linkers.

[0061] In some embodiments, the nucleotides in the overhang region of the dsRNA agent can be modified or unmodified nucleotides, including but not limited to 2'-sugar modified nucleotides such as 2'-F, 2'-O-methyl, 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), and any combination thereof. For example, TT(UU) can be the overhang sequence on either strand for either end. The overhang can form a mismatch with the target mRNA, can be complementary to the gene sequence to be targeted, or can be other sequences.

[0062] The 5'-overhang or 3'-overhang in the sense strand, antisense strand, or both strands of dsRNA agent may be phosphorylated.In some embodiments, the overhang region contains two nucleotides, with phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesyl phosphoramidate between the two nucleotides, and the two nucleotides may be the same or different.In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands.In one embodiment, the 3'-overhang is present in the antisense strand.In one embodiment, the 3'-overhang is present in the sense strand.

[0063] dsRNA agent may only comprise a single overhang, which may enhance the interference activity of dsRNA without affecting its overall stability.For example, the single-stranded overhang is located at the 3' end of the sense strand, or alternatively, at the 3' end of the antisense strand.dsRNA may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa.Generally, the antisense strand of dsRNA has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without being bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are suitable for loading the guide strand into RISC.For example, the single overhang comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0064] In some embodiments, the dsRNA agent may also have two blunt ends, on either end of the dsRNA duplex.

[0065] In some embodiments, every nucleotide in the sense strand and antisense strand of dsRNA agent may be modified.Each nucleotide is modified by the same or different modification, which may include one or more changes of one or both of non-linked phosphate oxygens and / or one or more of linked phosphate oxygens; modification of the components of ribose sugar, for example, the two hydroxyls on ribose sugar; complete replacement of phosphate moiety with "dephosphorylation" linker; modification or replacement of naturally occurring base; and replacement or modification of ribose-phosphate backbone.

[0066] In some embodiments, all or some of the bases in the 3'-overhang or 5'-overhang are modified, for example, by modifications described herein. Modifications can include, for example, the use of modifications at the 2'-position of the ribose sugar, by modifications known in the art, for example, the use of 2'-deoxy-2'-fluoro (2'-F) modified deoxyribonucleotides or 2'-O-methyl modified deoxyribonucleotides in place of the ribosugar of the nucleobase, and modifications in the phosphate group, for example, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesyl phosphoramidate modifications. The overhang does not have to be homologous to the target sequence.

[0067] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. A strand may contain more than one modification. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0068] In some embodiments, at least two different modifications are typically present on the sense strand and the antisense strand. These two modifications may be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, or other modifications.

[0069] In one embodiment, the sense and antisense strands each contain two differently modified nucleotides selected from 2'-fluoro, 2'-O-methyl, or 2'-deoxy.

[0070] dsRNA agent can further comprise at least one phosphorothioate, phosphorodithioate, phosphonic acid, phosphoramidate, mesyl phosphoramidate or methylphosphonic acid internucleotide linkage.Phosphorothioate, phosphorodithioate, phosphonic acid, phosphoramidate, mesyl phosphoramidate or methylphosphonic acid internucleotide linkage modification can occur on any nucleotide of sense strand or antisense strand or both at any position of chain.For example, internucleotide linkage modification can occur on every nucleotide of sense strand and / or antisense strand; each internucleotide linkage modification can occur on sense strand or antisense strand in alternating pattern; both sense strand or antisense strand comprise internucleotide linkage modification in alternating pattern. The alternating pattern of modifications of the internucleotide linkages on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of modifications of the internucleotide linkages on the sense strand may have a shift compared to the alternating pattern of modifications of the internucleotide linkages on the antisense strand.

[0071] In some embodiments, dsRNA agent comprises phosphorothioate, phosphorodithioate, phosphonic acid, phosphoramidate, mesyl phosphoramidate or methylphosphonic acid internucleotide linkage modification in overhanging region.For example, overhanging region comprises two nucleotides, and between two nucleotides, phosphorothioate, phosphorodithioate, phosphonic acid, phosphoramidate, mesyl phosphoramidate or methylphosphonic acid internucleotide linkage is.Internucleotide linkage modification is also applied to link overhanging nucleotide with terminal pairing nucleotide in double stranded region. For example, at least two, three, four or all of the protruding nucleotides are linked via phosphorothioate, phosphorodithioate, phosphonic acid, phosphoramidate, mesyl phosphoramidate or methylphosphonic acid internucleotide linkages, but optionally there can be additional phosphorothioate, phosphorodithioate, phosphonic acid, phosphoramidate, mesyl phosphoramidate or methylphosphonic acid internucleotide linkages that link the protruding nucleotide with the paired nucleotide adjacent to the protruding nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between three terminal nucleotides, where two of the three nucleotides are protruding nucleotides and the third nucleotide is the paired nucleotide adjacent to the protruding nucleotide.In some embodiments, these three terminal nucleotides can be at the 3' end of antisense strand.

[0072] In some embodiments, dsRNA compositions are linked by modified bases or nucleoside analogs as described in U.S. Patent No. 7,427,672, which is incorporated herein by reference.In some embodiments, modified bases or nucleoside analogs are referred to as linker or L in the formulas described herein.

[0073] In some embodiments, the modified base or nucleoside analog has the structure shown in Formula I, and salts thereof: [ka] [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 by 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 by a protecting group for nucleic acid synthesis, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a functional molecular unit substituent, m represents an integer of 0 to 2, and n represents an integer of 0 to 3. In some embodiments, m and n are 0.

[0074] In some embodiments, the modified base or nucleoside analog has a structure represented by formula I, and salts thereof, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic sulfonyl group, or an aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a lower alkyl, a lower alkoxy, a halogen, or a cyano group, or a methyl group substituted with one to three aryl groups having an aryl ring substituted with a silyl group.

[0075] In some embodiments, the modified base or nucleoside analog has a structure as shown in formula I, and salts thereof, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a trityl group, a dimethoxytrityl group, a monomethoxytrityl group, or a tert-butyldiphenylsilyl group.

[0076] In some embodiments, the modified base or nucleoside analog has a structure shown in formula I, and salts thereof, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic sulfonyl group, or an aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a lower alkyl, a lower alkoxy, a halogen, or a cyano group, a silyl group, a phosphoramidite group, a phosphonyl group, a phosphate group, or a phosphate group protected by a protecting group for nucleic acid synthesis.

[0077] In some embodiments, the modified base or nucleoside analog has the structure shown in formula I, and salts 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.

[0078] In some embodiments, the modified base or nucleoside analog has a structure shown in formula I, and salts 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 1 to 3 aryl groups, a lower aliphatic or aromatic sulfonyl group such as methanesulfonyl or p-toluenesulfonyl, 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.

[0079] In some embodiments, the modified base or nucleoside analog has a structure as shown in formula I, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeled molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear transport signal peptide, and salts thereof.

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

[0081] In some embodiments, the modified base or nucleoside analog has the chemical formula I, where the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected by 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 by a protecting group for nucleic acid synthesis 1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl with the amino group protected by a protecting group for nucleic acid synthesis] and salts thereof.

[0082] In some embodiments, the modified base or nucleoside analog has the structure shown in formula I, wherein m is 0 and n is 1, and salts thereof.

[0083] In some embodiments, the modified base or nucleoside analogue is an oligonucleotide analogue in which the linkage between each nucleoside is a phosphodiester bond [-OP(O2 - )O-], one or two or more phosphorothioate bonds [-OP(O)(S - )O-], phosphorodithioate bond [-O2PS2-], phosphonate bond [-PO(OH)2-], phosphoramidate bond [-O=P(OH)2-], or mesylphosphoramidate bond [-OP(O)(N)(SO2)(CH3)O-], and when one or more types of two or more of these structures are contained, the base may be one of these structures: [ka] [wherein the 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; m represents an integer of 0 to 2; and n represents an integer of 0 to 3], and may be the same or different between them. The nucleoside analogue has a structure shown in formula II, or a pharma- ceutically acceptable salt thereof, and is a DNA oligonucleotide analogue or an RNA oligonucleotide analogue that contains one or two or more structures of one or more types of unit structures of the nucleoside analogue. In some embodiments, m and n are 0.

[0084] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has a structure shown in formula II, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic sulfonyl group, or an aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a lower alkyl, a lower alkoxy, a halogen, or a cyano group, or a methyl group substituted with one to three aryl groups having an aryl ring substituted with a silyl group.

[0085] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has a structure shown in 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.

[0086] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has a structure shown in formula II, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic sulfonyl group, or an aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a lower alkyl, a lower alkoxy, a halogen, or a cyano group, a silyl group, a phosphoramidite group, a phosphonyl group, a phosphate group, or a phosphate group protected by a protecting group for nucleic acid synthesis.

[0087] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in formula II, where R2 is a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a tert-butyldiphenylsilyl group, -P(OCHCN)(N(i-Pr)), -P(OCH)(N(i-Pr)), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphate group.

[0088] In some embodiments, the oligonucleotide analog, or a pharma- ceutically acceptable salt thereof, has a structure shown in 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 1 to 3 aryl groups, a lower aliphatic or aromatic sulfonyl group such as methanesulfonyl or p-toluenesulfonyl, 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.

[0089] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has a structure shown in formula II, wherein the functional molecular unit substituent as R3 is a fluorescently or chemiluminescently labeled molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear transport signal peptide.

[0090] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the structure shown in Formula II, wherein the base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group, or a 2-oxopyrimidin-1-yl group, having a substituent selected from the following α groups: a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected by a protecting group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

[0091] In some embodiments, the oligonucleotide analog or a pharma- ceutically acceptable salt thereof has the chemical formula II, where the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl having a protected amino group, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected by a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl -9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected by 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 by a protecting group for nucleic acid synthesis. 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl with the amino group protected by a protecting group for nucleic acid synthesis.

[0092] In some embodiments, the oligonucleotide analog, or a pharma- ceutically acceptable salt thereof, has the structure shown in Formula II,

[0093] In some embodiments, the compositions described herein further comprise a polymer (polymer moiety C). In some cases, the polymer is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of monomers in two or three dimensions. In some cases, the polymer comprises a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some cases, the at least one polymer includes, but is not limited to, alpha-dihydroxyl polyethylene glycol, omega-dihydroxyl 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), polytetramethylene glycol (PTG), or polyurethane, as well as mixtures thereof. Mixtures as used herein refer to the use of different polymers in the same compound, as well as block copolymers. In some cases, a block copolymer is a polymer in which at least one segment of the polymer is constructed from a monomer of another polymer. In some cases, the polymer comprises a polyalkylene oxide. In some cases, the polymer comprises PEG. In some cases, the polymer comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0094] In some cases, C is a PEG moiety. In some cases, the PEG moiety is conjugated at the 5' end of the oligonucleotide molecule, while the 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, while the binding moiety is conjugated at the 5' end of the oligonucleotide molecule. In some cases, the PEG moiety is conjugated to an internal site of the oligonucleotide molecule. In some cases, the PEG moiety, the binding moiety, or a combination thereof is conjugated to an internal site of the oligonucleotide molecule. In some cases, the conjugation is direct conjugation. In some cases, the conjugation is conjugation via natural ligation.

[0095] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, polydisperse materials include a bidisperse distribution of materials with different molecular weights, characterized by size by average weight (weight average) and bidispersity. In some cases, monodisperse PEG includes molecules of one size. In some embodiments, C is a polydispersed polyalkylene oxide (e.g., PEG) or a monodispersed polyalkylene oxide (e.g., PEG), and the listed molecular weight represents the average molecular weight of the polyalkylene oxide molecules, e.g., PEG molecules.

[0096] 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, 270 0, 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.

[0097] 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, having a molecular weight of 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 290 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 800 Da. In some cases, the molecular weight of C is about 900 Da. In some cases, the molecular weight of C is about 1000 Da. In some cases, the molecular weight of C is about 1100 Da. In some cases, the molecular weight of C is about 1200 Da. In some cases, the molecular weight of C is about 1300 Da. In some cases, the molecular weight of C is about 1400 Da. In some cases, the molecular weight of C is about 1450 Da.In some cases, the molecular weight of C is about 1500 Da. In some cases, the molecular weight of C is about 1600 Da. In some cases, the molecular weight of C is about 1700 Da. In some cases, the molecular weight of C is about 1800 Da. In some cases, the molecular weight of C is about 1900 Da. In some cases, the molecular weight of C is about 2000 Da. In some cases, the molecular weight of C is about 2100 Da. In some cases, the molecular weight of C is about 2200 Da. In some cases, the molecular weight of C is about 2300 Da. In some cases, the molecular weight of C is about 2400 Da. In some cases, the molecular weight of C is about 2500 Da. In some cases, the molecular weight of C is about 2600 Da. In some cases, the molecular weight of C is about 2700 Da. In some cases, the molecular weight of C is about 2800 Da. In some cases, the molecular weight of C is about 2900 Da. In some cases, the molecular weight of C is about 3000 Da. In some cases, the molecular weight of C is about 3250 Da. In some cases, the molecular weight of C is about 3350 Da. In some cases, the molecular weight of C is about 3500 Da. In some cases, the molecular weight of C is about 3750 Da. In some cases, the molecular weight of C is about 4000 Da. In some cases, the molecular weight of C is about 4250 Da. In some cases, the molecular weight of C is about 4500 Da. In some cases, the molecular weight of C is about 4600 Da. In some cases, the molecular weight of C is about 4750 Da. In some cases, the molecular weight of C is about 5000 Da. In some cases, the molecular weight of C is about 5500 Da. In some cases, the molecular weight of C is about 6000 Da. In some cases, the molecular weight of C is about 6500 Da. In some cases, the molecular weight of C is about 7000 Da. In some cases, the molecular weight of C is about 7500 Da. In some cases, the molecular weight of C is about 8000 Da. In some cases, the molecular weight of C is about 10,000 Da. In some cases, the molecular weight of C is about 12,000 Da. In some cases, the molecular weight of C is about 20,000 Da. In some cases, the molecular weight of C is about 35,000 Da. In some cases, the molecular weight of C is about 40,000 Da. In some cases, the molecular weight of C is about 50,000 Da. In some cases, the molecular weight of C is about 60,000 Da.In some cases, the molecular weight of C is about 100,000 Da.

[0098] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, which is a polymeric PEG that contains more than one repeating ethylene oxide unit. In some cases, the discrete PEG (dPEG) contains 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some cases, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some cases, the dPEG contains about 2 or more repeating ethylene oxide units. In some cases, the dPEG contains about 3 or more repeating ethylene oxide units. In some cases, the dPEG contains about 4 or more repeating ethylene oxide units. In some cases, 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, the dPEG contains about 17 or more repeating ethylene oxide units. In some cases, the dPEG contains about 18 or more repeating ethylene oxide units.In some cases, 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 stepwise from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some cases, dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is dPEG manufactured by Quanta Biodesign, LMD.

[0099] In some embodiments, C is an albumin binding domain. In certain aspects, the albumin binding domain specifically binds to serum albumin, for example, human serum albumin (HSA), such that the albumin binding domain extends the half-life of the associated or linked domain or another therapeutic agent. In some embodiments, the human serum albumin binding domain comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the human serum albumin binding domain comprises a cysteine ​​(Cys) linked to the C-terminus or N-terminus of the domain. The addition of the N-terminus Met and / or C-terminus Cys can facilitate expression and / or conjugation with another molecule, which can be another half-life extending molecule, such as PEG, Fc region, etc.

[0100] In some embodiments, the albumin binding domain comprises the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, as set forth in Table 8. In some embodiments, the albumin binding domain (protein) is an isolated albumin binding domain (protein). In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or a percentage 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 is selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, provided that the protein has a substitution corresponding to position 10 of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. , 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or percent identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NOIn some embodiments, the isolated albumin binding domain comprises an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions when compared to the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the substitutions are at a position corresponding to position 10 of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the provided FN3 domains comprise a cysteine ​​residue at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, 88, or 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, or 93 of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or at the C-terminus. Although a series of positions are listed, each position is also individually selected. In some embodiments, the cysteine ​​is at a position corresponding to 6, 53, or 88. In some embodiments, further examples of albumin binding domains are found in U.S. Patent No. 10,925,932, incorporated herein by reference.

[0101] [Table 1]

[0102] In some embodiments, the dsRNA agent comprises a mismatch with the target in the duplex, or a combination thereof. The mismatch may occur in the overhang region or may occur in the duplex region. The base pairs are ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation for a particular pairing, nearest neighbor analysis or similar analysis may also be used, but the simplest approach is to consider pairs on an individual pair basis). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or pairings other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; pairings that include universal bases are preferred over canonical pairings.

[0103] In some embodiments, a dsRNA agent may comprise a phosphorus-containing group at the 5'-end of the sense strand or antisense strand. The 5'-end phosphorus-containing group may be 5'-end phosphate (5'-P), 5'-end phosphorothioate (5'-PS), 5'-end phosphorodithioate (5'-PS2), 5'-end vinylphosphonic acid (5'-VP), 5'-end methylphosphonic acid (MePhos), 5'-end mesyl phosphoramidate (5'MsPA), or 5'-deoxy-5'-C-malonyl. When the 5'-end phosphorus-containing group is 5'-end vinylphosphonic acid (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. Representative structures of these modifications can be found, for example, in U.S. Patent No. 10,233,448, which is incorporated herein by reference in its entirety.

[0104] In some embodiments, the nucleotide analog or synthetic nucleotide base comprises a nucleic acid with a modification at the 2' hydroxyl group of the ribose moiety. In some cases, the modification includes H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include those with chain lengths of C1 to C6. 10 and includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, both linear and branched. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazine, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some cases, the alkyl moiety further comprises an additional heteroatom, such as O, S, N, Se, each of which is further substituted by an alkyl group as described above. In some cases, the carbon of the heterocyclic group is substituted by nitrogen, oxygen, or sulfur. In some cases, the heterocyclic substitution comprises, but is not limited to, morpholino, imidazole, and pyrrolidino.

[0105] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification, or a 2'-O-methoxyethyl (2'-O-MOE) modification. Exemplary chemical structures of a 2'-O-methyl modification of an adenosine molecule and a 2'-O-methoxyethyl modification of a uridine are illustrated below.

[0106] [ka]

[0107] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group containing a propyl linker connects the amine group to the 2' oxygen. In some cases, this modification neutralizes the overall negative charge of the oligonucleotide molecule from the phosphate by introducing one positive charge per sugar from the amine group, thereby improving the cell uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below.

[0108] [ka]

[0109] In some cases, the modification at the 2' hydroxyl group is a locked ribose modification or a bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxymethylene linked bicyclic ribonucleotide monomer. An exemplary chemical structure of LNA is illustrated below. The representation shown on the left highlights the degree of chemical connectivity 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.

[0110] [ka]

[0111] In some cases, the modification at the 2' hydroxyl group includes, for example, ethylene nucleic acid (ENA), such as 2'-4'-ethylene bridged nucleic acid, which locks the sugar conformation into a C3'-endo sugar puckering conformation. ENA is part of the bridged nucleic acid class of modified nucleic acid, which also includes LNA. Exemplary chemical structures of ENA and bridged nucleic acid are illustrated below.

[0112] [ka]

[0113] In some embodiments, further modifications at the 2' hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0114] In some embodiments, the nucleotide analogs are 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2 propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5 position, such as 5-(2-amino)propyluridine, 5-halocytidine, 5-(2-amino)propylur ... -Deazanucleotides such as halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, 7-deazaadenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, 2-thiouridine and 4- Included are modified bases such as, but not limited to, other thio bases, such as thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, alkeuosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines, such as N6-methyladenosine, phenyl and modified phenyl groups, such as 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof 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 nucleotides known in the art as universal bases, by way of example and without limitation, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0115] In some embodiments, the nucleotide analogue further comprises morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro-N3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof. Morpholino, or phosphorodiamidate morpholino oligo (PMO), comprises synthetic molecules whose structure mimics natural nucleic acid structure by deviation from normal sugar and phosphate structures. In some cases, the five-membered ribose ring is replaced with a six-membered morpholino ring, containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked by phosphorodiamidate groups rather than phosphate groups. In such cases, the backbone modification removes all positive and negative charges, resulting in a morpholino, a neutral molecule capable of passing through cell membranes without the aid of an intracellular delivery agent, such as that used by charged oligonucleotides.

[0116] [ka]

[0117] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar rings or phosphate linkages, and the bases are joined and appropriately spaced by oligoglycine-like molecules, thus eliminating the backbone charge.

[0118] [ka]

[0119] In some embodiments, one or more modifications may occur at the internucleotide linkage. In some cases, the modified internucleotide linkage may be phosphorothioate, mesyl phosphoramidate, phosphorodithioate, methyl phosphonate, 5'-alkylene phosphonate, 5'-methyl phosphonate, 3'-alkylene phosphonate, boron trifluoridate, 3'-5' or 2'-5' linked boranophosphate and selenophosphate, phosphotriester, thionoalkyl phosphotriester, hydrogen phosphonate linkage, alkyl phosphonate, alkyl phosphonothioate, aryl phosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkyl phosphoramidate, aminoalkyl phosphoramidate, thionophosphoramidate, phosphoropiperazidate, phosphoroanilothioate, phosphoro ... These include, but are not limited to, oates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethylhydrazo, formacetals, thioformacetals, oximes, methyleneimino, methylenemethylimino, thioamidates, linkages with riboacetyl groups, aminoethylglycines, silyl or siloxane linkages, saturated or unsaturated and / or substituted and / or containing heteroatoms, e.g., alkyl or cycloalkyl linkages with or without heteroatoms, of 1 to 10 carbons, morpholino structures, amides, polyamides, in which the bases are directly or indirectly joined to the aza nitrogen backbone, and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides that contain phosphorothioate linkages. Mesyl phosphoramidate antisense oligonucleotides (MsPA ASOs) are antisense oligonucleotides that contain a mesyl phosphoramidate linkage.

[0120] In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate modification, a mesyl phosphoramidate modification, or a thiolphosphonate modification. In some cases, the modified nucleotide includes, but is not limited to, a 2'-fluoro-N3-P5'-phosphoramidite.

[0121] In some cases, modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)).

[0122] In some embodiments, the one or more modifications may further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications with nucleotide analogs at the 3'-end or 5'-end. For example, the 3'-end may include a 3' cationic group, or an inversion of the nucleoside at the 3'-end via a 3'-3' linkage. In another alternative, the 3'-end may be conjugated with an amino alkyl group, such as a 3'-C5-amino alkyl dT. In a further alternative, the 3'-end may be conjugated with an abasic site, such as an apurinic or apyrimidinic site. In some cases, the 5'-end may be conjugated with an amino alkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5'-end may be conjugated with an abasic site, such as an apurinic or apyrimidinic site.

[0123] In some embodiments, oligonucleotide molecules comprise one or more of the synthetic nucleotide analogs described herein.In some cases, oligonucleotide molecules comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more of the synthetic nucleotide analogs described herein. In some embodiments, synthetic nucleotide analogs include LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro-N3-P5'-phosphoramidites, or combinations thereof, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some cases, the oligonucleotide molecule may be 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-methylacetonitrile. The nucleic acid sequence may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, twenty, twenty-five or more synthetic nucleotide analogs selected from amide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro-N3-P5'-phosphoramidites, or combinations thereof.In some cases, an oligonucleotide molecule comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, twenty, twenty-five, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some cases, an 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.

[0124] 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.

[0125] 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.

[0126] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications.

[0127] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications.

[0128] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications.

[0129] In some cases, the oligonucleotide molecule comprises at least one of about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications.

[0130] In some cases, the oligonucleotide molecules contain at least one of about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0131] In some cases, the oligonucleotide molecules contain at least one of about 10% to about 30% modifications and about 20% to about 30% modifications.

[0132] In some cases, the oligonucleotide molecules contain about 10% to about 20% modifications.

[0133] In some cases, the oligonucleotide molecules contain from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

[0134] In further cases, the oligonucleotide molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0135] In some embodiments, the oligonucleotide molecule comprises at least about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, about ten, about eleven, 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.

[0136] 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.

[0137] In some cases, about 5 to about 100% of the 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 the oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 5% of the oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 10% of the oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 15% of the oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 20% of the oligonucleotide molecules include synthetic nucleotide analogs as described herein. In some cases, about 25% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 30% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 35% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 40% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 45% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 50% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 55% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 60% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 65% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 70% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein.In some cases, about 75% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 80% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 85% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 90% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 95% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 96% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 97% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 98% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 99% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some cases, about 100% of the oligonucleotide molecules contain synthetic nucleotide analogs as described herein. In some embodiments, synthetic nucleotide analogs include LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro-N3-P5'-phosphoramidites, or combinations thereof, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0138] In some embodiments, the oligonucleotide molecule comprises from about 1 to about 25 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 1 modification, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 2 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 3 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 4 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 5 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 6 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 7 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 8 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 9 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 10 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 11 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 12 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 13 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 14 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 15 modifications, including synthetic nucleotide analogs, as described herein.In some embodiments, the oligonucleotide molecule comprises about 16 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 17 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 18 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 19 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 20 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 21 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 22 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 23 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 24 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 25 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 26 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 27 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 28 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 29 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 30 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 31 modifications, including synthetic nucleotide analogs, as described herein.In some embodiments, the oligonucleotide molecule comprises about 32 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 33 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 34 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 35 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 36 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 37 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 38 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 39 modifications, including synthetic nucleotide analogs, as described herein. In some embodiments, the oligonucleotide molecule comprises about 40 modifications, including synthetic nucleotide analogs, as described herein.

[0139] In some embodiments, oligonucleotide molecule is assembled from two separate polynucleotides, one polynucleotide comprises the sense strand of oligonucleotide molecule, and the second polynucleotide comprises the antisense strand.In other embodiments, the sense strand is connected to the antisense strand via a linker molecule, which is optionally a polynucleotide linker or a non-nucleotide linker.

[0140] 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 the purine nucleotides present in the sense strand comprise 2'-deoxy purine nucleotides.

[0141] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense strand are 2'-O-methyl purine nucleotides.

[0142] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and wherein the purine nucleotides present in the antisense strand comprise 2'-deoxy purine nucleotides.

[0143] 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, the 2'-O-methyl or 2'-deoxy-2'-fluoro modified consecutive nucleotides are located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, the 2'-O-methyl or 2'-deoxy-2'-fluoro modified consecutive nucleotides are located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the sense strand of the oligonucleotide molecule comprises at least four, at least five, at least six, 2'-O-methyl modified consecutive nucleotides at its 5'-end and / or 3'-end, or both. Optionally, in such embodiments, the sense strand of the oligonucleotide molecule comprises at least one, at least two, at least three, at least four, 2'-deoxy-2'-fluoro modified nucleotides at the 3'-end of at least four, at least five, at least six, 2'-O-methyl modified consecutive nucleotides at the 5'-end of the polynucleotide, or at least four, at least five, at least six, 2'-O-methyl modified consecutive nucleotides at the 3'-end of the polynucleotide. Also optionally, such at least two, at least three, at least four, 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides.

[0144] 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.

[0145] 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 2'-deoxy-2'-fluoro modified consecutive nucleotides located at the 5'-end, while the other strand has at least two 2'-O-methyl modified consecutive nucleotides located at the 5'-end. In some embodiments, where the strand has at least two 2'-deoxy-2'-fluoro modified consecutive nucleotides located at the 5'-end, the strand also comprises at least two, at least three 2'-O-methyl modified consecutive nucleotides at the 3'-end of the at least two 2'-deoxy-2'-fluoro modified consecutive 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 2'-O-methyl modified consecutive nucleotides linked to 2'-deoxy-2'-fluoro modified nucleotides at its 5'-end and / or 3'-end. In some embodiments, one of the sense and antisense strands has at least four, at least five nucleotides that have alternating 2'-O-methyl and 2'-deoxy-2'-fluoro modified nucleotides.

[0146] In some embodiments, an oligonucleotide molecule, such as a siRNA, has Formula I: N1N2N3N4N5N6N7N8N9N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 : Sense strand (SS) 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) wherein each nucleotide, represented by N, is independently A, U, C, or G, or a modified nucleotide base, such as a modified nucleotide base provided herein. Formula I has the formula as exemplified in Formula I. The nucleotide N1 of sense strand and antisense strand represents the 5' end of each strand.For clarity, Formula I utilizes N1, N2, N3, etc. in both sense strand and antisense strand, but the nucleotide bases do not need to be, and are not intended to be, the same in both sense strand and antisense strand.The siRNA exemplified in Formula I will be complementary to target sequence.

[0147] For example, in some embodiments, the sense strand comprises 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones at N1 and N2, N3, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23, N24, N25, N26, N27, N28, N29, N30, N31, N32, N33, N34, N35, N36, N37, N38, N39, N40, N41, N42, N43, N44, N45, N46, N47, N48, N49, N50, N51, N52, N53, N54, N55, N56, N57, N58, N59, N60 12 , and N 17 2'-fluoro modified nucleotides, as well as N4, N5, N6, N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N18 , and N 19 The 2'O-methyl modified nucleotides are

[0148] In some embodiments, the antisense strand comprises a vinylphosphonate moiety conjugated to N1, a 2'-fluoro modified nucleotide with a phosphorothioate (PS) modified backbone at N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23, 10 , N 11 , N 12 , N 13 , N 15 , N 16 , N 17 , N 18 , and N 19 2'O-methyl modified nucleotide, N 14 2'-fluoro modified nucleotides, as well as N 20 and N 21 The nucleotide sequence of the present invention comprises 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones.

[0149] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, where the sense strand comprises a terminal cap moiety at the 5'-terminus, the 3'-terminus, or both the 5'-terminus and the 3'-terminus of the sense strand, hi other embodiments, the terminal cap moiety is an inverted deoxy abasic moiety.

[0150] In some embodiments, an oligonucleotide molecule comprises a sense strand and an antisense strand, where the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

[0151] In some embodiments, the oligonucleotide molecule is an oligonucleotide molecule in which the sense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, or at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand. 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 modified nucleotides, 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, a terminal cap molecule; The strand has from about 1 to about 10, or more specifically, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or one or more nucleotides at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand. comprises sense and antisense strands that include 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 modified nucleotides, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally, a terminal cap molecule.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or terminal cap molecules, either within the same strand or within different strands.

[0152] In some embodiments, the oligonucleotide molecule is an oligonucleotide molecule in which the sense strand has from about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and 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 modified nucleotides, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally, a terminal cap molecule; In some embodiments, the antisense strand has from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or one or more thereof, at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand. The sense and antisense strands include 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 modified nucleotides, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally, terminal cap molecules.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are 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, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 16, 17, 18, 19, 20 or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without terminal capping molecules, within the same strand or in different strands.

[0153] In some embodiments, the sense strand is nucleotide-linked with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioates, phosphorodithioates, phosphonates, phosphoramidates, or mesylphosphoramidates at the 3' end, the 5' end, or both the 3' and 5' ends of the sense strand. inter-nucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro modified nucleotides, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides. In some embodiments, the antisense strand has about 1 to about 10, or more specifically, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or none at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the antisense strand. 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 modified nucleotides, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally, an end cap molecule.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more pyrimidine nucleotides of the sense and / or antisense strand are chemically modified at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or terminal cap molecules, either within the same strand or within different strands.

[0154] In some embodiments, the oligonucleotide molecule is an oligonucleotide molecule in which the sense strand is substituted with about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesyl phosphoramidate nucleotides at the 3' end, the 5' end, or both the 3' and 5' ends of the sense strand. inter-base 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; The antisense strand can have from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesylphosphoramidate internucleotide linkages, and / or one or more, at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand. The sense and antisense strands include 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 modified nucleotides, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally, terminal cap molecules.In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus with about 1 to about 5, e.g., about 1, 2, 3, 4, 5, or more, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesyl phosphoramidate internucleotide linkages and / or terminal cap molecules, either within the same strand or within different strands.

[0155] In some embodiments, the oligonucleotide molecules described herein are chemically modified short interfering nucleic acid molecules having 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 phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, or mesyl phosphoramidate internucleotide linkages in each strand of the oligonucleotide molecule. In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and the antisense strand comprises a phosphate backbone modification at the 3' end of the antisense strand. Alternatively and / or in addition, the oligonucleotide molecule comprises a sense strand and an antisense strand, and the sense strand comprises a phosphate backbone modification at the 5' end of the antisense strand. In some cases, the phosphate backbone modification is phosphorothioate. In some cases, the phosphate backbone modification is phosphorodithioate. In some cases, the phosphate backbone modification is phosphonate. In some cases, the phosphate backbone modification is phosphoramidate. In some cases, the phosphate backbone modification is mesyl phosphoramidate. In some embodiments, the sense strand or antisense strand has three consecutive nucleosides coupled via two phosphorothioate backbones. In some embodiments, the sense strand or antisense strand has three consecutive nucleosides coupled via two phosphorodithioate backbones. In some embodiments, the sense strand or antisense strand has three consecutive nucleosides coupled via two phosphonate backbones. In some embodiments, the sense strand or antisense strand has three consecutive nucleosides coupled via two phosphoramidate backbones. In some embodiments, the sense strand or antisense strand has three consecutive nucleosides coupled via two mesyl phosphoramidate backbones.

[0156] In another embodiment, the oligonucleotide molecule described herein comprises 2'-5' internucleotide linkage.In some cases, the 2'-5' internucleotide linkage is at the 3' end, 5' end, or both 3' end and 5' end of one or both sequence strands.In further cases, the 2'-5' internucleotide linkage is present at other various positions in one or both sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more internucleotide linkages comprise 2'-5' internucleotide linkages, including any internucleotide linkages of pyrimidine nucleotides in one or both strands of the oligonucleotide molecule, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more internucleotide linkages comprise 2'-5' internucleotide linkages, including any internucleotide linkages of purine nucleotides in one or both strands of the oligonucleotide molecule.

[0157] In some embodiments, the oligonucleotide molecule is a single-stranded molecule that mediates RNAi activity in a cell or in a reconstituted in vitro system, where the oligonucleotide molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, where one or more pyrimidine nucleotides present in the oligonucleotide molecule are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., where all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, where a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and any purine nucleotides present in the oligonucleotide molecule are 2'-deoxy purine nucleotides (e.g., where all purine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides). the plurality of purine nucleotides are 2'-deoxypurine nucleotides, or alternatively, the plurality of purine nucleotides are 2'-deoxypurine nucleotides), the terminal cap modification is optionally present at the 3' end, the 5' end, or both the 3' and 5' ends of the antisense sequence, the oligonucleotide molecule optionally further comprises from about one to about four (e.g., about one, two, three, or four) terminal 2'-deoxynucleotides at the 3' end of the oligonucleotide molecule, the terminal nucleotides further comprising one or more (e.g., about one, two, three, or four) phosphorothioate or mesyl phosphoramidate internucleotide linkages, and the oligonucleotide molecule optionally further comprises a terminal phosphate group, such as a 5' terminal phosphate group.

[0158] In some cases, one or more of the synthetic nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonuclease and 3'-5' exonuclease, when compared to naturally occurring polynucleic acid molecules and endonucleases. In some cases, the 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) Synthetic nucleotide analogs, including modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro-N3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some cases, the 2'-O-methyl modified oligonucleotide molecules are nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, 2'O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, 2'-O-aminopropyl modified oligonucleotide molecules are nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant).In some cases, 2'-deoxy modified oligonucleotide molecules are nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules are nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules are nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some cases, LNA modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some cases, ENA modified oligonucleotide molecules are nuclease-resistant (e.g., RNase H-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant).In some cases, the HNA modified oligonucleotide molecule is nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, the morpholino is nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, the PNA modified oligonucleotide molecule is resistant to nucleases (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, the methylphosphonate nucleotide modified oligonucleotide molecule is nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, the thiol phosphonate nucleotide modified oligonucleotide molecule is nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, the oligonucleotide molecule comprising 2'-fluoro-N3-P5'-phosphoramidite is nuclease resistant (e.g., RNase H resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some cases, the 5' side conjugate described herein inhibits 5'-3' exonuclease cleavage. In some cases, the 3' side conjugate described herein inhibits 3'-5' exonuclease cleavage.

[0159] In some embodiments, one or more of the synthetic nucleotide analogues described herein have increased binding affinity for their mRNA targets compared to a comparable naturally occurring polynucleic acid molecule. One or more of the synthetic nucleotide analogs, including LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA), have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some cases, 2'-O-methyl modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-deoxy modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules.In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, LNA modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, ENA modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, PNA modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, HNA modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, morpholino modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, methylphosphonate nucleotide modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, thiolphosphonate nucleotide modified oligonucleotide molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules.In some cases, oligonucleotide molecules containing 2'-fluoro-N3-P5'-phosphoramidites have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, increased affinity is exemplified by a lower Kd, an increased melting temperature (Tm), or a combination thereof.

[0160] In some embodiments, the oligonucleotide molecules described herein are chirally pure (or stereochemically pure) polynucleic acid molecules or polynucleic acid molecules that contain a single enantiomer. In some cases, the oligonucleotide molecules contain L-nucleotides. In some cases, the oligonucleotide molecules contain D-nucleotides. In some cases, the oligonucleotide molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror image enantiomer. In some cases, the oligonucleotide molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of racemic mixture.

[0161] In some embodiments, the oligonucleotide molecule described herein is further modified to include aptamer conjugate portion.In some cases, the aptamer conjugate portion is a DNA aptamer conjugate portion.In some cases, the aptamer conjugate portion is Alphamer, which includes the aptamer portion that recognizes specific cell surface target and specific cell surface portion, and presents specific epitope for binding to circulating antibody.

[0162] In further embodiments, the oligonucleotide molecule described herein is modified to increase its stability.In some embodiments, the oligonucleotide molecule is RNA (e.g., siRNA).In some cases, the oligonucleotide molecule is modified by one or more of the modifications described above to increase its stability. In some cases, the oligonucleotide molecule is modified at the 2' hydroxyl position by 2'-O-methyl modification, 2'-O-methoxyethyl (2'-O-MOE) modification, 2'-O-aminopropyl modification, 2'-deoxy modification, 2'-deoxy-2'-fluoro modification, 2'-O-aminopropyl (2'-O-AP) modification, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modification, 2'-O-dimethylaminopropyl (2'-O-DMAP) modification, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modification, or 2'-ON-methylacetamide (2'-O-NMA) modification, or by locked ribose conformation or bridged ribose conformation (e.g., LNA or ENA). In some cases, the oligonucleotide molecule is modified by 2'-O-methyl ribose and / or 2'-O-methoxyethyl ribose. In some cases, oligonucleotide molecule also comprises morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoro-N3-P5'-phosphoramidite to increase its stability.In some cases, oligonucleotide molecule is chirally pure (or stereochemically pure) oligonucleotide molecule.In some cases, chirally pure (or stereochemically pure) oligonucleotide molecule is modified to increase its stability.Those skilled in the art will know the modification of RNA that is suitable for increasing stability for delivery.

[0163] In some embodiments, the oligonucleotide molecule comprises a 2' modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 3, 7, 8, 9, 12, and 17 from the 5' end of the sense strand are not modified with a 2'O-methyl modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 3, 7, 8, 9, 12, and 17 from the 5' end of the sense strand are modified with a 2'fluoro modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 2 and 14 from the 5' end of the antisense strand are not modified with a 2'O-methyl modification. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 2 and 14 from the 5' end of the antisense strand are modified with a 2'fluoro modification. In some embodiments, any of the nucleotides may further comprise a modification with a 5'-phosphorothioate group. In some embodiments, the nucleotides of the oligonucleotide molecule at positions 1 and 2 from the 5' end of the sense strand are modified with a 5'-phosphorothioate group. 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. In some embodiments, the 5' end of the sense strand or the antisense strand of the oligonucleotide molecule can 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.

[0164] In some cases, oligonucleotide molecule is a double-stranded polynucleotide molecule that comprises self-complementary sense and antisense regions, where antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in target nucleic acid molecule or part thereof, and sense region has nucleotide sequence that corresponds to target nucleic acid sequence or part thereof.In some cases, oligonucleotide molecule is assembled from two individual polynucleotides, where one strand is sense strand and the other strand is antisense strand, and antisense strand and sense strand are self-complementary (e.g., antisense strand and sense strand form a duplex or double-stranded structure, where each strand comprises nucleotide sequence that is complementary to nucleotide sequence in other strand, such as when double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in target nucleic acid molecule or part thereof, and sense strand comprises nucleotide sequence that corresponds to target nucleic acid sequence or part thereof. Alternatively, the oligonucleotide molecule can be assembled from a single oligonucleotide, in which the self-complementary sense and antisense regions of the oligonucleotide molecule are linked by a nucleic acid based or non-nucleic acid based linker.

[0165] In some cases, oligonucleotide molecule is a polynucleotide with a secondary structure of double strand, asymmetric double strand, hairpin or asymmetric hairpin, with self-complementary sense and antisense regions, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence in individual target nucleic acid molecule or a part thereof, and sense region comprises the nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof.In other cases, oligonucleotide molecule is a circular single-stranded polynucleotide with two or more loop structures and stems, comprising self-complementary sense and antisense regions, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence in target nucleic acid molecule or a part thereof, and sense region comprises the nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof, and circular polynucleotide is processed in vivo or in vitro to produce an active oligonucleotide molecule that can mediate RNAi. In further cases, the oligonucleotide molecule also includes a single-stranded polynucleotide having a nucleotide sequence that is complementary to a nucleotide sequence within a target nucleic acid molecule or a portion thereof (e.g., where such an oligonucleotide molecule does not require the presence within the oligonucleotide molecule of a nucleotide sequence that corresponds to the target nucleic acid sequence or a portion thereof), in which case the single-stranded polynucleotide further includes a terminal phosphate group, such as a 5' phosphate, or a 5' diphosphate, a 3' diphosphate, etc.

[0166] In some cases, the asymmetric hairpin is a linear oligonucleotide molecule that includes an antisense region, a loop portion that includes nucleotides or non-nucleotides, and a sense region that includes fewer nucleotides than the antisense region, so long as the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with the loop. For example, the asymmetric hairpin oligonucleotide molecule includes an antisense region that has a length (e.g., about 19 to about 22 nucleotides) sufficient to mediate RNAi in a cell or in vitro system, a loop region that includes about 4 to about 8 nucleotides, and a sense region that is complementary to the antisense region and has about 3 to about 18 nucleotides. In some cases, the asymmetric hairpin oligonucleotide molecule also includes a chemically modified 5'-terminal phosphate group. In further cases, the loop portion of the asymmetric hairpin oligonucleotide molecule includes nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0167] In some embodiments, an asymmetric duplex is an oligonucleotide molecule having two separate strands, including a sense region and an antisense region, where the sense region contains fewer nucleotides than the antisense region, so long as the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex oligonucleotide molecule includes an antisense region having a length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 19 to about 22 nucleotides), and a sense region having about 3 to about 19 nucleotides that are complementary to the antisense region.

[0168] In some cases, universal base refers to a nucleotide base analog that forms base pairs with each of the natural DNA / RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl, and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole known in the art.

[0169] In some embodiments, the dsRNA agent is 5' phosphorylated or includes a phosphoryl analog at the 5' prime end. 5' phosphate modifications include modifications that are compatible with RISC-mediated gene silencing. Suitable modifications include 5' monophosphate ((HO2(O)PO-5'); 5' diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5' triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5' guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N O-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'; 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); phosphorodithioate [-O2PS2-]; phosphonate [-PO(OH)2-]; phosphoramidate [-O=P(OH)2-]; mesyl phosphoramidate (CH3)(SO2)(N)P(O) 2-O-5'; any further combination of monophosphates, diphosphates, and triphosphates replaced with oxygen / sulfur (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). In some embodiments, the modifications are made in the antisense strand of the dsRNA.

[0170] 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 within GYS1, where the first nucleotide of the target sequence begins at any nucleotide within the GYS1 mRNA transcript in the coding region, or within the 5' untranslated region (UTR) or 3'UTR. For example, the first nucleotide of the target sequence is selected to start at nucleic acid position (nal; a number starting from the 5' end of the full-length GYS1 mRNA, e.g., the first nucleotide at the 5' end is nal 1) 1, nal 2, nal 3, nal 4, nal 5, nal 6, nal 7, nal 8, nal 9, nal 10, nal 11, nal 12, nal 13, nal 14, nal 15, nal 15, nal 16, nal 17, or any other nucleic acid position within the coding or non-coding region (5' untranslated region or 3' untranslated region) of GYS1 mRNA. In some embodiments, the first nucleotide of the target sequence is selected from the group consisting of nal 10 to nal 15, nal 10 to nal 20, nal 50 to nal 60, nal 55 to nal 65, nal 75 to nal 85, nal 95 to nal 105, nal 135 to nal 145, nal 155 to nal 165, nal 225 to nal 235, nal 265 to nal 275, nal 275 to nal 245, nal 245 to nal 255, nal 285 to nal 335, nal 335 to nal 345, nal 385 to nal 395, nal 515 to nal 525, nal 665 to nal 675, nal 675 to nal 685, nal 695 to nal 705, nal 705 to nal 715, nal 875 to nal 885, nal 885~nal 895, nal 895~nal 905, nal 1035~nal 1045, nal 1045~nal 1055, nal 1125~nal1135, nal 1135~nal 1145, nal 1145~nal 1155, nal 1155~nal 1165, nal 1125~nal 1135, nal 1155~nal 1165, nal 1225~nal 1235, nal 1235~nal 1245, nal 1275~nal 1245, nal 1245~nal 1255, nal 1265~nal 1275, nal 1125~nal 1135, nal 1155~nal 1165, nal 1225~nal 1235, nal 1235~nal 1245, nal 1275~nal 1245, nal 1245~nal 1255, nal 1265~nal 1275, nal 1275~nal 1285, nal 1335~nal 1345, nal 1345~nal 1355, nal 1525~nal 1535, nal 1535~nal 1545, nal 1605~nal 1615, nal 1615~c.1625, nal 1625~nal 1635, nal 1635~1735, nal 1735~1835, nal 1835~1935, nal 1836~1856, nal 1935~2000, nal 2000~2100, nal 2100~2200, nal 2200~2260, nal 2260~2400, nal 2400~2500, nal 2500~2600, nal The GYS1 mRNA sequence is selected to begin at or between 2600-2700, 2700-2800, 2800-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2860, etc. In some embodiments, the sequence of GYS1 mRNA is presented as NCBI Reference Sequence: NM_002103.

[0171] In some embodiments, the antisense strand of the dsRNA is 100% complementary to the target RNA so as to hybridize therewith and inhibit its expression via RNA interference. The target RNA can be any RNA expressed in a cell. In another embodiment, the cell is a tumor cell, a liver cell, a muscle cell, an immune cell, a dendritic cell, a cardiac cell, or a central nervous system cell. In another embodiment, the antisense strand of the dsRNA is at least 99%, at least 98%, at least 97%, at least 96%, 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA. In some embodiments, the target RNA is GYS1 RNA. In some embodiments, the siRNA molecule is an siRNA that reduces the expression of GYS1. In some embodiments, the siRNA molecule is an siRNA that reduces expression of GYS and does not reduce expression of other RNAs by more than 50% in the assays described herein at a concentration of 200 nM or less, as described herein.

[0172] The siRNA is targeted to any gene or RNA (eg, mRNA) transcript of interest.

[0173] Other modifications and modification patterns can be found, for example, in US Pat. No. 10,233,448, which is incorporated herein by reference.

[0174] Other modifications and modification patterns can be found, for example, in Anderson et al., Nucleic Acids Research, 2021, 49(16), 9026-9041, which is incorporated herein by reference.

[0175] Other modifications and modification patterns can be found, for example, in PCT Publication No. WO2021 / 030778, which is incorporated by reference herein.

[0176] Other modifications and modification patterns can be found, for example, in PCT Publication No. WO2021 / 030763, which is incorporated by reference herein.

[0177] In some embodiments, siRNA is linked to a protein such as FN3 domain. siRNA is 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 linking of the sense strand to the FN3 domain.

[0178] In some embodiments, the present invention relates to a compound represented by the formula (X1): n -(X2) q -(X3) yCompositions are provided having the formula: -L-X4, where X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is a nucleic acid molecule, such as but not limited to an siRNA molecule; n, q, and y are each independently 0 or 1. In some embodiments, X1, X2, and X3 bind to different target proteins. In some embodiments, y is 0. In some embodiments, n is 1, q is 0, and y is 0. In some embodiments, n is 1, q is 1, and y is 0. In some embodiments, n is 1, q is 1, and y is 0. In some embodiments, n is 1, q is 1, and y is 1. In some embodiments, the third FN3 domain extends the half-life of the molecule as a whole compared to the molecule without X3. In some embodiments, the half-life extension moiety is an FN3 domain that binds to albumin. Examples of such FN3 domains include, but are not limited to, the FN3 domains described in US Patent Publication No. 20170348397 and US Patent No. 9,156,887, which are incorporated herein by reference in their entirety. The FN3 domain may incorporate other subunits, for example, via covalent interactions. In some embodiments, the FN3 domain further comprises a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin binding proteins and / or albumin binding domains, transferrin and fragments and analogs thereof, and Fc regions. The amino acid sequence of human Fc regions is well known and includes the Fc regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. 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 of the half-life extending moieties described herein. In some embodiments, the second FN3 domain binds to albumin, albumin variants, albumin binding proteins and / or albumin binding domains, and fragments and analogs thereof.

[0179] In some embodiments, compositions are provided herein having the formula (X1)-(X2)-L-(X4), where X1 is a first FN3 domain; X2 is a second FN3 domain; L is a linker; and X4 is a nucleic acid molecule. In some embodiments, X4 is a siRNA molecule. In some embodiments, X1 is a FN3 domain that binds to one of CD71. In some embodiments, X2 is a FN3 domain that binds to one of CD71. In some embodiments, X1 and X2 do not bind to the same target protein. In some embodiments, X1 and X2 bind to the same target protein, but at different binding sites on the protein. In some embodiments, X1 and X2 bind to the same target protein. In some embodiments, X1 and X2 are FN3 domains that bind to CD71. In some embodiments, the composition does not include (e.g., is free of) compounds or proteins that bind to ASGPR.

[0180] In some embodiments, C-(X1) n -(X2) q [L-X4]-(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; C is a polymer; and n, q, and y are each independently 0 or 1.

[0181] In some embodiments, the present invention relates to a compound represented by the formula (X1): n -(X2) q [L-X4]-(X3) y 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; C is a polymer; and n, q, and y are each independently 0 or 1.

[0182] In some embodiments, C-(X1) n -(X2) q [L-X4]L-(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; X4 is an oligonucleotide molecule; C is a polymer; and n, q, and y are each independently 0 or 1.

[0183] In some embodiments, the present invention relates to a compound represented by the formula (X1): n -(X2) q [L-X4]L-(X3) y 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; C is a polymer; and n, q, and y are each independently 0 or 1.

[0184] In some embodiments, the formula of A1-B1, wherein A1 is C-L1-X s B1 has the formula: AS -L2-F1, C is a polymer such as PEG; L1 and L2 are each independently a linker; X S is a 5'-3' oligonucleotide that is the sense strand of the double-stranded siRNA molecule; X AS is a 3'-5' oligonucleotide that is the antisense strand of the double-stranded siRNA molecule; F1 is a polypeptide containing at least one FN3 domain; X S and X AS forms a double-stranded oligonucleotide molecule to form a composition / complex. A composition or complex is provided having the following structure:

[0185] In some embodiments, C can be a molecule that extends the half-life of a molecule. Such moieties are described herein. In some embodiments, C can also be Endoporter, INF-7, TAT, polyarginine, polylysine, or an amphipathic peptide. These moieties are used instead of or in addition to other half-life extending moieties provided herein. In some embodiments, C can be a molecule that delivers the complex to a cell, endosome, or ER, said molecule being selected from the peptides listed in Table 9:

[0186] [Table 2]

[0187] In some embodiments, the formula of A1-B1, wherein A1 is X s B1 has the formula: AS -L2-F1].

[0188] In some embodiments, the formula of A1-B1, wherein A1 is C-L1-X s B1 has the formula: AS The composition or complex has the formula:

[0189] In some embodiments, the sense strand is a sense strand presented herein.

[0190] In some embodiments, the antisense strand is an antisense strand presented herein.

[0191] In some embodiments, the sense strand and the antisense strand form a double-stranded siRNA molecule that targets GYS1. In some embodiments, the double-stranded oligonucleotide is about 21-23 nucleotide base pairs in length. In certain embodiments, C is optional.

[0192] In some embodiments, the formula of A1-B1, wherein A1 is F1-L1-X s B1 has the formula: AS -L2-C, F1 is a polypeptide containing at least one FN3 domain; L1 and L2 are each independently a linker; C is a polymer such as PEG; X S is a 5'-3' oligonucleotide that is the sense strand of the double-stranded siRNA molecule; X AS is a 3'-5' oligonucleotide that is the antisense strand of the double-stranded siRNA molecule; X S and X AS forms a double-stranded oligonucleotide molecule to form a composition / complex. In certain embodiments, C is optional. In some embodiments, the formula of A1-B1, wherein A1 is X s B1 has the formula: AS -L2-C]. In some embodiments, the formula of A1-B1, wherein A1 is F1-L1-X s B1 has the formula: AS The composition or complex has the formula:

[0193] In some embodiments, C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of monomers in two or three dimensions. In some cases, the polymer comprises a polysaccharide, lignin, rubber, or a polyalkylene oxide, which may be, for example, polyethylene glycol. In some cases, the at least one polymer includes, but is not limited to, alpha-dihydroxyl polyethylene glycol, omega-dihydroxyl 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), polytetramethylene glycol (PTG), or polyurethane, as well as mixtures thereof. Mixtures as used herein refer to the use of different polymers in the same compound, as well as block copolymers. In some cases, a block copolymer is a polymer in which at least one segment of the polymer is constructed from a monomer of another polymer. In some cases, the polymer comprises a polyalkylene oxide. In some cases, the polymer comprises PEG. In some cases, the polymer comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0194] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, polydisperse materials include a bidisperse distribution of materials with different molecular weights, characterized by size by average weight (weight average) and bidispersity. In some cases, monodisperse PEG includes molecules of one size. In some embodiments, C is a polydispersed polyalkylene oxide (e.g., PEG) or a monodispersed polyalkylene oxide (e.g., PEG), and the listed molecular weight represents the average molecular weight of the polyalkylene oxide molecules, e.g., PEG molecules.

[0195] 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, 270 0, 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.

[0196] 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, having a molecular weight of 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 290 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 800 Da. In some cases, the molecular weight of C is about 900 Da. In some cases, the molecular weight of C is about 1000 Da. In some cases, the molecular weight of C is about 1100 Da. In some cases, the molecular weight of C is about 1200 Da. In some cases, the molecular weight of C is about 1300 Da. In some cases, the molecular weight of C is about 1400 Da. In some cases, the molecular weight of C is about 1450 Da.In some cases, the molecular weight of C is about 1500 Da. In some cases, the molecular weight of C is about 1600 Da. In some cases, the molecular weight of C is about 1700 Da. In some cases, the molecular weight of C is about 1800 Da. In some cases, the molecular weight of C is about 1900 Da. In some cases, the molecular weight of C is about 2000 Da. In some cases, the molecular weight of C is about 2100 Da. In some cases, the molecular weight of C is about 2200 Da. In some cases, the molecular weight of C is about 2300 Da. In some cases, the molecular weight of C is about 2400 Da. In some cases, the molecular weight of C is about 2500 Da. In some cases, the molecular weight of C is about 2600 Da. In some cases, the molecular weight of C is about 2700 Da. In some cases, the molecular weight of C is about 2800 Da. In some cases, the molecular weight of C is about 2900 Da. In some cases, the molecular weight of C is about 3000 Da. In some cases, the molecular weight of C is about 3250 Da. In some cases, the molecular weight of C is about 3350 Da. In some cases, the molecular weight of C is about 3500 Da. In some cases, the molecular weight of C is about 3750 Da. In some cases, the molecular weight of C is about 4000 Da. In some cases, the molecular weight of C is about 4250 Da. In some cases, the molecular weight of C is about 4500 Da. In some cases, the molecular weight of C is about 4600 Da. In some cases, the molecular weight of C is about 4750 Da. In some cases, the molecular weight of C is about 5000 Da. In some cases, the molecular weight of C is about 5500 Da. In some cases, the molecular weight of C is about 6000 Da. In some cases, the molecular weight of C is about 6500 Da. In some cases, the molecular weight of C is about 7000 Da. In some cases, the molecular weight of C is about 7500 Da. In some cases, the molecular weight of C is about 8000 Da. In some cases, the molecular weight of C is about 10,000 Da. In some cases, the molecular weight of C is about 12,000 Da. In some cases, the molecular weight of C is about 20,000 Da. In some cases, the molecular weight of C is about 35,000 Da. In some cases, the molecular weight of C is about 40,000 Da. In some cases, the molecular weight of C is about 50,000 Da. In some cases, the molecular weight of C is about 60,000 Da.In some cases, the molecular weight of C is about 100,000 Da.

[0197] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, which is a polymeric PEG that contains more than one repeating ethylene oxide unit. In some cases, the discrete PEG (dPEG) contains 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some cases, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some cases, the dPEG contains about 2 or more repeating ethylene oxide units. In some cases, the dPEG contains about 3 or more repeating ethylene oxide units. In some cases, the dPEG contains about 4 or more repeating ethylene oxide units. In some cases, 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, the dPEG contains about 17 or more repeating ethylene oxide units. In some cases, the dPEG contains about 18 or more repeating ethylene oxide units.In some cases, 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 stepwise from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some cases, dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is dPEG manufactured by Quanta Biodesign, LMD.

[0198] In some embodiments, L1 is a polymer that is linked to C and to a sense strand, X S or the polypeptide of F1 is linked to the sense strand, X S In some embodiments, L1 is an optional linker used to link [ka] [wherein X S , X AS , and F1 is as defined above.

[0199] 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 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-cleaved peptide, such as, but not limited to, Val-Cit, Val-Ala.

[0200] In some embodiments, L2 binds the polypeptide of F1 to the antisense strand, X AS or linking the polymer C to the antisense strand X AS is an optional linker used to link

[0201] In some embodiments, L2 is [ka] [wherein X AS and F1 is as defined above.

[0202] 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 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-cleaved peptide, such as, but not limited to, Val-Cit, Val-Ala.

[0203] In some embodiments, the linker is: [ka] In some embodiments, A1-B1 is covalently attached to F1 via a cysteine ​​residue present on F1, exemplified as follows: [ka] wherein C is a polymer such as PEG, Endoporter, INF-7, TAT, polyarginine, polylysine, an amphipathic peptide, or a peptide listed in Table 9 provided herein; and X S is a 5'-3' oligonucleotide that is the sense strand of the double-stranded siRNA molecule; X AS is a 3'-5' oligonucleotide that is the antisense strand of a double-stranded siRNA molecule; F1 is a polypeptide containing at least one FN3 domain; X S and X AS form a double-stranded siRNA molecule. Sense and antisense strands are represented by "N" notation, where each nucleotide represented by N is independently A, U, C, or G, or modified nucleobases, such as modified nucleobases presented herein. Sense and antisense strand nucleotide N1 represents the 5' end of each strand. For clarity, Formula I utilizes N1, N2, N3, etc., in both sense and antisense strands, but the nucleotide bases do not need to be, and are not intended to be, the same. The siRNA exemplified by Formula I will be complementary to target sequence.

[0204] For example, in some embodiments, the sense strand comprises 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones at N1 and N2, N3, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23, N24, N25, N26, N27, N28, N29, N30, N31, N32, N33, N34, N35, N36, N37, N38, N39, N40, N41, N42, N43, N44, N45, N46, N47, N48, N49, N50, N51, N52, N53, N54, N55, N56, N57, N58, N59, N60 12 , and N 17 2'-fluoro modified nucleotides, as well as N4, N5, N6, N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 , and N 19 The 2'O-methyl modified nucleotides are

[0205] In some embodiments, the antisense strand comprises a vinylphosphonate moiety conjugated to N1, a 2'-fluoro modified nucleotide with a phosphorothioate (PS) modified backbone at N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23, 10 , N 11 , N 12 , N 13 , N 15 , N 16 , N 17 , N 18 , and N 19 2'O-methyl modified nucleotide, N 14 2'-fluoro modified nucleotides, as well as N 20 and N 21 The nucleotide sequence of the present invention comprises 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones.

[0206] In some embodiments herein, [ka] wherein F1 is a polypeptide comprising at least one FN3 domain and is conjugated to a linker; L1, L1 is a group represented by the formula: S Connected to X S is the 5'-3' oligonucleotide which is the sense strand of the double-stranded siRNA molecule, and X AS is a 3'-5' oligonucleotide that is the antisense strand of the double-stranded siRNA molecule; X S and X AS form double-stranded siRNA molecules]. The linkers exemplified above are non-limiting examples and other types of linkers may also be used.

[0207] In some embodiments, F1 is (X1) n -(X2) q -(X3) ywherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension; and n, q, and y are each independently 0 or 1, with the proviso that at least one of n, q, and y is 1. In some embodiments, n, q, and y are each 1. In some embodiments, n and q are 1 and y is 0. In some embodiments, n and y are 1 and q is 0.

[0208] In some embodiments, X1 is a CD71 binding domain of FN3, such as the CD71 binding domain of FN3 provided herein. In some embodiments, X2 is a CD71 binding domain of FN3. In some embodiments, X1 and X2 are different CD71 binding domains of FN3. In some embodiments, the binding domains are the same binding domain. In some embodiments, X3 is an FN3 domain that binds human serum albumin. In some embodiments, X3 is an Fc domain without effector functions that extend 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 an albumin binding domain of FN3. Examples of such polypeptides are provided herein and below. In some embodiments, C-(X1) n -(X2) q -(X3) y -L-X4, where C is a polymer such as PEG, Endoporter, INF-7, TAT, polyarginine, polylysine, an amphipathic peptide, or a peptide provided in Table 9; 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; n, q, and y are each independently 0 or 1.

[0209] In some embodiments, the present invention relates to a compound represented by the formula (X1): n-(X2) q -(X3) y X is a nucleic acid molecule; and C is a polymer; n, q, and y are each independently 0 or 1.

[0210] In some embodiments, X-L-(X) n -(X2) q -(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extension molecule; L is a linker; and X4 is a nucleic acid molecule; n, q, and y are each independently 0 or 1.

[0211] In some embodiments, C-X-L-(X1) n -(X2) q -(X3) y 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; n, q, and y are each independently 0 or 1.

[0212] In some embodiments, X-L-(X) n -(X2) q -(X3) y 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; n, q, and y are each independently 0 or 1.

[0213] In some embodiments, the GYS1 siRNA pair may have the following sequences: sense strand (5'-3') nsnsnnnnNfNfNfnnnnnnnnsnsa, and antisense strand (5'-3') UfsNfsnnnNfnnnnnnNfnNfnnnsusu, where (n) is 2'-O-Me (methyl), (Nf) is 2'-F (fluoro), and (s) is a phosphorothioate backbone modification. Each nucleotide is independently or in combination modified at the ribosugar position and the nucleobase position in both the sense and antisense strands.

[0214] In some embodiments, the siRNA molecule comprises a sequence pair from Table 1A or 1B.

[0215] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0216] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0217] In some embodiments, the polynucleotides exemplified above include polynucleotides that do not contain 2'-O-methylvinylphosphonate uridine as the 5' nucleotide on the antisense strand of the siRNA.

[0218] In some embodiments, the polynucleotide is as presented herein. In some embodiments, the polynucleotide comprises a first strand and a second strand for the portion that comprises a double strand. In some embodiments, the polynucleotide comprises a sense strand and an antisense strand. In some embodiments, the polynucleotide comprises the sequence illustrated in Table 1A or 1B. In some embodiments, the polynucleotide comprises the sequence illustrated in Table 1A or 1B, but without base modification. In some embodiments, the pharmaceutical composition comprises the siRNA pair presented herein. In some embodiments, the siRNA pair is not conjugated to FN3 domain.

[0219] In some embodiments, the oligonucleotide molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art.For example, oligonucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the oligonucleotide molecule and target nucleic acid.Alternatively, oligonucleotide molecules are biologically produced in antisense orientation (i.e., the RNA transcribed from the inserted oligonucleotide molecule has antisense orientation so that it targets the polynucleic acid molecule of interest) using expression vectors into which the oligonucleotide molecules are subcloned.

[0220] In some embodiments, the oligonucleotide molecules are synthesized via a tandem synthesis method in which both strands are synthesized as a single contiguous oligonucleotide fragment, or strand separated by a cleavable linker, and then cleaved to provide separate fragments or strands that hybridize and allow purification of the duplex.

[0221] In some cases, oligonucleotide molecules are also assembled from two distinct nucleic acid strands or fragments, where one fragment comprises the sense region and the second fragment comprises the antisense region of the molecule.

[0222] In some cases, when the chemical modification of the internucleotide linkage of oligonucleotide molecules is performed by phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate or mesyl phosphoramidate, the linkage improves stability.Excessive modification may cause toxicity or reduced activity.Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages may be minimized.In such cases, reducing the concentration of these linkages may reduce the toxicity of these molecules, increase their efficacy, and increase their specificity.

[0223] As described herein, in some embodiments, the nucleic acid molecule is modified to include a linker at the 5' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule is modified to include vinylphosphonic acid or modified vinylphosphonic acid at the 5' end of the antisense strand of the dsRNA. In some embodiments, the nucleic acid molecule is modified to include a linker at the 3' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule is modified to include vinylphosphonic acid at the 3' end of the antisense strand of the dsRNA. The linker is used to link the dsRNA to the FN3 domain. The linker can be covalently attached to a cysteine ​​residue on the FN3 domain, for example, naturally occurring or substituted as described herein, for example, in U.S. Patent No. 10,196,446, which is incorporated herein by reference in its entirety. Non-limiting examples of such modified strands of dsRNA are illustrated in Table 2.

[0224] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0225] In some embodiments, the siRNA pair of A-PPPP provided above comprises a linker at the 3'-end of the sense strand. In some embodiments, the siRNA pair of A-PPPP provided above comprises vinylphosphonic acid at the 5'-end of the sense strand.

[0226] The structure of the linker (L) is as shown in Table 3 below.

[0227] [Table 6]

[0228] Other linkers may also be used, such as linkers formed by click chemistry reactions, amide coupling, reductive amination, oxime coupling, enzymatic coupling such as transglutaminase conjugation and conjugation with sortase, etc. The linkers presented herein are exemplary in nature, and other linkers made by other such methods may also be used.

[0229] When attached to an siRNA, the structure L-(X4) is represented by the following formula: [ka]

[0230] Although certain siRNA sequences with certain modified nucleobases are exemplified herein, sequences without such modifications are also presented herein. That is, sequences can include sequences exemplified in the table presented herein without modifications. In some embodiments, unmodified siRNA sequences can still include a linker at the 5' end of the sense strand of dsRNA. In some embodiments, nucleic acid molecules are modified to include vinylphosphonic acid at the 5' end of the antisense strand of dsRNA. In some embodiments, nucleic acid molecules are modified to include a linker at the 3' end of the sense strand of dsRNA. In some embodiments, nucleic acid molecules are modified to include vinylphosphonic acid at the 3' end of the antisense strand of dsRNA. Linkers can be as presented herein.

[0231] In some embodiments, an FN3 protein is provided that comprises a polypeptide that binds to CD71. In some embodiments, the polypeptide comprises an FN3 domain that binds to CD71. In some embodiments, a polypeptide is provided that comprises a sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710. 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.

[0232] In some embodiments, the FN3 domain that binds CD71 is based on the Tencon sequence of SEQ ID NO: 1, or the Tencon 27 sequence of SEQ ID NO: 4 (LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT), optionally with substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponds to SEQ ID NO: 4).

[0233] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0234] 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 MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX12 X 13 VKGGX 14 X 15 SX 16 PLX 17 AX 18 FTT [In the formula, X8, X9, X 17 , and X 18 are each independently any amino acid other than methionine or proline; X1 is selected from D, F, Y, or H; X2 is selected from Y, G, A, or V; X3 is selected from I, T, L, A, or H; X4 is selected from S, Y, or P; X5 is selected from Y, G, Q, or R; X6 is selected from G or P; X7 is selected from A, Y, P, D, or S; X 10 is selected from W, N, S, or E; X 11 is selected from L, Y, or G; X 12 is selected from D, Q, H, or V; X 13 is selected from G or S; X 14 is selected from R, G, F, L, or D; X 15 is selected from W, S, P, or L; X 16 is selected from T, V, M, or S] It is.

[0235] In some embodiments: X1 is selected from D, F, Y, or H; X2 is selected from G, A, or V; X3 is selected from T, L, A, or H; X4 is selected from Y or P; X5 is selected from G, Q, or R; X6 is selected from G or P; X7 is selected from Y, P, D, or S; X 10 is selected from W, N, S, or E; X 11 is selected from L, Y, or G; X 12 is selected from Q, H, or V; X 13 is selected from G or S; X 14 is selected from G, F, L, or D; X 15 is selected from S, P, or L; X 16 is selected from V, M, or S.

[0236] In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 are as shown in the sequence of SEQ ID NO: 288. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 are as shown in the sequence of SEQ ID NO: 289. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 are as shown in the sequence of SEQ ID NO: 290. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X15 , and X 16 is as shown in the sequence of SEQ ID NO:291.

[0237] In some embodiments, X8, X9, X 17 , and X 18 are independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 17 , and X 18 are not independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. 17 , and X 18 are independently alanine. In some embodiments, X, X, 17 , and X 18 are independently arginine. In some embodiments, X, X, X 17 , and X 18 are independently asparagine. In some embodiments, X, X, X 17 , and X 18 are independently aspartic acid. 17 , and X 18 are independently cysteine. In some embodiments, X, X, 17 , and X 18 are independently glutamine. In some embodiments, X, X, 17 , and X 18 are independently glutamic acid. 17 , and X 18 are independently glycine. In some embodiments, X, X, X 17 , and X 18are independently histidine. In some embodiments, X, X, X 17 , and X 18 are independently isoleucine. In some embodiments, X, X, 17 , and X 18 are independently leucine. In some embodiments, X, X, 17 , and X 18 are independently lysine. In some embodiments, X, X, 17 , and X 18 are independently phenylalanine. In some embodiments, X, X, X 17 , and X 18 are independently serine. In some embodiments, X, X, 17 , and X 18 are independently threonine. In some embodiments, X, X, 17 , and X 18 are independently tryptophan. In some embodiments, X, X, X 17 , and X 18 are independently tyrosine. In some embodiments, X, X, X 17 , and X 18 are independently valine.

[0238] In some embodiments, the sequence is X8, X9, X 17 , and X 18 In some embodiments, X8 is not V, X9 is not T, and X is not V, except that the position corresponding to the position of 17 But it's not S, it's X. 18 is designated as shown in sequence SEQ ID NO:288, except that it is not I.

[0239] In some embodiments, the sequence is X8, X9, X 17 , and X 18 In some embodiments, X8 is not V, X9 is not T, and X is not V, except that the position corresponding to the position of17 But it's not S, it's X. 18 is designated as shown in sequence SEQ ID NO:289, except that it is not I.

[0240] In some embodiments, the sequence is X8, X9, X 17 , and X 18 In some embodiments, X8 is not V, X9 is not T, and X is not V, except that the position corresponding to the position of 17 But it's not S, it's X. 18 is designated as shown in sequence SEQ ID NO:290, except that it is not I.

[0241] In some embodiments, the sequence is X8, X9, X 17 , and X 18 In some embodiments, X8 is not V, X9 is not T, and X is not V, except that the position corresponding to the position of 17 But it's not S, it's X. 18 is designated as shown in sequence SEQ ID NO:291, except that it is not I.

[0242] 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.

[0243] Percent identity is determined using default parameters to align the two sequences using BlastP, available through the NCBI website.

[0244] In some embodiments, a fibronectin type III (FN3) domain is provided that binds or specifically binds to human CD71 protein (SEQ ID NO: 2 or 5). As provided herein, the FN3 domain can bind to CD71 protein. The domain can also specifically bind to CD71 protein, and is provided, even if not explicitly stated. Thus, for example, an FN3 domain that binds to CD71 would also encompass an FN3 domain protein that specifically binds to CD71. These molecules are used, for example, in therapeutic and diagnostic applications and imaging. In some embodiments, a polynucleotide encoding the FN3 domain, or its complementary nucleic acid, vector, host cell, and methods of making and using the same disclosed herein are provided.

[0245] In some embodiments, an isolated FN3 domain is provided that binds or specifically binds to CD71.

[0246] 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 a short peptide sequence described herein. For example, the linker can be a G / S linker.

[0247] In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker, such as those provided herein. Exemplary linkers include (GS)2, (SEQ ID NO: 720), (GGGS)2 (SEQ ID NO: 721), (GGGGS) 1-5 (Sequence number 1065 ), (AP) 1-20 (SEQ ID NO: 1066) (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 amino acid sequence of EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 730); APAPAPAPAP (SEQ ID NO: 731); or EAAAK (SEQ ID NO: 732).

[0248] In some embodiments, the FN3 domain is about 1×10 as determined by surface plasmon resonance or Kinexa as performed by one of skill in the art. -7 Less than M, e.g., about 1 x 10 -8 Less than M, approx. 1×10 -9 Less than M, approx. 1×10 -10 Less than M, approx. 1×10 -11 Less than M, approx. 1×10 -12 Less than M or about 1×10-13 Dissociation constant (K D ) can bind to CD71. The measured affinity of a particular FN3 domain-antigen interaction can vary when measured under different conditions (e.g., osmolality, pH). Thus, affinity and other antigen-binding parameters (e.g., K D , K on , K off ) is measured with normalized solutions of protein scaffold and antigen, as well as normalized buffers, such as those described herein.

[0249] In some embodiments, the FN3 domain may bind to CD71 in a standard solution ELISA assay at least 5-fold greater than the signal obtained for a negative control.

[0250] In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises a cysteine ​​(Cys) linked to the C-terminus of the FN3 domain. The addition of an N-terminal Met and / or a C-terminal Cys may facilitate expression and / or conjugation to extend the half-life of the molecule and provide other functions.

[0251] The FN3 domain may also contain cysteine ​​substitutions, such as those described in U.S. Pat. No. 10,196,446, which is incorporated herein by reference in its entirety. Briefly, in some embodiments, the polypeptides provided herein may contain 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:6 or SEQ ID NO:1 of U.S. Pat. No. 10,196,446, and at the equivalent position in a related FN3 domain. 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.

[0252] Cysteine ​​substitution at a position in a domain or protein comprises replacing an existing amino acid residue with a cysteine ​​residue.In some embodiments, cysteine ​​is inserted into the sequence adjacent to the position listed above, rather than being replaced.Other examples of cysteine ​​modification can be found, for example, in US Patent Publication No. 20170362301, the entirety of which is incorporated herein by reference.Sequence alignment is performed, for example, using BlastP using default parameters on the NCBI website.

[0253] In some embodiments, a cysteine ​​residue is inserted anywhere within the domain or protein.

[0254] In some embodiments, the FN3 domain that binds to CD71 is internalized into the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a detectable label or a therapeutic agent to the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a cytotoxic agent to the cell. The cytotoxic agent may act as a therapeutic agent. In some embodiments, internalization of the FN3 domain may facilitate delivery of any detectable label, therapeutic, and / or cytotoxic agent disclosed herein to the cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of an oligonucleotide to the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is a central nervous system cell. In some embodiments, the cell is a cardiac cell.

[0255] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 273, 288-291, 301-310, 312-572, 592-599, or 708-710.

[0256] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 301. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 302. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 305. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 306. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 310. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 312. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 313. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 314. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 315. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 316. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 317. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 318. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 319. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 320. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 321. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 322. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 323.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 324. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 325. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 326. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 327. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 328. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 329. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 330. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 331. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 332. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 333. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 334. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 336. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 337. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 338. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 339. In some embodiments, the isolated FN3 domain that binds 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 CD71 comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 347. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 348. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 349. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 350. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 351. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 352. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 353. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 354. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 355. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 356. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 357. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 358. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 359. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 360. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 361. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 362. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 363. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 364. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 365. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 366. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 367. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 368. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 369. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 371. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 372.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 373. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 374. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 375. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 376. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 377. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 378. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 379. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 380. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 381. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 382. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 383. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 384. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 385. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 386. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 387. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 388. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 389. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 390. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 391. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 392.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 393. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 394. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 395. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 396. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 397. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 398. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 399. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 400. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 401. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 402. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 405. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 407. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 409. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 410. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 412. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 414. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 415. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 416. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 418. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 422. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 423. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 424. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 425.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 427. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 429. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 430. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 431. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 432. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 433. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 434. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 435. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 436. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 437. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 438. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 439. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 440. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 441. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 443. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 444. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 445.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 446. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 447. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 448. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 449. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 450. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 451. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 452. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 453. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 455. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 456. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 457. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 458. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 459. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 461. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 463. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 464. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 465.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 467. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 469. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 471. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 473. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 475. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 479. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 481. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 483. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 484. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 485. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 487. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 489.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 490. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 491. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 492. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 493. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 495. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 497. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 499. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 501. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 503. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 504. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 505. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 507. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 508. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 509.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:510. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:511. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:512. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:513. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:514. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:515. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:516. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:517. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:518. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:519. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 521. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 522. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 523. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 524. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 525. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 527. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 528. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 529. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 530.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 531. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 532. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 533. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 534. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 535. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 536. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 537. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 538. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 539. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 540. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 541. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 542. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 543. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 544. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 545. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 546. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 547. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 548. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 549. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 550.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 551. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 552. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 553. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 554. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 555. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 556. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 557. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 558. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 559. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 560. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 561. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 562. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 563. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 564. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 565. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 566. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 567. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 568. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 569. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 570. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 571. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 572. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 708. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 709. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:710.

[0257] In some embodiments, the isolated FN3 domain that binds CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

[0258] 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 NOs: 273, 288-291, 301-310, 312-572, 592-599, or 708-710. Percent identity is determined using default parameters to align the two sequences using BlastP, available through the NCBI website. Sequences of FN3 domains that bind to CD71 are found, for example, in Table 4.

[0259] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11] [Table 7-12]

[0260] 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 below:

[0261] [Table 8]

[0262] 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 a short peptide sequence described herein. For example, the linker can be a G / S linker or a 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 (SEQ ID NO: 1067) , (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, other linkers may also be used. GGGGS (SEQ ID NO: 1068) Repeat or GGGGA (SEQ ID NO: 1069) The number of repeats can also be 1, 2, 3, 4, or 5. In some embodiments, the linker comprises one or more GGGGS repeats. (SEQ ID NO: 1068) Repeats and one or more GGGGA (SEQ ID NO: 1069)and repeats. In some embodiments, the linker comprises EAAAAKEAAAKEAAAKEAAAK (SEQ ID NO:729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:730); APAPAPAPAP (SEQ ID NO:731); or EAAAK (SEQ ID NO:732).

[0263] In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 592. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 593. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 594. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 595. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 596. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the amino acid sequence of SEQ ID NO: 597. In some embodiments, the FN3 domain comprising two FN3 domains connected by a linker has the 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.

[0264] In some embodiments, the FN3 domain has a molecular weight of about 1×10 as determined by surface plasmon resonance or Kinexa methods as performed by one of skill in the art, if applicable. -7 Less than M, e.g., about 1 x 10 -8 Less than M, approx. 1×10 -9 Less than M, approx. 1×10 -10 Less than M, approx. 1×10 -11 Less than M, approx. 1×10 -12Less than M or about 1×10 -13 Dissociation constant (K D ) can bind to CD71. The measured affinity of a particular FN3 domain-antigen interaction can vary when measured under different conditions (e.g., osmolality, pH). Thus, affinity and other antigen-binding parameters (e.g., K D , K on , K off ) is measured with normalized solutions of protein scaffold and antigen, as well as normalized buffers, such as those described herein.

[0265] In some embodiments, the FN3 domain may bind to its target protein in a standard solution ELISA assay at least 5-fold greater than the signal obtained for a negative control.

[0266] In some embodiments, the FN3 domain that binds or specifically binds to its target protein comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to its target protein comprises a cysteine ​​(Cys) linked to the C-terminus of the FN3 domain. The addition of the N-terminal Met and / or C-terminal Cys can facilitate expression and / or conjugation of half-life extension molecules.

[0267] FN3 domains may also contain cysteine ​​substitutions, such as those described in U.S. Patent No. 10,196,446, which is incorporated by reference in its entirety. Briefly, in some embodiments, a polypeptide comprising an FN3 domain may have an FN3 domain having residues substituted with cysteines, referred to as a cysteine ​​engineered fibronectin type III (FN3) domain. In some embodiments, the FN3 domain comprises at least one cysteine ​​substitution at a position selected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the FN3 domain based on SEQ ID NO: 1 (LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT) of U.S. Patent No. 10,196,446, which is incorporated herein by reference in its entirety, and at equivalent positions in related FN3 domains. A cysteine ​​substitution at a position within a domain or protein comprises the replacement of an existing amino acid residue with a cysteine ​​residue. Other examples of cysteine ​​modifications can be found, for example, in US Patent Publication No. 20170362301, which is incorporated by reference in its entirety. Sequence alignments are performed, for example, using BlastP on the NCBI website using default parameters.

[0268] In some embodiments, the FN3 domain that binds to the target protein is internalized into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a detection label or a therapeutic agent to the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent to the cell. The cytotoxic agent can act as a therapeutic agent. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of any detection label, therapeutic, and / or cytotoxic agent disclosed herein to the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell, a lung cell, a muscle cell, an immune cell, a dendritic cell, a CNS cell, or a cardiac cell. In some embodiments, the therapeutic agent is an siRNA molecule as provided herein. The CD71-binding FN3 domain conjugated to a detection label is used to assess the expression of CD71 on a sample, such as a tumor tissue, in vivo or in vitro. CD71-binding FN3 domains conjugated to a detectable label are used to assess CD71 expression on blood, immune, muscle, or dendritic cell samples in vivo or in vitro.

[0269] As presented herein, the different FN3 domains linked to siRNA molecules are also conjugated or linked to another FN3 domain that binds to different targets.This will allow the molecule to be multispecific (e.g., bispecific, trispecific, etc.), so that it binds to a first target and another, e.g., target.In some embodiments, the first FN3 binding domain is linked to another FN3 domain that binds to an antigen (tumor antigen) expressed by tumor cells.

[0270] In some embodiments, the FN3 domains are 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 one, two, three, or four G / S repeats. A G / S repeat unit consists of four glycines followed by a serine, e.g., GGGGS. (SEQ ID NO: 1068) Other examples of linkers are provided herein and may also be used.

[0271] In some embodiments, the linker is selected from the group consisting of (GS), (SEQ ID NO: 720), (GGGS) (SEQ ID NO: 721), (GGGGS) (SEQ ID NO: 722), (AP) 2-20 (Sequence number 1067 ), (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, other linkers may also be used. (SEQ ID NO: 1068) Repeat or GGGGA (SEQ ID NO: 1069) The number of repeats can also be 1, 2, 3, 4, or 5. In some embodiments, the linker comprises one or more GGGGS repeats. (SEQ ID NO: 1068) Repeats and one or more GGGGA (SEQ ID NO: 1069) In some embodiments, the linker comprises one or more GGGGS repeats. (SEQ ID NO: 1068) Repeat and one or more EAAAK (SEQ ID NO:732) In some embodiments, the linker comprises one or more GGGGS repeats. (SEQ ID NO: 1068) repeat and one or more "AP" repeats. In some embodiments, the linker comprises EAAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 730); APAPAPAPAP (SEQ ID NO: 731); or EAAAK (SEQ ID NO: 732).

[0272] Without being bound to any particular theory, in some embodiments, the FN3 domain linked to the nucleic acid molecule is used in the targeted delivery of therapeutic agents to cells that express one or more binding partners of the FN3 domain (e.g., tumor cells), resulting in intracellular accumulation of the nucleic acid molecule therein. This allows the siRNA molecule to properly interact with the cellular machinery to inhibit the expression of target genes, improving efficacy, and in some embodiments, avoiding the toxicity that may occur from non-targeted administration of the same siRNA molecule.

[0273] The FN3 domains described herein that bind to their specific target proteins are produced as monomers, dimers, or multimers, for example, to increase the binding valency to the target molecule, thereby increasing the binding avidity to the target molecule, or as a means to create bispecific or multispecific scaffolds that bind to two or more different target molecules simultaneously. Dimers and multimers are produced by linking monospecific, bispecific, or multispecific protein scaffolds, for example, by incorporating 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 (SEQ ID NO: 1067) , (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 amino acid sequence of EAAAKEAAAKEAAAKEAAAK (SEQ ID NO:729); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:730); APAPAPAPAP (SEQ ID NO:731); or EAAAK (SEQ ID NO:732).

[0274] The dimers and multimers are linked together in the NC direction. The use of naturally occurring and synthetic peptide linkers to connect polypeptides with newly linked fusion polypeptides is well known in the literature (Hallewell et al., J Biol Chem, 264, 5260-5268, 1989; Alfthan et al., Protein Eng., 8, 725-731, 1995; Robinson and Sauer, Biochemistry, 35, 109-116, 1996; U.S. Patent No. 5,856,456). The linkers described in this paragraph are also used to link the formulas presented herein and the domains presented above.

[0275] Half-life extension moiety In some embodiments, the FN3 domain may 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 binding protein domains, transferrin and fragments and analogs thereof, and Fc regions. The amino acid sequences of human Fc regions are well known and include the Fc regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. In some embodiments, the FN3 domain binds to albumin, albumin variants, albumin binding proteins and / or albumin binding domains, and fragments and analogs thereof, and extends the half-life of the entire molecule.

[0276] 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 an isolated albumin binding domain (protein). In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or a percentage 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 is selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, provided that the protein has a substitution corresponding to position 10 of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. , 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or percent identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NOIn some embodiments, the isolated albumin binding domain comprises an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions when compared to the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the substitutions are at a position corresponding to position 10 of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the provided FN3 domains comprise a cysteine ​​residue at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, 88, or 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, or 93 of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or at the C-terminus. Although a series of positions are listed, each position is also individually selected. In some embodiments, the cysteine ​​is at a position corresponding to 6, 53, or 88. In some embodiments, further examples of albumin binding domains are found in U.S. Patent No. 10,925,932, incorporated herein by reference.

[0277] All or part of the antibody constant region may be joined to the FN3 domain to confer antibody-like properties, particularly properties associated with the Fc region, such as Fc effector functions, such as binding to C1q, complement dependent cytotoxicity (CDC), binding to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors (e.g., B cell receptor; BCR), and may be further modified by modifying residues within the Fc that contribute to these activities (for review, see Strohl, Curr Opin Biotechnol., 20, 685-691, 2009).

[0278] For desired properties, additional moieties are also incorporated into the FN3 domain, such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, such as lauric acid, myristic acid, stearic acid, arachidic acid, behenic acid, oleic acid, arachidonic acid, octanedioic acid, tetradecanediic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides). These moieties may be direct fusions with the coding sequence of the protein scaffold, and may be produced by standard cloning and expression methods. Alternatively, well-known chemical coupling methods are used to join moieties to the recombinantly produced molecules disclosed herein.

[0279] A PEG moiety may be added to an FN3 domain, for example, by incorporating a cysteine ​​residue into the C-terminus of the molecule, or by engineering a cysteine ​​into a residue position away from the binding face of the molecule and conjugating a PEG group to the cysteine ​​using well-known methods.

[0280] The FN3 domains incorporating additional moieties are compared for functionality by several well-known assays. For example, the properties that are altered by the incorporation of Fc domains and / or Fc domain variants may be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI receptors, FcγRII receptors, FcγRIII receptors, or FcRn receptors, or may be assayed using well-known cell-based assays that measure, for example, ADCC or CDC, or assess the pharmacokinetic properties of the molecules disclosed herein in in vivo models.

[0281] The compositions presented herein are prepared by preparing FN3 proteins and nucleic acid molecules and linking them together. Techniques for linking proteins to nucleic acid molecules are known and any method may be used. For example, in some embodiments, a nucleic acid molecule is modified with a linker, such as the linkers presented herein, and then the protein is mixed with a nucleic acid molecule containing the linker to form a composition. For example, in some embodiments, an FN3 domain is conjugated to an siRNA cysteine ​​using thiol-maleimide chemistry. In some embodiments, a cysteine-containing FN3 domain can be reduced, for example, in phosphate buffered saline (or any other suitable buffer) with a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) to provide a free thiol. Then, in some embodiments, the free thiol containing FN3 domain is mixed with the siRNA duplex modified with maleimide linkage and incubated under conditions to form a linked complex. In some embodiments, the mixture is incubated at room temperature for 0-5 hours, or for about 1, 2, 3, 4, or 5 hours. The reaction is quenched, for example, with N-ethylmaleimide. In some embodiments, the conjugate is purified using affinity chromatography and ion exchange. Other methods can also be used, but this is just one non-limiting embodiment.

[0282] Methods for making FN3 proteins are known, and any method may be used to make the protein. Examples are provided in the references incorporated herein by reference.

[0283] In some embodiments, an FN3 domain that specifically binds CD71 comprises the amino acid sequence of SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, where a histidine tag is appended to the N-terminus or C-terminus of the polypeptide to facilitate purification. In some embodiments, the histidine tag (His tag) is a sequence of six histidine residues. (SEQ ID NO: 1070) In further embodiments, the His tag is connected to the FN3 domain by at least one glycine residue, or about two to about four glycine residues. Thus, after purification of the FN3 domain and cleavage of the His tag from the polypeptide, one or more glycines may be left on the N-terminus or C-terminus. In some embodiments, when the His tag is removed from the N-terminus, all glycines are removed. In some embodiments, when the His tag is removed from the C-terminus, one or more of the glycines are retained.

[0284] 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 following purification of the FN3 domain.

[0285] kit In some embodiments, a kit is provided that includes the compositions described herein.

[0286] The kits are used for therapeutic use and as diagnostic kits.

[0287] In some embodiments, the kit comprises an FN3 domain conjugated to a nucleic acid molecule.

[0288] Use of conjugated FN3 domains The compositions provided herein are used to diagnose, monitor, modulate, treat, alleviate, prevent the occurrence of, or relieve symptoms of human disease, or specific pathologies, in cells, tissues, organs, body fluids, or generally in a host.

[0289] In some embodiments, a method is provided for selectively reducing GYS1 mRNA and GYS1 protein in skeletal muscle. In certain embodiments, GYS1 mRNA and GYS1 protein are not reduced in liver and / or kidney.

[0290] In some embodiments, the reduction in GYS1 mRNA and GYS1 protein is maintained for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or more than 5 weeks after administration of a conjugate described herein.

[0291] In some embodiments, the FN3 domain may facilitate delivery to CD71 positive tissues (eg, skeletal muscle, smooth muscle) for the treatment of muscle diseases.

[0292] In some embodiments, the FN3 domain may facilitate delivery to activated lymphocytes, activated dendritic cells, or other activated immune cells for the treatment of immune diseases.

[0293] In some embodiments, the polypeptide that binds CD71 is directed to an immune cell. In some embodiments, the polypeptide that binds CD71 is directed to a dendritic cell. In some embodiments, a method of treating an autoimmune disease in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide as presented herein linked or conjugated to a therapeutic agent. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, and immune thrombocytopenic purpura.

[0294] In some embodiments, a method of treating a subject having Pompe disease (GSD2; acid alpha-glucosidase (GAA) deficiency) is provided, comprising administering to the subject a composition provided herein. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent.

[0295] In some embodiments, a method of treating glycogen storage disease in a subject in need thereof is provided, comprising administering a composition as 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, Andersen's disease (GSD4; glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7; muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1; von Gierke disease), Haas disease (GSD6; hepatic glycogen phosphorylase (PYGL) deficiency or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11; glucose transporter (GLUT2) deficiency), aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0296] In some embodiments, the present composition is used in the preparation of a pharmaceutical composition or medicament for treating cancer.In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia, myelodysplastic syndrome, gastric cancer, clear cell renal cell carcinoma, clear cell breast cancer, clear cell endometrial cancer, clear cell ovarian cancer, clear cell uterine cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, soft tissue cancer, Ewing's sarcoma, and non-small cell lung cancer.

[0297] In some embodiments, the CD71 cells are cells involved in CNS diseases, inflammatory / immune diseases, such as multiple sclerosis (MS) and infectious diseases of the brain. In some embodiments, the polypeptide that binds to CD71 is directed to the central nervous system. In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds to CD71. In some embodiments, the polypeptide is an FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide presented herein linked or conjugated to a therapeutic agent. In some embodiments, the brain tumor is selected from the group consisting of a non-malignant brain tumor, a benign brain tumor, and a malignant brain tumor. In some embodiments, the neurological condition is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Lafora's disease, Pompe's disease, adult polyglucosan body disease, stroke, spinal cord injury, ataxia, Bell's palsy, cerebral aneurysm, epilepsy, seizures, Guillain-Barre syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndromes, migraine headaches, encephalitis, sepsis, and myasthenia gravis.

[0298] In some embodiments, a method of treating a subject having cancer is provided, comprising administering to the subject a composition provided herein. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds to CD71. In some embodiments, the polypeptide is an FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide provided herein linked or conjugated to a therapeutic agent.

[0299] In some embodiments, the subject has a solid tumor.

[0300] In some embodiments, the solid tumor is a melanoma.

[0301] 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.

[0302] In some embodiments, the solid tumor is renal cell carcinoma (RCC).

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

[0304] In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).

[0305] In some embodiments, the solid tumor is colorectal cancer.

[0306] In some embodiments, the solid tumor is prostate cancer. In some embodiments, the solid tumor is castration-resistant prostate cancer.

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

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

[0309] In some embodiments, the solid tumor is gastric cancer.

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

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

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

[0313] In some embodiments, the solid tumor is head and neck squamous cell carcinoma.

[0314] In some embodiments, the solid tumor is an esophageal or gastrointestinal cancer.

[0315] In some embodiments, the solid tumor is breast cancer.

[0316] In some embodiments, the solid tumor is fallopian tube cancer.

[0317] In some embodiments, the solid tumor is brain cancer.

[0318] In some embodiments, the solid tumor is a urethral cancer.

[0319] In some embodiments, the solid tumor is a genitourinary cancer.

[0320] In some embodiments, the solid tumor is endometriosis.

[0321] In some embodiments, the solid tumor is cervical cancer.

[0322] In some embodiments, the solid tumor is a metastatic lesion of a cancer.

[0323] In some embodiments, the subject has a hematological malignancy.

[0324] In some embodiments, the hematological malignancy is lymphoma, myeloma, or leukemia. In some embodiments, the hematological malignancy is B-cell lymphoma. In some embodiments, the hematological malignancy is Burkitt's lymphoma. In some embodiments, the hematological malignancy is Hodgkin's lymphoma. In some embodiments, the hematological malignancy is non-Hodgkin's lymphoma.

[0325] In some embodiments, the hematological malignancy is a myelodysplastic syndrome.

[0326] In some embodiments, the hematological malignancy is acute myeloid leukemia (AML). In some embodiments, the hematological malignancy is chronic myelogenous leukemia (CML). In some embodiments, the hematological malignancy is chronic myelomonocytic leukemia (CMML).

[0327] In some embodiments, the hematological malignancy is multiple myeloma (MM).

[0328] In some embodiments, the hematological malignancy is plasmacytoma.

[0329] In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the soft tissue cancer is Ewing's sarcoma.

[0330] In some embodiments, a method for treating cancer in a subject in need thereof is provided. In some embodiments, the method comprises administering any of the compositions provided herein to the subject. In some embodiments, the use of the compositions provided herein in the preparation of a pharmaceutical composition or medicament for treating cancer is provided. In some embodiments, the composition is used to treat cancer.

[0331] In some embodiments, a method of treating Pompe disease (GSD2; Acid alpha-glucosidase (GAA) deficiency) in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject any of the compositions presented herein. In some embodiments, a use of the compositions presented herein in the preparation of a pharmaceutical composition or medicament for treating Pompe disease (GSD2; Acid alpha-glucosidase (GAA) deficiency) is provided. In some embodiments, the composition is used to treat Pompe disease (GSD2; Acid alpha-glucosidase (GAA) deficiency).

[0332] In some embodiments, a method of treating glycogen storage disease in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject any of the compositions provided herein. In some embodiments, a use of the compositions provided herein in the preparation of a pharmaceutical composition or medicament for treating glycogen storage disease is provided. In some embodiments, the composition is used to treat glycogen storage disease.

[0333] In some embodiments, a method of treating glycogen storage disease in a subject in need thereof is provided, comprising administering a composition as 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, Andersen's disease (GSD4; glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7; muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1; von Gierke disease), Haas disease (GSD6; hepatic glycogen phosphorylase (PYGL) deficiency or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11; glucose transporter (GLUT2) deficiency), aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0334] In some embodiments, the polypeptide that binds CD71 is directed to the central nervous system. In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide presented herein linked or conjugated to a therapeutic agent. In some embodiments, the brain tumor is selected from the group consisting of a non-malignant brain tumor, a benign brain tumor, and a malignant brain tumor. In some embodiments, the neurological condition is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Lafora's disease, Pompe's disease, adult polyglucosan body disease, stroke, spinal cord injury, ataxia, Bell's palsy, cerebral aneurysm, epilepsy, seizures, Guillain-Barre syndrome, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, migraine, encephalitis, sepsis, and myasthenia gravis. In some embodiments, a method of treating a neurological condition and / or brain tumor in a subject is provided, comprising administering to the subject an FN3 domain that binds to CD71, wherein the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide such as an siRNA, an ASO, an FN3 domain that binds to another target, etc.).

[0335] In some embodiments, a method of treating Pompe disease in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds to CD71. In some embodiments, the polypeptide is a FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NO: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide as presented herein linked or conjugated to a therapeutic agent. In some embodiments, a method of treating Pompe disease in a subject is provided, comprising administering to the subject a FN3 domain that binds to CD71, where the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide such as an siRNA, an ASO, an FN3 domain that binds to another target, etc.).

[0336] In some embodiments, the polypeptide that binds CD71 is directed to an immune cell. In some embodiments, the polypeptide that binds CD71 is directed to a dendritic cell. In some embodiments, a method of treating an autoimmune disease in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is a FN3 domain that binds CD71. In some embodiments, the polypeptide comprises a sequence such as SEQ ID NOs: 301-301, 310, 312-519, 521-572, 592-599, or 708-710, or a polypeptide as presented herein linked or conjugated to a therapeutic agent. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, 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 is provided, comprising administering to the subject an FN3 domain that binds to CD71, where the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide such as an siRNA, an ASO, an FN3 domain that binds to another target, etc.).

[0337] In some embodiments, a method for reducing the expression of a target gene in a cell is provided. In some embodiments, the method comprises delivering a composition or pharmaceutical composition as provided herein to a cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an in vivo cell. In some embodiments, the target gene is GYS1. However, the target gene can be any target gene, as evidence provided herein supports that siRNA molecules are efficiently delivered when conjugated to an FN3 domain. In some embodiments, the siRNA that targets GYS1 is linked to an FN3 domain. In some embodiments, the FN3 polypeptide (domain) is an FN3 polypeptide (domain) that binds to CD71. In some embodiments, the FN3 polypeptide is as described herein or as described in PCT Application No. PCT / US20 / 55509, US Application No. 17 / 070,337, PCT Application No. PCT / US20 / 55470, or US Application No. 17 / 070,020, each of which is incorporated herein by reference in its entirety. In some embodiments, the siRNA is not conjugated to the FN3 domain.

[0338] In some embodiments, a method of reducing expression of a target gene in a cell is provided. In some embodiments, the method comprises delivering a composition or pharmaceutical composition provided herein to the cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an in vivo cell. In some embodiments, the method of reducing expression of a target gene results in about 99%, 90-99%, 50-90%, or 10-50% reduction in expression of the target gene.

[0339] In some embodiments, a method of reducing the expression of GYS1 is provided. In some embodiments, the expression that is reduced is the expression (amount) of GYS1 mRNA. In some embodiments, the method of reducing the expression of GYS1 results in a reduction of about 99%, 90-99%, 50-90%, or 10-50% of the expression of GYS1. In some embodiments, the expression that is reduced is the expression (amount) of GYS1 protein. In some embodiments, the protein that is reduced 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 comprises an siRNA molecule as provided herein that targets GYS1. In some embodiments, the siRNA is conjugated to an FN3 domain. In some embodiments, the FN3 domain is an FN3 domain that binds to CD71. In some embodiments, the FN3 domain is as provided herein. In some embodiments, the FN3 domain is a dimer of two FN3 domains that bind to CD71. In some embodiments, the FN3 domain is the same FN3 domain. In some embodiments, the two FN3 domains are different, i.e., bind to different regions or amino acid residues of CD71, i.e., bind to different epitopes. In some embodiments, the method comprises administering to a subject (patient) a GYS1 siRNA molecule, such as the GYS1 siRNA molecule provided herein. In some embodiments, the GYS1 siRNA administered to the subject is conjugated or linked to an FN3 domain. In some embodiments, the FN3 domain is an FN3 domain that binds to CD71. In some embodiments, the FN3 domain is as provided herein. In some embodiments, the FN3 domain is a dimer with two FN3 domains that bind to CD71. In some embodiments, the FN3 domain is the same FN3 domain. In some embodiments, the two FN3 domains are different, i.e., bind to different regions or amino acid residues of CD71, i.e., bind to different epitopes. In some embodiments, the CD71-binding domain is a polypeptide provided herein.

[0340] In some embodiments, a method is provided for delivering siRNA molecules to cells in a subject. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising the composition presented herein. In some embodiments, the cell is a CD71 positive cell. The term "positive cell" referring to a protein refers to a cell that expresses the protein. In some embodiments, the protein is expressed on the cell surface. In some embodiments, the cell is a tumor cell, a liver cell, an immune cell, a dendritic cell, a cardiac cell, a muscle cell, a CNS cell, or a cell inside the blood-brain barrier. In some embodiments, the siRNA downregulates the expression of a target gene in the cell. In some embodiments, the target gene is GYS1.

[0341] In some embodiments, the compositions provided herein are used to diagnose, monitor, modulate, treat, alleviate, prevent the occurrence of, or alleviate symptoms of human diseases or specific pathologies in cells, tissues, organs, body fluids, or generally in a host, and also exhibit the property of being able to cross 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 junctions, so that the passage of small and large substances from the blood to the central nervous system is controlled by the BBB. This structure makes it difficult to treat and manage patients with neurological diseases and disorders (e.g., brain cancer), as many therapeutic agents are not delivered across the BBB with the desired efficiency. Further conditions involving the disruption of the BBB include stroke, diabetes, seizures, hypertensive encephalopathy, acquired immune deficiency syndrome, traumatic brain injury, multiple sclerosis, Lafora's disease, Pompe's disease, adult polyglucosan body disease, Parkinson's disease (PD), and Alzheimer's disease. This ability is useful for treating brain cancer, including, for example, astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; or spinal cancer, for example, neurofibroma, meningioma, glioma, and sarcoma, among others. This ability is also useful in treating Pompe's disease and / or glycogen storage disease. In certain embodiments, the compositions presented herein are used to deliver therapeutic or cytotoxic agents, for example, across the blood-brain barrier. In certain embodiments, the compositions presented herein are used to deliver therapeutic or cytotoxic agents, for example, into muscle.

[0342] In some embodiments, a method of treating glycogen storage disease in a subject in need thereof is provided, comprising administering a composition as 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, Andersen's disease (GSD4; glycogen debranching enzyme (GBE1) deficiency), Tarui's disease (GSD7; muscle phosphofructokinase (PFKM) deficiency), and adult polyglucosan body disease. In some embodiments, the glycogen storage disease is glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1; von Gierke disease), Haas disease (GSD6; hepatic glycogen phosphorylase (PYGL) deficiency or muscle phosphoglycerate mutase (PGAM2) deficiency), phosphorylase kinase (PHKA2 / PHKB / PHKG2 / PHKA1) deficiency syndrome (GSD9), phosphoglycerate mutase (PGAM2) deficiency (GSD10), muscle lactate dehydrogenase (LDHA) deficiency (GSD11), Fanconi-Bickel syndrome (GSD11; glucose transporter (GLUT2) deficiency), aldolase A deficiency (GSD12), beta-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

[0343] In some embodiments, the compositions or pharmaceutical compositions presented herein are used to treat a muscle disease, such as a muscular dystrophy, which is Duchenne muscular dystrophy (DMD).

[0344] In some embodiments, the compositions or pharmaceutical compositions presented herein are administered alone or in combination with other therapeutic agents, i.e., simultaneously or sequentially. In some embodiments, the other or additional therapeutic agents are other anti-tumor or therapeutic agents. Different tumor types and tumor stages may require the use of various supplementary compounds useful in the treatment of cancer. For example, the compositions presented herein are used in combination with various chemotherapeutic agents, such as taxol, tyrosine kinase inhibitors, leucovorin, fluorouracil, irinotecan, phosphatase inhibitors, MEK inhibitors, among others. The compositions are also used in combination with drugs that modulate the immune response against tumors, such as anti-PD-1 or anti-CTLA-4, among others. The additional treatment may be an agent that modulates the immune system, such as an antibody that targets PD-1 or PD-L1.

[0345] In some embodiments, a composition or pharmaceutical composition presented herein is administered in combination with GAA enzyme replacement therapy (ERT).

[0346] "Treating" or "treatment" refers to therapeutic and prophylactic measures in which the objective is to prevent or slow (reduce) an undesired physiological change or disorder, such as the onset or spread of cancer. In some embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of disease, stabilization of the disease state (i.e., non-progression), delay or slowing of disease progression, improvement or relief of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to life expectancy in the absence of treatment. Patients in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0347] "Therapeutically effective amount" refers to an amount effective at the dosage required and for the period of time required to achieve the desired therapeutic result. 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 effective amount are improvement of patient's well-being, reduction or regression of tumor size, cessation or slowing of tumor growth, and / or absence of metastasis of cancer cells to other locations in the body.

[0348] Administration / Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided by the composition presented herein and a pharma- ceutically acceptable carrier. For therapeutic use, the composition is prepared as a pharmaceutical composition containing an effective amount of the domain or molecule as an active ingredient in a pharma- ceutical carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine are used. These solutions are sterile and generally free of particulate matter. These solutions are sterilized by conventional, well-known sterilization methods (e.g., filtration). The composition can contain pharma- ceutical acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and colorants. The concentration of the molecules disclosed herein in such pharmaceutical formulations can vary widely, i.e., less than about 0.5% of body weight, typically at least about 1% to as much as 15 or 20%, and will be selected primarily based on the required dose, fluid volumes, viscosities, etc., according to 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, edited by Troy, DB, Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, "Pharmaceutical Manufacturing", pages 691-1092 (see, inter alia, 958-989).

[0349] Modes of administration for therapeutic use of the compositions disclosed herein can be any suitable route that delivers the agent to the host, such as parenteral, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary administration, using tablet, capsule, solution, powder, gel, particle formulations; transmucosal (oral, nasal, vaginal, rectal) administration, contained within a syringe, implantable device, osmotic pump, cartridge, micropump; or other means well known in the art and understood by the skilled artisan. Site-specific administration is achieved, for example, by intra-articular, intrabronchial, intraperitoneal, intra-articular capsule, intrachondral, intracavity, intraluminal, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, intravaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

[0350] The pharmaceutical composition is provided as a kit comprising a container containing the pharmaceutical composition described herein.The pharmaceutical composition may be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that is reconstituted before injection.Alternatively, such a kit may comprise a dry powder disperser, liquid aerosol generator, or nebulizer for administering the pharmaceutical composition.Such a kit may further comprise written information on the indication and use of the pharmaceutical composition.

[0351] Working Example The following examples illustrate the embodiments disclosed herein.These examples are presented for illustrative purposes only, and the embodiments should not be considered to be limited to these examples in any way, but should be considered to include any and all variations that are evidenced as a result of the teachings presented herein.Those skilled in the art will easily recognize various parameters that are not critical and can be changed or modified to produce essentially similar results. EXAMPLES

[0352] Identification and characterization of GYS1 siRNA sequences Computational Screening of siRNAs: Computational siRNA screening was performed to identify human siRNAs complementary to human GYS1 mRNA (see FIG. 1). All possible 19-mer antisense sequences were generated from the human GYS1 siRNA sequence (NM_001161587), and each 19-mer was evaluated for activity against other human GYS1 isoforms as well as potential cross-reactivity between mouse, rat, and Cynomolgus macaque. Using dbSNP (b155 v2), human siRNA target sites were evaluated for common human SNPs (minor allele frequency (MAF) >1%). Sequences targeting common alleles were discarded. Next, siRNA off-target genes were evaluated for sense and antisense strands in all relevant model organisms. This selection resulted in 200 potential candidate sequences, which were further defined by an in vitro knockdown screen. The top siRNA candidate sequences are shown in Tables 1A and 1B. The GYS1 siRNA linker for conjugation with cysteine ​​engineered centrin was prepared as described in Table 3.

[0353] HEK293T cells were lipofected with 10 nM ABXO-HHH (siRNA paired with HHH) for 24 hours (six replicates per treatment group) along with untreated control cells. mRNA was polyA+ mRNA selected from cells and subjected to non-stranded 2x150bp paired-end sequencing on an Illumina HiSeq platform with an average depth of >30 million reads.

[0354] Figure 2: Volcano plot showing DESeq2 results for ABXO-HHH treated cells versus untreated cells. The x-axis represents the log2 fold change in gene expression (gene expression by ABXO-HHH treated cells divided by gene expression by untreated cells) for all genes. The y-axis represents the negative log 10 The corrected P values ​​are shown, transformed by α = 0. Colored dots represent genes with significant complementarity to ABXO-HHH as detected via BLAST.

[0355] RNA-seq library quality was examined using FastQC. Libraries were pseudo-aligned against the GrCH38 human transcriptome using Kallisto. We achieved exceptional read alignment, with an average of >90% read alignment against the transcriptome. Differential expression was then assessed using DESeq2, comparing ABXO-HHH-treated cells with cells treated with Lipofectamine alone.

[0356] DESeq results showed substantial and significant knockdown of the GYS1 target. GYS1 expression in ABXO-HHH treated samples was downregulated to 31% of untreated samples (Figure 2). GYS1 was the most downregulated protein-coding gene of all differentially expressed (DE) genes under ABXO-HHH treatment. GYS1 was also by far the most differentially expressed gene with the smallest p-value, 1×10 87.7 The corrected p-value of was more than 30 orders of magnitude lower than the nearest minimum corrected p-value.

[0357] Computational prediction of potential off-target effects was performed using BLAST. The sense and antisense sequences of ABXO-HHH were aligned by BLAST against an internal BLAST database constructed from the GRCh38 transcriptome. Of all potential off-target effects identified by BLAST, only RAP2C was significantly downregulated in the presence of ABXO-HHH (Figure 2) and did not exceed the DE threshold of <-1 log2 fold change.

[0358] Taken together, these data confirm that ABXO-HHH is a highly specific siRNA, even at relatively high concentrations in the human transcriptome.

[0359] Protocol for oligonucleotide synthesis, deprotection, and annealing: Synthesis of oligonucleotides was carried out on a Mermade 12 synthesizer using standard phosphoramidite chemistry on 500 Å controlled pore glass (CPG) with a phosphoramidite concentration of 0.1 M in acetonitrile. I2 (0.02 M) in THF / pyridine / water was the oxidizing agent with 0.6 M ETT (5-ethylthiotetrazole) as the activating agent. N,N-Dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)methanimidamide (DDTT), 0.09 M in pyridine, was used as the sulfurizing reagent to introduce phosphorothioate (PS) bonds. Dichloroacetic acid, 3% (v / v) in dichloromethane was used as the deprotecting solution. All single strands without maleimide were purified by ion exchange chromatography (IEX) with 20 mM phosphate at pH 8.5 as buffer A and 20 mM phosphate pH 8.5 and 1 M sodium bromide as buffer B. After purification, oligonucleotide fractions were pooled, concentrated and desalted. The desalted samples were then lyophilized and stored at -20°C.

[0360] Deprotection of the antisense strand After synthesis, the support was washed with acetonitrile (ACN), dried in a column under vacuum, transferred to a sealable 1 mL screw-capped vial, and shaken with a 5% solution of diethylamine in aqueous ammonia for 5 h at 65° C. Cleavage and deprotection of the crude oligos were confirmed by liquid chromatography-mass spectrometry (LC-MS) and then purified by IEX-HPLC.

[0361] Synthesis and deprotection of maleimide-containing oligonucleotides Maleimide-containing oligos were made using 3' amino-modified CPG solid supports or 5' amino-modified phosphoramidites. The supports were transferred to sealable 1 mL vials and incubated with 50 / 50 v / v 40% aqueous methylamine / ammonia (AMA) at room temperature for 2 hours or at 65°C for 10 minutes for cleavage and deprotection. Single strands were purified by IEX chromatography and desalted under the same conditions as the antisense before maleimide addition.

[0362] About 20mg / mL of amine-modified sense strand was prepared in 0.05M phosphate buffer at pH 7.1, and 10 molar equivalents of maleimide N-hydroxysuccinimide (NHS) ester dissolved in ACN was added to it. NHS ester solution was added to aqueous oligonucleotide solution and shaken at room temperature for 3 hours. Thus, the maleimide-conjugated oligonucleotide was purified by reverse phase chromatography (20mM triethylammonium acetate with 80% acetonitrile in buffer B) to prevent hydrolysis of maleimide under ion exchange buffer conditions.

[0363] After purification, the oligonucleotide fractions were pooled, concentrated and desalted.

[0364] To avoid hydrolysis of the maleimide, duplexing of the sense and antisense strands was performed via lyophilization using equimolar amounts of each desalted single strand.

[0365] Conjugation of centirin to siRNA, purification and analysis of conjugates: Centilin was conjugated to siRNA via maleimide-mediated cysteine-specific chemistry. Cysteine-containing centirin at 50-200 μM in PBS was reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) at room temperature (30 min) to yield free thiols. The free thiols containing centirin were then immediately mixed with maleimide containing siRNA duplex in water at a molar ratio of centirin:siRNA of approximately 1.5:1. After incubation for 2 h at room temperature or 37°C, the reaction was quenched with N-ethylmaleimide (to give a final NEM concentration of 1 mM in the reaction mixture). The conjugates were purified in two steps. Step I: Immobilized metal affinity chromatography (for tagged proteins) or hydrophobic interaction chromatography (for untagged proteins) to remove unreacted siRNA linkers. Step II: Capto-DEAE to remove unreacted centrin. Fractions containing the conjugate were pooled and exchanged into HEPES-buffered saline (HBS) by desalting using dialysis and concentrated if necessary.

[0366] Analytical characterization of centirin-siRNA conjugates: Centirin-siRNA conjugates were characterized by a combination of analytical methods. SDS-PAGE was used to compare the amount of conjugates to the amount of free protein. For SDS-PAGE, 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels (BioRad) were run in SDS buffer at 100 V for 1 hour. Gels were visualized under UV light. Analytical SEC (Superdex-75 5 / 150 GL column; GE) was used to analyze the purity and aggregation state of centirin-siRNA conjugates. Liquid chromatography / mass spectrometry (LCMS) was used to confirm the identity and purity of the conjugates. Samples were analyzed using a Waters Acuity UPLC / Xevo G2-XS TOF mass spectrometer system. The instrument was operated in negative electrospray ionization mode and scanned over m / z of 200-3000. The conjugate was seen as two fragments: antisense-sentirin and sense-sentirin.

[0367] Conjugation of centirin to siRNA, purification and analysis of conjugates: Centilin was conjugated to siRNA via maleimide-mediated cysteine-specific chemistry. Cysteine-containing centirin at 50-200 μM in PBS was reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) at room temperature (30 min) to yield free thiols. The free thiols containing centirin were then immediately mixed with maleimide containing siRNA duplex in water at a molar ratio of centirin:siRNA of approximately 1.5:1. After incubation for 2 h at room temperature or 37°C, the reaction was quenched with N-ethylmaleimide (to give a final NEM concentration of 1 mM in the reaction mixture). The conjugates were purified in two steps. Step I: Immobilized metal affinity chromatography (for tagged proteins) or hydrophobic interaction chromatography (for untagged proteins) to remove unreacted siRNA linkers. Step II: Capto-DEAE, to remove unreacted centrin. Fractions containing the conjugate were pooled and exchanged into HBS by desalting using dialysis and concentrated if necessary.

[0368] Figure 6: IMAC chromatography for tagged proteins. For IMAC, HisTrap HP columns (1 ml, 5 ml) from Cytiva were used. Histrap Buffer A (binding buffer): 50 mM Tris pH 7.4, 500 mM NaCl, and 10 mM imidazole in Type 1 H2O, and Histrap Buffer B (elution buffer): 50 mM Tris pH 7.4, 500 mM NaCl, and 250 mM imidazole in Type 1 H2O. The reaction sample was injected directly onto the column via the sample loop or sample pump. After sample application, the column was washed with 5-10 column volumes of Buffer A. Elution typically began with a step gradient (0-100%). Fractions were collected when the UV reading was above 50 mAU using a fraction collector and peak fragmentation.

[0369] Figure 7: HIC for untagged proteins (removal of excess siRNA). For HIC, Cytiva HiTrap Butyl HP columns (1 ml, 5 ml) were used. HIC Buffer A (binding buffer) was 2M ammonium sulfate, 25 mM sodium phosphate pH 7.0 in Type 1 H2O, while HIC Buffer B (elution buffer): 25 mM sodium phosphate pH 7.0 in Type 1 H2O. The conjugation reaction sample was diluted in a 1:1 ratio with Buffer A. The sample prepared above was directly injected into the column via the sample loop or sample pump. After sample application, the column was washed with 5 column volumes of Buffer A. Elution typically started with 0.0% in B and then continued with the gradient in Table 10 below. Fractions were collected when the UV reading was 50 mAU or higher using a fraction collector and peak fragmentation.

[0370] [Table 9]

[0371] Ring opening: To avoid loss of cargo via retro-Michael reaction, hydrolysis of the maleimide ring is performed. Pooled fractions from Histrap (tagged protein) or HIC (untagged protein) were dialyzed into 25 mM Tris pH 8.9 buffer. Reactions in this buffer were placed in an incubator shaker at 37 degrees Celsius for 72 hours. Reactions were monitored for completion by LC-MS.

[0372] Figure 8: Ion exchange chromatography (IEX) for tagged and untagged proteins (removal of excess unreacted centrin). For IEX chromatography, HiTrap Capto DEAE columns (1 ml, 5 ml) from Cytiva were used. Capto DEAE Buffer A (binding buffer) was 25 mM Tris pH 8.8 in Type 1 H2O, whereas Capto DEAE Buffer B (elution buffer) used was 25 mM Tris pH 8.8, 1 M NaCl in Type 1 H2O.

[0373] The samples after ring opening were injected directly onto the column via the sample loop or sample pump. After sample application, the column was washed with 5-10 column volumes of Buffer A. Elution typically started with 0.0% in B and then continued with the gradient in Table 11 below.

[0374] Unreacted protein was eluted in the flow-through, where it was typically the first peak and the second peak was typically the pure conjugate. All fractions were collected and pooled. The concentration of the pool was determined by measuring A260 using Nanodrop for yield calculation.

[0375] [Table 10]

[0376] Figure 9: Example of analytical SEC for centirin-oligonucleotide conjugates. Centirin-siRNA conjugates were characterized by a combination of analytical methods. Analytical SEC was used to analyze the purity and aggregation state of centirin-siRNA conjugates. A Waters H-class UPLC with a Waters ACQUITY UPLC Protein BEH SEC Column, 125 Å, 1.7 μm, 4.6×150 mm, was used for routine SEC analysis. Typically, 2-5 ul samples were injected using an autosampler, the flow rate was 0.25 ml per minute, and the mobile phase used was 1× PBS or 100 mM, pH 7.2 phosphate buffer.

[0377] Figure 10: Example of SDS PAGE gel for conjugates. The amount of conjugate was compared to the amount of free protein using SDS-PAGE. An Invitrogen Mini Gel tank.PowerEase™ Touch 600W Power Supply and 115V AC were used to run the SDS PAGE gel. Invitrogen - NuPAGE™ 4-12% Bis-Tris Protein Gel, 1.0mm was used with 20x NuPAGE® MES SDS Running Buffer and SeeBlue™ Pre-stained Protein Standard. Samples were normalized to approximately 0.5mg / mL with ultrapure water. For non-reduced gels, 10μL of normalized sample was mixed with 10μL of 2x Laemmli Sample Buffer in a 1:1 ratio. For reduced gel, 2x Laemmli Sample Buffer was mixed with β-mercaptoethanol in a 95:1 ratio, then mixed with 10 μL of sample in a 1:1 ratio. The resulting sample mixture was boiled at 95°C for 5 minutes, then cooled in a thermocycler. 15 μL of sample and protein ladder were loaded into appropriate wells, the voltage was set to 200V, and the run time was set to about 30 minutes. When the gel was finished, it was visualized by Coomassie Blue or SYBRGreen or methylene blue. The leftmost lane is the protein ladder, the next lane is centilin itself, and the last lane is centilin-siRNA conjugate.

[0378] Candidate siRNA sequences were transfected into human cells (H358) at a range of concentrations. 24 hours after transfection, RNA was harvested and analyzed for GYS1 levels via quantitative reverse transcription polymerase chain reaction (RT-PCR). 18S ribosomal RNA was used as an RT-PCR endogenous control gene. Knockdown levels were compared to untreated cells. Graphpad Prism software was used to calculate EC50 values, and Emax values ​​represent the maximum percent knockdown observed for GYS1 mRNA (Table 5).

[0379] [Table 11] Other siRNAs were also examined as described above and their EC50s are presented in Table 6.

[0380] [Table 12]

[0381] The selectivity of the candidate siRNA sequences was assessed by transfection of a range of siRNA concentrations into human cells (HEK-293). Cell viability was assessed 72 hours after transfection using CellTiterGlo. Emax values ​​were reported as the maximum percent reduction in cell viability at the highest siRNA concentration tested (10 nM) (Table 7).

[0382] [Table 13] EXAMPLES

[0383] Selection of fibronectin type III (FN3) domains that bind to CD71 Panning and biochemical screening methods to identify FN3 domains that bind to CD71 without inhibiting transferrin binding to CD71: To screen for FN3 domains that specifically bind to CD71 without inhibiting transferrin binding to CD71, streptavidin-coated Maxisorp plates (Nunc; model number: 436110) were blocked in Starting Block T20 (Pierce) for 1 hour, and then coated with biotinylated CD71 (using the same antigen as the antigen in the panning method) or negative control (unrelated Fc fusion recombinant protein and human serum albumin) for 1 hour in the presence of transferrin or with FN3 protein that binds to the transferrin binding site of CD71. The concentration of transferrin is up to 35 μM. Without being bound to any particular theory, the incorporation of FN3 protein that binds to transferrin or the transferrin binding site of CD71 forces the selection of FN3 domains to obtain FN3 domains that do not compete with or inhibit the binding of transferrin to CD71. The plate was rinsed with TBST, and the diluted lysate was applied to the plate for 1 hour. Following further rinsing, the wells were treated with HRP-conjugated anti-V5tag antibody (Abcam; ab1325) for 1 hour, and then assayed with peroxidase (POD) (Roche; 11582950001). Sequencing DNA from FN3 domain lysates with ELISA signals at least 10 times higher than the ELISA signal for streptavidin control resulted in FN3 domain sequences isolated from the screening. EXAMPLES

[0384] Selection of fibronectin type III (FN3) domains that bind to CD71 but do not compete with transferrin A biased CIS display strategy was designed to identify CD71-binding FN3 domains that are not competitive with transferrin or that are minimally competitive. Briefly, the output collected after five rounds of panning on the extracellular domain (ECD) of human CD71 was used (Example 3). Further rounds of off-rate selection were also performed as described in Example 3, but with the addition of 1) a washing step with human holotransferrin, which elutes centrin bound to the same site as transferrin, before the final elution step; or 2) elution of FN3 domain binders with monoclonal antibody OKT9. FN3 domains recovered from the transferrin washing strategy and the OKT9 elution strategy were PCR amplified and cloned into pET vectors as previously described. 228 FN3 domains that specifically bound to huCD71 were confirmed for binding to huCD71 ECD by ELISA. A subset of unique binders were analyzed by SEC, conjugated to monomethylauristatin F (MMAF), and assessed for internalization via cell viability assays in SKBR-3 cells with and without human holo-transferrin. The polypeptides were found to be internalized by the receptor.

[0385] Integral Molecular conducted a membrane proteome array (MPA) assay to profile the specificity of ABX1198 (SEQ ID NO: 509) and ABX1100 (siRNA pair with SEQ ID NO: 509 + linker number OOOO) against a library of human membrane proteins. The MPA library contained over 6000 human membrane proteins, including all single-pass transmembrane proteins, multi-pass transmembrane proteins, and 94% of GPI-anchored proteins, ion channels, and transporters, including G protein-coupled receptors (GPCRs), with each membrane protein uniquely expressed in a chicken QT6 cell background. Flow cytometry was used to directly detect ligands (FN3 domains) binding to individually expressed membrane proteins in unfixed cells.

[0386] ABX1198 (SEQ ID NO:509) and ABX1100 (siRNA pair with SEQ ID NO:509 + linker number OOOO) were screened against MPA at concentrations with optimal signal / background noise ratios of 1.25ug / ml, 1.25ug / ml, and 0.31ug / ml, respectively. Membrane protein targets identified in the screen were followed up in a validation procedure using serial dilutions of the ligand and cells individually transfected with the identified targets. EXAMPLES

[0387] Knockdown of mRNA in muscle cells using CD71 FN3 domain-oligonucleotide conjugates The FN3 domain that binds to muCD71 is conjugated to an siRNA oligonucleotide or antisense oligonucleotide (ASO) using maleimide chemistry via a cysteine ​​specifically engineered into the FN3 domain. The cysteine ​​substitution can be a cysteine ​​substitution such as those presented herein and also in US Patent Publication No. 20150104808, which is incorporated herein by reference in its entirety. The siRNA or ASO is modified in vitro with standard chemical modifications to confirm that it allows knockdown of the target mRNA. The FN3 domain-oligonucleotide conjugate was administered intravenously to mice with a maximum dose of 10 mg / kg of oligonucleotide payload. At various time points after administration, the mice were sacrificed and skeletal muscle, cardiac muscle, and various other tissues were harvested and stored in RNAlater™ (Sigma Aldrich) until required. Standard qPCR ΔΔC T Using the method and primers specific for the target and control genes, knockdown of the target gene was assessed. The target gene was found to be knocked down in muscle, and such knockdown was enhanced by conjugating siRNA or ASO to the CD71-binding domain of FN3.

[0388] The FN3-siRNA conjugates tested are as described in Table 8.

[0389] [Table 14]

[0390] FIG. 4A demonstrates knockdown of GYS1 mRNA in mouse gastrocnemius muscle using three different FN3 domain-siRNA conjugates compared to vehicle alone. For efficacy studies, male GAA- / - mice (age 4-5 weeks) were obtained from Jackson Laboratories. All animals were treated according to IACUC protocols. Five animals received a single tail vein bolus injection of 5.4 mg / kg of three different FN3 domain-siRNA conjugates (3 mg / kg Gys1 siRNA) or vehicle. Four weeks after the single dose, mice were euthanized and gastrocnemius muscles were harvested in RNAlater, stored overnight at 4°C, and frozen at -80°C. Total RNA was isolated from the calves using the RNeasy Fibrous Tissue kit from Qiagen. Expression levels of target Gys1 and endogenous controls (Pgk1, Ubc, Hprt1, and Aha1) were analyzed using real-time quantitative PCR. Data were analyzed using normalization by the ΔΔCt method to control animals that received vehicle alone. Percentage knockdown of Gys1 mRNA in FN3 domain-siRNA conjugate treated groups was determined by subtracting the percentage of remaining Gys1 mRNA levels from 100. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison test in GraphPad Prism software. Statistical significance is indicated on the figures with an asterisk: *** Presented with p<0.001.

[0391] FIG. 4B demonstrates knockdown of GYS1 protein in mouse gastrocnemius muscle using three different FN3 domain-siRNA conjugates compared to vehicle alone. Quantification of Gys1 protein in the calf was performed by homogenizing the calf in RIPA buffer. Protein concentration in the calf was measured using a Bradford assay. Gys1 levels were quantified using the manufacturer's standard method for 12-230 kDa Jess separation modules (SM-W004). Proteins were separated by immobilization on capillaries using Protein Simple's proprietary light-activated capture chemistry. Anti-Gys1 primary antibody was used at a dilution of 1:100. Chemiluminescence was established using peroxide / Luminol-S. Digital images of the capillary chemiluminescence were captured using Protein Simple's Compass for Simple Western software, which automatically measures height (chemiluminescence intensity), area, and signal / noise ratio. An internal system was incorporated into each trial. Peak area values ​​of FN3 domain-siRNA conjugate treatment groups were normalized to vehicle-treated tissues, and the percentage knockdown of Gys1 protein within treatment groups was determined by subtracting the percentage of remaining Gys1 protein levels from 100. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison test in GraphPad Prism software. Statistical significance is indicated on the figures with an asterisk: *** Presented with p<0.001.

[0392] Figure 5 confirms that GYS1 knockdown using three different FN3 domain-siRNA conjugates and compared with siRNA against a different target (AHA-1) is highly specific to skeletal muscle. Male GAA- / - mice (8-9 weeks old) were obtained from Jackson Laboratories. All animals were treated according to IACUC protocols. Three animals received a single tail vein bolus injection of 17.9 mg / kg of three different FN3 domain-siRNA conjugates (10 mg / kg Gys1 siRNA), 17.9 mg / kg of one FN3 domain-siRNA conjugate (10 mg / kg Aha1 siRNA) or vehicle. Two weeks after the single dose, the mice were euthanized. Gastrocnemius, quadriceps, diaphragm, heart, liver, and kidney tissues were collected in RNAlater, stored overnight at 4°C, and frozen at -80°C. Total RNA was isolated from tissues using the RNeasy Fibrous Tissue kit from Qiagen. Expression levels of targets Gys1 / Aha1 and endogenous control (Pgk1) were analyzed using real-time quantitative PCR. Data were analyzed using normalization by the ΔΔCt method against control animals receiving vehicle alone. EXAMPLES

[0393] RNA-seq experiments comparing ABX-HHH treated and untreated cells mRNA sequencing was performed on polyA+ selected mRNA after lipofection of HEK293T cells with 10 nM ABXO-HHH for 24 hours. Six replicates of mRNA per treatment group were sequenced to an average depth of >30 million reads. Libraries were pseudoaligned against the GRCh38 transcriptome using Kallisto with a mapping rate of >90%. Values ​​in this Volcano plot were generated using DESeq2 comparing ABXO-HHH treated RNA-seq libraries versus untreated cells. Each dot in the plot in Figure 2 represents the change in expression measured within a gene. The x-axis represents the log2 fold change in gene expression (gene expression from ABXO-HHH treated cells divided by gene expression from untreated cells) for all genes. The y-axis represents the negative log 10 The black arrow indicates GYS1 on the plot. EXAMPLES

[0394] Binding specificity of CD71 FN3 domain-siRNA conjugates Integral Molecular (www.integralmolecular.com) performed a proprietary membrane proteome array (MPA) assay to profile the specificity of CD71 FN3 domain and CD71 FN3 domain-siRNA conjugates against a library of human membrane proteins (Figure 3). The MPA contained over 6000 human membrane proteins, spanning 94% of GPI-anchored proteins, ion channels, and transporters, including all single-pass transmembrane proteins, multi-pass transmembrane proteins, and G protein-coupled receptors (GPCRs), with each membrane protein uniquely expressed in a chicken QT6 cell background. Flow cytometry was used to directly detect FN3 domain binding to individually expressed membrane proteins in unfixed cells.

[0395] FN3 domain and FN3 domain-siRNA conjugates were screened against MPA at concentrations of 1.25ug / ml or 0.31ug / ml, respectively, with optimal signal / background noise ratio. Membrane protein targets identified in the screen were validated using serial dilutions of the ligand on cells that uniquely express the identified targets.

[0396] General method Standard methods in molecular biology are described (Sambrook, Fritsch, and Maniatis (1982 and 1989, 2nd ed.; 2001, 3rd ed.) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001), Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993), Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also found in Ausbel et al. (2001), Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc., New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0397] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization have been described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis of proteins, chemical modification, post-translational modifications, generation of fusion proteins, and glycosylation are described (see, e.g., Coligan et al. (2000), Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001), Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001), Products for Life Science Research, St. Louis, MO, pp. 45-89; Amersham Pharmacia Biotech (2001), BioDirectory, Piscataway, NJ, pp. 384-391). The production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan et al. (2001), Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999), Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al. (2001), Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0398] All references cited herein are incorporated by reference to the same extent as if each individual publication, database accession number (e.g., Genbank sequence or Gene ID accession number), patent application, or patent was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended to refer to each individual publication, database accession number (e.g., Genbank sequence or Gene ID accession number), patent application, or patent, and any individual citations thereof, each of which is clearly identified by the applicants in accordance with 37 CFR § 1.57(b)(2), even if such citation is not made in close proximity to the exclusive statement of incorporation by reference, in accordance with 37 CFR § 1.57(b)(1). The incorporation of an exclusive statement of incorporation by reference herein, if any, does not in any way nullify this general statement of incorporation by reference. Citation of references herein is not intended as an admission that the references are pertinent prior art, nor does it constitute any admission as to the contents or publication dates of these publications or documents.

[0399] The present embodiments are not intended to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the embodiments in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0400] The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the embodiments, and various modifications of the embodiments in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

1. 1. An siRNA molecule comprising a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence of SEQ ID NO:704 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:705; The sense strand comprises the nucleotide sequence of SEQ ID NO:614 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:615; The sense strand comprises the nucleotide sequence of SEQ ID NO:632 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:633; The sense strand comprises the nucleotide sequence of SEQ ID NO: 10, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 11; The sense strand comprises the nucleotide sequence of SEQ ID NO:12 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:13; The sense strand comprises the nucleotide sequence of SEQ ID NO: 14 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 15; The sense strand comprises the nucleotide sequence of SEQ ID NO: 16, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 17; The sense strand comprises the nucleotide sequence of SEQ ID NO:18 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:19; The sense strand comprises the nucleotide sequence of SEQ ID NO:20 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:21; The sense strand comprises the nucleotide sequence of SEQ ID NO:22 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:23; The sense strand comprises the nucleotide sequence of SEQ ID NO:24 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:25; The sense strand comprises the nucleotide sequence of SEQ ID NO:26 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:27; The sense strand comprises the nucleotide sequence of SEQ ID NO:28 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:29; The sense strand comprises the nucleotide sequence of SEQ ID NO: 30, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 31; The sense strand comprises the nucleotide sequence of SEQ ID NO:32 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:33; The sense strand comprises the nucleotide sequence of SEQ ID NO:34 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:35; The sense strand comprises the nucleotide sequence of SEQ ID NO:36 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:37; The sense strand comprises the nucleotide sequence of SEQ ID NO:38 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:39; The sense strand comprises the nucleotide sequence of SEQ ID NO: 40 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 41; The sense strand comprises the nucleotide sequence of SEQ ID NO: 42 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 43; The sense strand comprises the nucleotide sequence of SEQ ID NO: 44 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 45; The sense strand comprises the nucleotide sequence of SEQ ID NO:46 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:47; The sense strand comprises the nucleotide sequence of SEQ ID NO:48 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:49; The sense strand comprises the nucleotide sequence of SEQ ID NO:50 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:51; The sense strand comprises the nucleotide sequence of SEQ ID NO:52 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:53; The sense strand comprises the nucleotide sequence of SEQ ID NO:54 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:55; The sense strand comprises the nucleotide sequence of SEQ ID NO:56 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:57; The sense strand comprises the nucleotide sequence of SEQ ID NO:58 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:59; The sense strand comprises the nucleotide sequence of SEQ ID NO:60 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:61; The sense strand comprises the nucleotide sequence of SEQ ID NO:62 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:63; The sense strand comprises the nucleotide sequence of SEQ ID NO:64 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:65; The sense strand comprises the nucleotide sequence of SEQ ID NO:66 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:67; The sense strand comprises the nucleotide sequence of SEQ ID NO:68 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:69; The sense strand comprises the nucleotide sequence of SEQ ID NO:600 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:601; The sense strand comprises the nucleotide sequence of SEQ ID NO:602 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:603; The sense strand comprises the nucleotide sequence of SEQ ID NO:604 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:605; The sense strand comprises the nucleotide sequence of SEQ ID NO:606 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:607; The sense strand comprises the nucleotide sequence of SEQ ID NO:608 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:609; The sense strand comprises the nucleotide sequence of SEQ ID NO:610, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:611; The sense strand comprises the nucleotide sequence of SEQ ID NO:612 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:613; The sense strand comprises the nucleotide sequence of SEQ ID NO:616 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:617; The sense strand comprises the nucleotide sequence of SEQ ID NO:618 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:619; The sense strand comprises the nucleotide sequence of SEQ ID NO:620, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:621; The sense strand comprises the nucleotide sequence of SEQ ID NO:622 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:623; The sense strand comprises the nucleotide sequence of SEQ ID NO:624 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:625; The sense strand comprises the nucleotide sequence of SEQ ID NO:626 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:627; The sense strand comprises the nucleotide sequence of SEQ ID NO:628 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:629; The sense strand comprises the nucleotide sequence of SEQ ID NO:630, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:631; The sense strand comprises the nucleotide sequence of SEQ ID NO:634 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:635; The sense strand comprises the nucleotide sequence of SEQ ID NO:636 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:637; The sense strand comprises the nucleotide sequence of SEQ ID NO:638 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:639; The sense strand comprises the nucleotide sequence of SEQ ID NO:640 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:641; The sense strand comprises the nucleotide sequence of SEQ ID NO:642 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:643; The sense strand comprises the nucleotide sequence of SEQ ID NO:644 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:645; The sense strand comprises the nucleotide sequence of SEQ ID NO:646 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:647; The sense strand comprises the nucleotide sequence of SEQ ID NO:648 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:649; The sense strand comprises the nucleotide sequence of SEQ ID NO:650 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:651; The sense strand comprises the nucleotide sequence of SEQ ID NO:652 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:653; the sense strand comprises the nucleotide sequence of SEQ ID NO:654 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:655; or The sense strand comprises the nucleotide sequence of SEQ ID NO: 656, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 657; siRNA molecule.

2. The siRNA molecule of claim 1 , wherein the siRNA molecule comprises one or more nucleotide modifications or one or more internucleotide linkage modifications.

3. The siRNA molecule of claim 1 or 2, wherein the siRNA further comprises a linker covalently attached to a sense strand of the siRNA, the linker being attached to the 3' end of the sense strand.

4. The siRNA molecule of any one of claims 1 to 3, wherein the siRNA further comprises a vinylphosphonic acid modification on the antisense strand, the vinylphosphonic acid modification being conjugated to the 5' end of the antisense strand.

5. the sense strand comprises a nucleic acid molecule of SEQ ID NO:706 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:707; the sense strand comprises a nucleic acid molecule of SEQ ID NO:660 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:661; the sense strand comprises a nucleic acid molecule of SEQ ID NO:678 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:679; The sense strand comprises a nucleic acid molecule of SEQ ID NO: 70 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 71; the sense strand comprises a nucleic acid molecule of SEQ ID NO:72 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:73; the sense strand comprises a nucleic acid molecule of SEQ ID NO:74 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:75; the sense strand comprises a nucleic acid molecule of SEQ ID NO:76 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:77; the sense strand comprises a nucleic acid molecule of SEQ ID NO:78 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:79; The sense strand comprises a nucleic acid molecule of SEQ ID NO:80 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:81; The sense strand comprises a nucleic acid molecule of SEQ ID NO:82 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:83; the sense strand comprises a nucleic acid molecule of SEQ ID NO:84 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:85; The sense strand comprises a nucleic acid molecule of SEQ ID NO:86 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:87; the sense strand comprises a nucleic acid molecule of SEQ ID NO:658 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:659; the sense strand comprises a nucleic acid molecule of SEQ ID NO:662 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:633; the sense strand comprises a nucleic acid molecule of SEQ ID NO:664 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:665; the sense strand comprises a nucleic acid molecule of SEQ ID NO:666 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:667; the sense strand comprises a nucleic acid molecule of SEQ ID NO:668 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:669; the sense strand comprises a nucleic acid molecule of SEQ ID NO:670 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:671; the sense strand comprises a nucleic acid molecule of SEQ ID NO:672 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:673; the sense strand comprises a nucleic acid molecule of SEQ ID NO:674 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:675; the sense strand comprises a nucleic acid molecule of SEQ ID NO:676 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:677; The sense strand comprises a nucleic acid molecule of SEQ ID NO:680 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:681; The sense strand comprises the nucleic acid molecule of SEQ ID NO:682 and the antisense strand comprises the nucleic acid molecule of SEQ ID NO:683 Contains molecules; the sense strand comprises a nucleic acid molecule of SEQ ID NO:684 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:685; the sense strand comprises a nucleic acid molecule of SEQ ID NO:686 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:687; the sense strand comprises a nucleic acid molecule of SEQ ID NO:688 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:689; the sense strand comprises a nucleic acid molecule of SEQ ID NO:690 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:691; the sense strand comprises a nucleic acid molecule of SEQ ID NO:692 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:693; the sense strand comprises a nucleic acid molecule of SEQ ID NO:694 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:695; the sense strand comprises a nucleic acid molecule of SEQ ID NO:696 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:697; the sense strand comprises a nucleic acid molecule of SEQ ID NO:698 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:699; the sense strand comprises a nucleic acid molecule of SEQ ID NO: 700 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 701; the sense strand comprises a nucleic acid molecule of SEQ ID NO:702 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:703; the sense strand comprises a nucleic acid molecule of SEQ ID NO:711 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:712; the sense strand comprises a nucleic acid molecule of SEQ ID NO: 1053 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 1054; the sense strand comprises a nucleic acid molecule of SEQ ID NO: 1055 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 1056; the sense strand comprises a nucleic acid molecule of SEQ ID NO: 1057 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 1058; or the sense strand comprises a nucleic acid molecule of SEQ ID NO: 1059 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 1060; The siRNA molecule according to any one of claims 1 to 4.

6. A composition comprising one or more FN3 domains conjugated to an siRNA comprising a sense strand and an antisense strand, wherein the one or more FN3 domains comprise an FN3 domain that binds to CD71, the siRNA is an siRNA that reduces expression of GYS1, and the FN3 domain is linked to the sense strand of the siRNA.

7. The composition according to claim 6, wherein the siRNA molecule is an siRNA molecule according to any one of claims 1 to 5.

8. The composition of claim 6 or 7, wherein the FN3 domain is conjugated to the siRNA molecule via a cysteine ​​on the FN3 domain, and the cysteine ​​in the FN3 domain is at a position corresponding to residue 54, 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 59, 60, 62, 64, 70, 88, 89, 90, 91, or 93 of the FN3 domain based on SEQ ID NO:

405.

9. The FN3 domain is Mal-C ２ H ４ C(O)(NH)-(CH2) ６ The composition of claim 8, wherein the amino acid sequence is linked to the sense strand by a linker having the formula:

10. The FN3 polypeptide domain comprises: an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:509; Amino acid sequence of SEQ ID NO:509; an amino acid sequence that is at least 95% identical to the amino acid sequence of one of SEQ ID NOs: 273, 301-310, 312-572, 592-599, or 708-710; or The amino acid sequence of any one of SEQ ID NOs: 273, 301-310, 312-572, 592-599, or 708-710. The composition according to any one of claims 7 to 9, comprising:

11. The siRNA molecule is the sense strand comprises the nucleotide sequence of SEQ ID NO: 704, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 705; the sense strand comprises the nucleotide sequence of SEQ ID NO: 614, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 615; the sense strand comprises the nucleotide sequence of SEQ ID NO: 632, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 633; the sense strand comprises a nucleic acid molecule of SEQ ID NO: 706 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 707; the sense strand comprises a nucleic acid molecule of SEQ ID NO:660 and the antisense strand comprises a nucleic acid molecule of SEQ ID NO:661; or The sense strand comprises a nucleic acid molecule of SEQ ID NO: 678, and the antisense strand comprises a nucleic acid molecule of SEQ ID NO: 679; The composition according to any one of claims 7 to 10.

12. A pharmaceutical composition comprising an siRNA molecule according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 11.

13. A composition for use in a method of treating a subject having a Gys1 mediated disease, such as glycogen storage disease, comprising an siRNA molecule according to any one of claims 1 to 5.

14. Glycogen storage diseases include Corey or Forbes disease (GSD3; glycogen debranching enzyme (AGL) deficiency), McArdle disease (GSD5; muscle glycogen phosphorylase (PYGM) deficiency), type II diabetes mellitus / diabetic nephropathy, aldolase A deficiency (GSD12), Lafora disease, hypoxia, Andersen disease (GSD4; glycogen debranching enzyme (GBE1) deficiency), Tarui disease (GSD7; muscle phosphofructokinase (PFKM) deficiency), adult polyglucosan body disease, glycogen synthase (GYS2) deficiency (GSD0), glucose-6-phosphatase (G6PC / SLC37A4) deficiency (GSD1; von Gierke disease), and Haas disease (GSD6).

14. The composition of claim 13, wherein the deficiency is selected from the group consisting of liver glycogen phosphorylase (PYGL) deficiency 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 (GSD11; glucose transporter (GLUT2) deficiency), aldolase A deficiency (GSD12), β-enolase (ENO3) deficiency (GSD13), and glycogenin-1 (GYG1) deficiency (GSD15).

15. A pharmaceutical composition according to claim 12 for use in reducing the expression of Gys1 in a cell.

16. A diagnostic kit comprising an siRNA molecule according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 11, and instructions for use.

Images