Compositions and methods for treating facioscapulohumeral muscular dystrophy

Abstract

Polynucleic acid molecules, pharmaceutical compositions, and methods for treating facioscapulohumeral muscular dystrophy are provided. [Solution] A conjugate comprising (i) an anti-transferrin receptor antibody or an antigen-binding fragment thereof, (ii) an siRNA comprising a guide strand and a passenger strand, and (iii) a linker, as well as a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable excipient are provided.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 992,071, filed March 19, 2020, and U.S. Provisional Patent Application No. 63 / 066,655, filed August 17, 2020, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Background of the Invention Gene suppression through RNA-induced gene silencing provides multiple levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA downregulation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) exerts long-lasting effects that span multiple cell divisions. Therefore, RNAi represents a viable method for drug target validation, gene function analysis, pathway analysis, and disease treatment.

[0003] Incorporation by Reference I All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0004] In certain embodiments, disclosed herein are polynucleic acid molecules and pharmaceutical compositions for regulating genes associated with muscle wasting disorders, particularly facioscapulohumeral muscular dystrophy (FSHD). Also described herein, in some embodiments, are methods for treating muscle wasting disorders, particularly FSHD, with the polynucleic acid molecules or polynucleic acid molecule conjugates disclosed herein.

[0005] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DUX4, wherein the polynucleic acid molecule conjugate hybridizes to a target sequence of DUX4. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a non-human antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is an anti-transferrin receptor antibody or antigen-binding fragment thereof.

[0006] In certain embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand and / or the antisense strand each independently comprise at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In certain embodiments, the polynucleotide hybridizes to at least 8 consecutive bases of a target sequence of DUX4. In certain embodiments, the polynucleotide is about 8 to about 50 nucleotides in length, or about 10 to about 30 nucleotides in length. In certain embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% of the target sequence. The polynucleic acid molecule is at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 1-70 or 141-210. Alternatively and / or additionally, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from SEQ ID NOs: 71-140 or SEQ ID NOs: 211-280.

[0007] In some embodiments, the polynucleotide comprises at least one 2'-modified nucleotide, and further, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modified nucleotide. In some embodiments, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA), or a combination thereof. In certain embodiments, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In certain embodiments, the polynucleic acid molecule comprises three or more 2'-modified nucleotides selected from 2'-O-methyl and 2'-deoxy-2'-fluoro. In certain embodiments, the polynucleic acid molecule comprises a 5'-terminal vinylphosphonate-modified nucleotide, such as those described in U.S. Patent Application Publication No. 2019 / 0192681.

[0008] In certain embodiments, the 2'-modified nucleotide is a 2'-O-methyl modified nucleotide, and the 2'-O-methyl modified nucleotide is at the 5'-end of the sense strand and / or the antisense strand.In some embodiments, the 2'-O-methyl modified nucleotide is a purine nucleotide, or the 2'-O-methyl modified nucleotide is a pyridine nucleotide.In certain embodiments, the sense strand and / or the antisense strand comprises at least two, three, or four consecutive 2'-O-methyl modified nucleotides at the 5'-end.

[0009] In certain embodiments, the polynucleic acid molecule conjugate includes a linker connecting the target cell binding moiety to the polynucleic acid moiety. In such embodiments, the linker is a C1-C6 alkyl linker, or the linker is a homobifunctional or heterobifunctional linker and includes a maleimide group, a dipeptide moiety, a benzoic acid group, or derivatives thereof. Alternatively and / or additionally, the linker is a cleavable or non-cleavable linker. In certain embodiments, the ratio between the polynucleic acid moiety and the target cell binding moiety is about 1:1, 2:1, 3:1, or 4:1.

[0010] In certain embodiments, the polynucleic acid portion mediates RNA interference against human DUX4 and alleviates symptoms of muscular dystrophy in a subject. In some embodiments, the RNA interference comprises reducing the expression of the mRNA transcript of the DUX4 gene by at least 50%, at least 60%, or at least 70% or more compared to the amount of the mRNA transcript of the DUX4 gene in untreated cells. Alternatively and / or additionally, the RNA interference comprises affecting the expression of a marker gene in the cell, the marker gene being selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX. In some embodiments, affecting the expression of the marker gene reduces the expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or more. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0011] In certain embodiments, the polynucleic acid molecule conjugate comprises a molecule of formula (I): AXB, where A is an antibody or antigen-binding fragment thereof, B is a polynucleic acid molecule that hybridizes to a target sequence of DUX4, and X is a bond or non-polymeric linker that is conjugated to a cysteine ​​residue of A.

[0012] In some embodiments, pharmaceutical compositions are disclosed herein, comprising a polynucleic acid molecule conjugate described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, or intravenous, subcutaneous, or intrathecal administration.

[0013] Symptoms of FSHD include effects on skeletal muscle. Skeletal muscles affected by FSHD include those around the eyes and mouth, shoulder muscles, upper arm muscles, lower leg muscles, abdominal muscles, and hip muscles. In some cases, FSHD symptoms also affect vision and hearing. In some cases, FSHD symptoms also affect heart and lung function. In some cases, FSHD symptoms include muscle weakness, muscle atrophy, muscular dystrophy, pain inflammation, contractures, scoliosis, lordosis, hypoventilation, retinal abnormalities, exposure to keratitis, mild hearing loss, and EMG abnormalities. As used herein, the term muscle atrophy refers to the broad range of muscle-related effects of FSHD.

[0014] In certain embodiments, disclosed herein is a method for treating muscular dystrophy in a subject in need thereof by providing a polynucleic acid conjugate described herein and administering the polynucleic acid conjugate to the subject in need thereof to treat the muscular dystrophy. The polynucleic acid conjugate reduces the amount of human DUX4 mRNA transcripts. In some embodiments, the polynucleic acid moiety mediates RNA interference against human DUX4, thereby regulating muscle atrophy in the subject. In certain embodiments, the RNA interference involves affecting the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX in cells affected by muscular dystrophy. Preferably, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0015] Disclosed herein, in certain embodiments, is the use of a polynucleic acid molecule conjugate or pharmaceutical composition described herein for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD). Also disclosed herein, in certain embodiments, is the use of a polynucleic acid molecule conjugate or pharmaceutical composition described herein for the manufacture of a medicament for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

[0016] Disclosed herein, in certain embodiments, are kits comprising the polynucleic acid molecule conjugates or pharmaceutical compositions described herein. [Brief explanation of the drawings]

[0017] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings. The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] Illustrates a diagram of FSHD pathology. [Figure 2] 1 shows a flow chart diagram of in silico selection of DUX4 siRNA. [Figure 3] The location and number of selected DUX4 siRNAs in the DUX4 mRNA transcript are shown. [Figure 4] Immunofluorescence detection of DUX4 expression in myonuclei. [Figure 5] 1 shows a bar graph of siRNA-mediated reduction of DUX4 target biomarker gene expression. [Figure 6]6A-B show graphs of siRNA-mediated reduction of DUX4 target biomarker gene expression in cultured FSHD primary myotubes and FSHD complex of siRNA-mediated reduction of DUX4 target biomarker gene expression in cultured FSHD primary myotubes. [Figure 7] A bar graph of ACTA1 gene expression in myotubes treated with DUX4 siRNA at a concentration of 10 nM is shown. [Figure 8] FSHD complex expression in myotubes treated with DUX4 siRNA at a concentration of 10 nM is shown. [Figure 9] FSHD complex expression in myotubes treated with DUX4 siRNA at a concentration of 0.5 nM is shown. [Figure 10] 1 shows a flow chart diagram of the selection of the top 28 DUX4 siRNAs based on data from the initial screen. [Figure 11] Figures 11A-B show FSHD complex expression in two FSHD primary myotubes upon treatment with DUX4 siRNA at concentrations of 10 nM and 0.5 nM. [Figure 12] 1 shows a KD correlation analysis identifying DUX4 siRNAs effective in one or more FSHD primary myotubes. [Figure 13] ACTA1 gene expression in patient-derived myotubes treated with DUX4 siRNA at a concentration of 10 nM is shown. [Figure 14] FSHD complex expression in patient-derived myotubes treated with DUX4 siRNA at a concentration of 10 nM. [Figure 15] FSHD complex expression in myotubes from six patients treated with DUX4 siRNA is shown. [Figure 16A] 1 shows a graph of FSHD complex expression in myotubes from three patients treated with 14 selected DUX4 siRNAs. [Figure 16B] 1 shows a graph of FSHD complex expression in myotubes from three patients treated with 14 selected DUX4 siRNAs. [Figure 16C]1 shows a graph of FSHD complex expression in myotubes from three patients treated with 14 selected DUX4 siRNAs. [Figure 17A] 1 shows a graph of FSHD complex expression in myotubes from three FSHD patients treated with eight selected DUX4 siRNAs. [Figure 17B] 1 shows a graph of FSHD complex expression in myotubes from three FSHD patients treated with eight selected DUX4 siRNAs. [Figure 17C] 1 shows a graph of FSHD complex expression in myotubes from three FSHD patients treated with eight selected DUX4 siRNAs. [Figure 18] Figures 18A-B show graphs of FSHD complex expression in cultured FSHD primary myotubes treated with eight antibody DUX4-siRNA conjugates without vinyl phosphonate (DUX4-AOC) or eight antibody DUX4-siRNA conjugates with vinyl phosphonate. [Figure 19] 1 shows a graph of AOC-mediated in vivo reduction of nuclear-localized Inc-RNA Malat1 levels in mouse skeletal muscle. [Figure 20] 1 shows a graph of sustained SSB-AOC-mediated in vivo reduction of SSB mRNA levels in mouse skeletal muscle over an 8-week period with a single dose of 3 mg / kg siRNA. DETAILED DESCRIPTION OF THE INVENTION

[0018] Muscle atrophy is the loss of muscle mass or the progressive weakening and degeneration of muscles, such as skeletal or voluntary muscles that control movement, cardiac muscle, and smooth muscle. Various pathophysiological diseases, including disuse, cancer, diabetes, and renal failure, or treatment with glucocorticoids, cause muscle atrophy and loss of strength. The phenotypic effects of muscle atrophy are caused by various molecular events, including inhibition of muscle protein synthesis, increased muscle protein turnover, abnormal regulation of satellite cell differentiation, and abnormal muscle fiber type conversion.

[0019] FSHD is a rare, progressively disabling disease with no approved treatment. It is one of the most common forms of muscular dystrophy, affecting both men and women equally and typically occurring in teenagers and young adults. FSHD is characterized by progressive skeletal muscle loss, initially causing muscle weakness in the face, shoulders, arms, and trunk, and progressing to muscle weakness in the lower limbs and pelvic girdle. Skeletal muscle weakness results in severe physical limitations. This includes progressive loss of facial muscles, which can lead to an inability to smile or communicate, difficulty using the arms for daily activities, and difficulty getting out of bed and using a wheelchair for daily mobility activities. The majority of patients with FSHD report experiencing chronic pain, anxiety, and depression.

[0020] FSHD is caused by abnormal expression of the gene DUX4 in skeletal muscle, resulting in inappropriate expression of the DUX4 protein. DUX4 itself is a transcription factor that induces the expression of other genes, and these inappropriately expressed downstream genes cause muscle pathology. Normally, DUX4-driven gene expression is restricted to germline and early stem cell development. In FSHD patients, the DUX4 protein in skeletal muscle regulates other gene products, some of which are toxic to muscle. Evidence of abnormal DUX4-driven gene expression is a key molecular feature that distinguishes FSHD-affected muscle tissue from healthy muscle. The result of abnormal DUX4 expression in FSHD is muscle death and its replacement by fat, leading to skeletal muscle weakness and progressive disability. Data suggest that reducing the expression of the DUX4 gene and its downstream transcriptional program may provide a disease-modifying therapeutic approach to treat the underlying cause of FSHD.

[0021] There are two ways in which the DUX4 gene can be desilencing or derepressing. FSHD1, which accounts for approximately 95% of FSHD patients, harbors a series of DNA shortening mutations in a region near the end of the long arm of chromosome 4 known as D4Z4, which contains repeats in the subtelomeric region of the chromosome. The D4Z4 region is abnormally shortened, containing 1–10 repeats instead of the usual 11–100. This contraction results in hypomethylation of the D4Z4 region and derepression of DUX4. FSHD2 patients do not have a significant D4Z4 repeat contraction, but instead have mutations in a regulatory gene known as SMCHD1, which normally contributes to DUX4 gene repression via DNA methylation. Mutations in the SMCHD1 gene cause hypomethylation of the D4Z4 region, resulting in loss of repression, leading to inappropriate expression of DUX4 and disease progression. Figure 1 provides an illustration of FSHD pathology.

[0022] Nucleic acid (e.g., RNAi) therapy is a targeted therapy that boasts high selectivity and specificity. However, in some cases, nucleic acid therapy is also hindered by poor cellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications of nucleic acid compositions have been explored, such as novel linkers for better stabilization and / or lower toxicity, optimization of binding moieties for increased target specificity and / or targeted delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0023] In some embodiments, the arrangement or order of the various components comprising the nucleic acid composition further affects cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, when the nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide), the order or arrangement of the binding moiety, polymer, and / or polynucleic acid molecule (or polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule) further affects cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0024] In some embodiments, the present disclosure includes polynucleic acid molecules and polynucleic acid molecule conjugates for the treatment of muscle atrophy, particularly facioscapulohumeral muscular dystrophy or associated muscle atrophy. In some instances, the polynucleic acid molecule conjugates described herein enhance intracellular uptake, stability, and / or efficacy. In some instances, the polynucleic acid molecule conjugates include an antibody or antigen-binding fragment conjugated to a polynucleic acid molecule. In some instances, the polynucleic acid molecule hybridizes to the target sequence of DUX4, preferably human DUX4.

[0025] Further embodiments described herein include methods of treating FSHD comprising administering to a subject a polynucleic acid molecule or polynucleic acid molecule conjugate described herein.

[0026] Polynucleic acid molecule In certain embodiments, the polynucleic acid molecule hybridizes to a target sequence of the double homeobox 4 (DUX4) gene. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of the human DUX4 gene (DUX4) and reduce DUX4 mRNA in muscle cells.

[0027] In some embodiments, the polynucleic acid molecule comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-70. In some embodiments, the polynucleic acid molecule comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 141-210. In some embodiments, the polynucleic acid molecule comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 71-140. In some embodiments, the polynucleic acid molecule comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 211-280.

[0028] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-70. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 71-140. In some cases, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 141-210. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 211-280.

[0029] In some embodiments, the polynucleic acid molecule comprises a sense strand (e.g., passenger strand) and an antisense strand (e.g., guide strand). In some cases, the sense strand (e.g., passenger strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-70. In some cases, the antisense strand (e.g., guide strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 71-140. In some embodiments, the polynucleic acid molecule comprises a sense strand (e.g., passenger strand) and an antisense strand (e.g., guide strand). In some cases, the sense strand (e.g., passenger strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 141-210. In some cases, the antisense strand (e.g., guide strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 211-280.

[0030] In some cases, the sense strand comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1, 2, 3, 6, 14, 36, 52, 56, 61, 62, 63, 65, and 66. In some cases, the antisense strand comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 71, 72, 73, 76, 84, 106, 122, 127, 131, 132, 133, 135, and 136. In some cases, the siRNA comprises the sense and antisense strands provided in Table 11.

[0031] In some cases, the sense strand comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 141, 142, 143, 146, 176, 192, 196, 201, 202, 203, 205, 206. The antisense strand comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 211, 212, 213, 216, 246, 262, 266, 271, 272, 273, 275, 276. In some cases, the siRNA comprises a sense strand and an antisense strand as presented in Table 12.

[0032] In some embodiments, the polynucleic acid molecules described herein comprise RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some instances, the RNA comprises small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some instances, the RNA comprises shRNA. In some instances, the RNA comprises miRNA. In some instances, the RNA comprises dsRNA. In some instances, the RNA comprises tRNA. In some instances, the RNA comprises rRNA. In some instances, the RNA comprises hnRNA. In some instances, the RNA comprises siRNA. In some instances, the polynucleic acid molecule comprises siRNA.

[0033] In some embodiments, the nucleic acid polymer is about 8 to about 50 nucleotides in length. In some embodiments, the nucleic acid polymer is about 10 to about 50 nucleotides in length. In some embodiments, the polynucleic acid molecule is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length.

[0034] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 19 nucleotides in length. In some instances, the polynucleic acid molecule is about 18 nucleotides in length. In some instances, the polynucleic acid molecule is about 17 nucleotides in length. In some instances, the polynucleic acid molecule is about 16 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 nucleotides in length. In some instances, the polynucleic acid molecule is about 14 nucleotides in length. In some instances, the polynucleic acid molecule is about 13 nucleotides in length. In some instances, the polynucleic acid molecule is about 12 nucleotides in length. In some instances, the polynucleic acid molecule is about 11 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 nucleotides in length. In some instances, the polynucleic acid molecule is about 8 nucleotides in length. In some instances, the polynucleic acid molecule is about 8 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 to about 30 nucleotides in length.In some instances, the polynucleic acid molecule is about 12 to about 30 nucleotides in length.

[0035] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide. In some instances, the polynucleic acid molecule comprises a second polynucleotide. In some instances, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide is a sense strand or passenger strand. In some instances, the second polynucleotide is an antisense strand or guide strand.

[0036] In some embodiments, the polynucleic acid molecule is a first polynucleotide. In some embodiments, the first polynucleotide is about 8 to about 50 nucleotides in length. The first polynucleotide is about 10 to about 50 nucleotides in length. In some embodiments, the first polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length.

[0037] In some instances, the first polynucleotide is about 50 nucleotides in length. In some instances, the first polynucleotide is about 45 nucleotides in length. In some instances, the first polynucleotide is about 40 nucleotides in length. In some instances, the first polynucleotide is about 35 nucleotides in length. In some instances, the first polynucleotide is about 30 nucleotides in length. In some instances, the first polynucleotide is about 25 nucleotides in length. In some instances, the first polynucleotide is about 20 nucleotides in length. In some instances, the first polynucleotide is about 19 nucleotides in length. In some instances, the first polynucleotide is about 18 nucleotides in length. In some instances, the first polynucleotide is about 17 nucleotides in length. In some instances, the first polynucleotide is about 16 nucleotides in length. In some instances, the first polynucleotide is about 15 nucleotides in length. In some instances, the first polynucleotide is about 14 nucleotides in length. In some examples, the first polynucleotide is about 13 nucleotides in length. In some examples, the first polynucleotide is about 12 nucleotides in length. In some examples, the first polynucleotide is about 11 nucleotides in length. In some examples, the first polynucleotide is about 10 nucleotides in length. In some examples, the first polynucleotide is about 8 nucleotides in length. In some examples, the first polynucleotide is about 8 to about 50 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 50 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 45 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 40 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 35 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 30 nucleotides in length.In some examples, the first polynucleotide is about 10 to about 25 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 20 nucleotides in length. In some examples, the first polynucleotide is about 15 to about 25 nucleotides in length. In some examples, the first polynucleotide is about 15 to about 30 nucleotides in length. In some examples, the first polynucleotide is about 12 to about 30 nucleotides in length.

[0038] In some embodiments, the polynucleic acid molecule is a second polynucleotide. In some embodiments, the second polynucleotide is about 8 to about 50 nucleotides in length. In some embodiments, the second polynucleotide is about 10 to about 50 nucleotides in length. In some embodiments, the second polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length.

[0039] In some instances, the second polynucleotide is about 50 nucleotides in length. In some instances, the second polynucleotide is about 45 nucleotides in length. In some instances, the second polynucleotide is about 40 nucleotides in length. In some instances, the second polynucleotide is about 35 nucleotides in length. In some instances, the second polynucleotide is about 30 nucleotides in length. In some instances, the second polynucleotide is about 25 nucleotides in length. In some instances, the second polynucleotide is about 20 nucleotides in length. In some instances, the second polynucleotide is about 19 nucleotides in length. In some instances, the second polynucleotide is about 18 nucleotides in length. In some instances, the second polynucleotide is about 17 nucleotides in length. In some instances, the second polynucleotide is about 16 nucleotides in length. In some instances, the second polynucleotide is about 15 nucleotides in length. In some instances, the second polynucleotide is about 14 nucleotides in length. In some instances, the second polynucleotide is about 13 nucleotides in length. In some instances, the second polynucleotide is about 12 nucleotides in length. In some instances, the second polynucleotide is about 11 nucleotides in length. In some instances, the second polynucleotide is about 10 nucleotides in length. In some instances, the second polynucleotide is about 8 nucleotides in length. In some instances, the second polynucleotide is about 8 to about 50 nucleotides in length. In some instances, the second polynucleotide is about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is about 10 to about 45 nucleotides in length. In some instances, the second polynucleotide is about 10 to about 40 nucleotides in length. In some instances, the second polynucleotide is about 10 to about 35 nucleotides in length. In some instances, the second polynucleotide is about 10 to about 30 nucleotides in length.In some examples, the second polynucleotide is about 10 to about 25 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 20 nucleotides in length. In some examples, the second polynucleotide is about 15 to about 25 nucleotides in length. In some examples, the second polynucleotide is about 15 to about 30 nucleotides in length. In some examples, the second polynucleotide is about 12 to about 30 nucleotides in length.

[0040] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the polynucleic acid molecule further comprises a blunt end, an overhang, or a combination thereof. In some instances, the blunt end is a 5' blunt end, a 3' blunt end, or both. Optionally, the overhang is a 5' overhang, a 3' overhang, or both. Optionally, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base paired nucleotides. Optionally, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base paired nucleotides. Optionally, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base paired nucleotides. Optionally, the overhang comprises 1, 2, 3, or 4 non-base paired nucleotides. Optionally, the overhang comprises 1 non-base paired nucleotide. Optionally, the overhang comprises 2 non-base paired nucleotides. Optionally, the overhang comprises 3 non-base paired nucleotides. Optionally, the overhang comprises 4 non-base paired nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises two non-base-pairing nucleotides at the 3' end as an overhang, while the sense strand has no overhang. Optionally, in such embodiments, the non-base-pairing nucleotides have the sequence TT, dTdT, or UU. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand has one or more nucleotides at the 5' end that are complementary to the antisense sequence.

[0041] In some embodiments, the sequence of the polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence of DUX4. In some embodiments, the target sequence of DUX4 is a nucleic acid sequence 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, wherein the first nucleotide of the target sequence starts at any nucleotide of the coding region or 5' or 3' untranslated region (UTR) of the DUX4 mRNA transcript. For example, the first nucleotide of the target sequence can be selected so that it starts at nucleic acid position (nal, a number starting from the 5' end of the entire length of the DUX mRNA, e.g., the first nucleotide at the 5' end is nal.1) 1, nal 2, nal 3, nal 4, nal 5, nal 6, nal 7, nal 8, nal 9, nal 10, nal 11, nal 12, nal 13, nal 14, nal 15, nal 16, nal 17, or any other nucleic acid position in the coding or non-coding region (5' or 3' untranslated region) of the DUX mRNA.In some embodiments, the first nucleotide of the target sequence is nal10-nal15, nal10-nal20, nal50-nal60, nal55-nal65, nal10-nal15, nal10-nal20, nal55-nal65, nal75-nal85, nal95-nal105, nal135-nal145, nal155-nal165, nal225-nal235, nal265-nal275, nal275-nal285, nal285-nal295, nal325-nal335, nal335-nal345, nal385-nal395, nal515-nal525, nal665-nal675, nal675-nal685, nal 695-nal 705, nal 705-nal 715, nal 875-nal 885, nal 885-nal 895, nal 895-nal 905, nal 1035-nal 1045, nal 1045-nal 1055, nal 1125-nal 1135, nal 1135-nal 1145, 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 1285, nal 1285-nal 1295, nal 1305-nal 1315, nal It can be selected to start at or between nal 1125-nal 1135, nal 1155-nal 1165, nal 1225-nal 1235, nal 1235-nal 1245, nal 1275-nal 1285, nal 1285-nal 1295, nal 1305-nal 1315, nal 1315-nal 1325, nal 1335-nal 1345, nal 1345-nal 1355, nal 1525-nal 1535, nal 1535-nal 1545, nal 1605-nal 1615, nal 1615-nal 1625, nal 1625-nal 1635.

[0042] In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some examples, the sequence of the polynucleic acid molecule is 100% complementary to a target sequence described herein.

[0043] In some embodiments, the sequence of the polynucleic acid molecule has five or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has four or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has three or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has two or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has one or fewer mismatches to the target sequences described herein.

[0044] In some embodiments, a group of polynucleic acid molecules that potentially bind to the DUX4 target sequence is selected from all polynucleic acid molecules to create a polynucleic acid molecule library. In certain embodiments, such a selection process is performed in silico via one or more steps to eliminate less desirable polynucleic acid molecules from the candidates. For example, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules with single nucleotide polymorphisms (SNPs) and / or MEF<-5. Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules with 0 and 1 mismatch (MM) in the human transcriptome (so that the only acceptable hits are DUX, DUX5, and DBET). Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules with 0 MM in human intragenic regions (so that the only acceptable hits are DUX1, DUX5, and DBET pseudogenes). Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules having a DUX4 human sequence from the MM used in the FLExDUX4 FSHD mouse model. Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules with a predicted survival rate <60. Alternatively and / or additionally, such a selection process includes carrying forward one or more polynucleic acid molecules with a predicted survival rate ≥60. Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules that match the seed region of a known miRNA1-1000. Alternatively and / or additionally, in some embodiments, the selection process includes removing one or more polynucleic acid molecules with a %GC content of 75 or greater. Alternatively and / or additionally, in some embodiments, the selection process includes selecting for no more than eight predicted off-target hits in 2MM. In some embodiments, no more than 12 predicted off-target hits in 2MM are allowed for the region 295-1132 (nal 295-1132).

[0045] In some embodiments, the selection process is performed in silico through one or more successive steps that eliminate less desirable polynucleic acid molecules from the candidates. For example, in some embodiments, the selection process begins by collecting candidate polynucleic acid molecules to generate a library. From the library, the first elimination step involves removing one or more polynucleic acid molecules with single nucleotide polymorphisms (SNPs) and / or MEF<-5. Next, the second elimination step involves removing one or more polynucleic acid molecules with 0 and 1 MM in the human transcriptome (so that the only allowed hits are DUX, DUX5, and DBET). Then, the third elimination step involves removing one or more polynucleic acid molecules with 0 MM in human intragenic regions (so that the only allowed hits are DUX1, DUX5, and DBET pseudogenes). Then, the next elimination step involves removing one or more polynucleic acid molecules with a human sequence from MM to DUX4, which is used in the FLExDUX4 FSHD mouse model. Next, the next step involves retaining only polynucleic acid molecules, or one or more polynucleic acid molecules, with a predicted survival rate of 60 or greater. Next, a pruning step involves removing one or more polynucleic acid molecules that match the seed region of known miRNAs 1-1000. This is followed by a pruning step that removes one or more polynucleic acid molecules with a %GC content of 75 or greater. The final selection process then involves no more than eight predicted off-target hits in 2MM, except for the region 295-1132, where a maximum of 12 hits is allowed.

[0046] In some embodiments, the specificity of a polynucleic acid molecule that hybridizes to a target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some instances, hybridization is under highly stringent hybridization conditions.

[0047] In some embodiments, the polynucleic acid molecule has reduced off-target effects. In some instances, "off-target" or "off-target effect" refers to any instance in which a polynucleic acid polymer directed against a given target causes an unintended effect by directly or indirectly interacting with another mRNA sequence, DNA sequence, cellular protein, or other moiety. In some instances, an "off-target effect" occurs when there is simultaneous degradation of other transcripts due to partial homology or complementarity between the other transcripts and the sense and / or antisense strands of the polynucleic acid molecule.

[0048] In some embodiments, polynucleic acid molecules contain natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules contain a combination of DNA, RNA, and / or nucleotide analogs. In some instances, synthetic or artificial nucleotide analogs or bases contain modifications at one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0049] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some examples, the modification includes H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some examples, the alkyl moiety further comprises a modification. In some examples, the modification includes 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, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some examples, the alkyl moiety further comprises a heterosubstitution. In some examples, a carbon of the heterocyclic group is replaced with nitrogen, oxygen, or sulfur. In some examples, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0050] In some cases, the modification of the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. 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. [ka]

[0051] In some embodiments, the modification of the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some instances, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving its cellular uptake properties due to its zwitterionic properties. A typical chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below. [ka]

[0052] In some instances, the modification of the 2' hydroxyl group is a locked or 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, thus forming a 2'-C,4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. Representative examples of the chemical structure of LNA are illustrated below. The representative example shown on the left highlights the chemical connectivity of the LNA monomer. The representative example shown on the right shows a locked 3'-endo ( 3 E) emphasizes structure. [ka]

[0053] In some embodiments, the modification at the 2' hydroxyl group locks the sugar structure into a 3'-endo sugar puckering conformation, e.g., ethylene nucleic acid (ENA), such as 2'-4'-ethylene bridged nucleic acid. ENA is part of the bridged nucleic acid class of modified nucleic acids, which also includes LNA. Typical chemical structures of ENA and bridged nucleic acids are illustrated below. [ka]

[0054] In some embodiments, additional modifications at the 2' hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0055] In some embodiments, nucleotide analogs include, but are not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, Included are 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, queusine, archaeosine, naphthyl and substituted naphthyl groups, O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine, 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides further include nucleotides modified at the sugar moiety, as well as nucleotides having non-ribosyl sugars or analogs thereof. 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 those known in the art as universal bases.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0056] In some embodiments, the nucleotide analog further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics natural nucleic acid structures by deviating from normal sugar and phosphate structures. In some instances, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some cases, backbone modification removes all positive and negative charges, making morpholino neutral molecules capable of crossing cell membranes without the aid of cellular delivery agents, such as those used by charged oligonucleotides. [ka]

[0057] In some embodiments, peptide nucleic acids (PNAs) contain no sugar backbone rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating backbone charge. [ka]

[0058] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some examples, the modified internucleotide bond includes, but is not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, borontrifluorides, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkylphosphonates, alkylphosphonothioates, arylphosphorothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, phosphoropipera Antisense oligonucleotides include didate, phosphoroanilothioate, phosphoroanilidate, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazo, methylenedimethyldimethylhydrazo, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, thioamidate, riboacetyl group bond, aminoethylglycine, silyl, or siloxane bond, e.g., saturated or unsaturated and / or substituted and / or heteroatom-containing alkyl or cycloalkyl bond of 1 to 10 carbon atoms with or without heteroatoms, morpholino bond, amide, polyamide in which bases are directly or indirectly bound to the aza nitrogen of the backbone, or combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate linkages. Exemplary PS ASOs are described below. [ka]

[0059] In some instances, the modification is a methyl or thiol modification, such as a methyl phosphonate or thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left) and methyl phosphonate nucleotides (right) are illustrated below. [ka]

[0060] In some examples, modified nucleotides include, but are not limited to, 2'-fluoro N3-P5'-phosphoramidites, exemplified as follows: [ka]

[0061] In some examples, modified nucleotides include, but are not limited to, hexitol nucleic acids (alternatively, 1',5'-anhydrohexitol nucleic acids (HNA)), exemplified as follows: [ka]

[0062] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications of the 3'- or 5'-terminal nucleotide analog. For example, the 3'-terminal optionally includes a 3' cationic group, or the nucleoside is inverted at the 3'-terminal including a 3'-3' linkage. In another alternative, the 3'-terminal is optionally linked to an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In a further alternative, the 3'-terminal is optionally linked to an abasic site, such as an apurinic or apyrimidinic acid site. In some instances, the 5'-terminal is linked to an aminoalkyl group, such as a 5'-O-aminoalkyl substituent. In some cases, the 5'-terminal is linked to an abasic site, such as an apurinic or apyrimidinic acid site.

[0063] In some embodiments, a polynucleic acid molecule comprises one or more artificial nucleotide analogs described herein. In some examples, a polynucleic acid molecule described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof. In some embodiments, the polynucleic acid molecule is 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 , modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or a combination thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogs selected from the group consisting of:In some embodiments, a polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methyl modified nucleotides. In some embodiments, a polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, a polynucleic acid molecule described herein 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.

[0064] In some examples, the polynucleic acid molecule comprises at least one of the following: 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.

[0065] In some cases, the polynucleic acid molecule comprises at least one of the following: 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.

[0066] In some cases, the polynucleic acid molecule comprises at least one of the following: 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.

[0067] In some examples, the polynucleic acid molecule comprises at least one of the following: 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.

[0068] In some examples, the polynucleic acid molecule comprises at least one of the following: 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.

[0069] In some cases, the polynucleic acid molecule comprises at least one of the following: 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.

[0070] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0071] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 30% modifications, and about 20% to about 30% modifications.

[0072] Optionally, the polynucleic acid molecule contains about 10% to about 20% modifications.

[0073] In some cases, the polynucleic acid molecule contains from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

[0074] In further instances, the polynucleic acid molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0075] In some embodiments, the polynucleic acid 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, or more modifications.

[0076] In some examples, the polynucleic acid 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, or more modified nucleotides.

[0077] In some examples, about 5 to about 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 10% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 15% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 20% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 25% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 30% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 35% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 40% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 45% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 50% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 55% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 60% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 65% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 70% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 75% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 80% of the polynucleic acid molecules contain an artificial nucleotide analog described herein.In some examples, about 85% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 90% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 95% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 96% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 97% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 98% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 99% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, -methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0078] In some embodiments, the polynucleic acid molecule comprises from about 1 to about 25 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 1 modification, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 2 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 3 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 4 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 5 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 6 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 7 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 8 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 9 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 10 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, wherein the modifications comprise an artificial nucleotide analog described herein.In some embodiments, the polynucleic acid molecule comprises about 15 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 16 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 17 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 18 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 19 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 20 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 21 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 22 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 23 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 24 modifications, wherein the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 25 modifications, wherein the modifications include artificial nucleotide analogs described herein.

[0079] In some embodiments, the polynucleic acid molecule is assembled from two separate polynucleotides, where one polynucleotide comprises the sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule, hi other embodiments, the sense strand is connected to the antisense strand by a linker molecule, which in some instances is a polynucleotide linker or a non-nucleotide linker.

[0080] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides in the sense strand comprise 2'-O-methylpyrimidine nucleotides and the purine nucleotides in the sense strand comprise 2'-deoxypurine nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides present in the sense strand comprise 2'-deoxypurine nucleotides.

[0081] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, are 2'-O-methyl purine nucleotides.

[0082] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, comprise 2'-deoxy-purine nucleotides.

[0083] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and at least one of the sense strand and the antisense strand has a plurality (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, etc.) of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, at least two of the plurality of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides. In some embodiments, the consecutive 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, the consecutive 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the sense strand of the polynucleic acid molecule comprises at least 4, at least 5, or at least 6 consecutive 2'-O-methyl modified nucleotides at its 5'-end and / or 3'-end, or both. Optionally, in such embodiments, the sense strand of the polynucleic acid molecule comprises at least one, at least two, at least three, or at least four 2'-deoxy-2'-fluoro modified nucleotides at the 5'-end of at least four, at least five, or at least six contiguous 2'-O-methyl modified nucleotides at the 5'-end of the polynucleotide, at the 3'-end of the polynucleotide, or at least two, at least three, or at least four contiguous 2'-O-methyl modified nucleotides at the 3'-end of the polynucleotide. Also optionally, such at least two, at least three, or at least four 2'-deoxy-2'-fluoro modified nucleotides are contiguous nucleotides.

[0084] In some embodiments, the polynucleic acid 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 pyridine nucleotide.

[0085] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand has two or more consecutive 2'-deoxy-2'-fluoro modified nucleotides at its 5'-end. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand has two or more consecutive 2'-O-methyl modified nucleotides at its 3'-end. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand has at least 2, 3, 4, 5, 6, or 7 consecutive 2'-O-methyl modified nucleotides.

[0086] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand comprises 5'-nsnsnnnnNfNfNfnnnnnnnnsnsa-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification). In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand comprises 5'-UfsNfsnnnNfnnnnnnNfnNfnnnsusu-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification). In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-nsnsnnnnNfNfNfnnnnnnnnsnsa-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification) and the antisense strand comprises 5'-UfsNfsnnnNfnnnnnnnnNfnNfnnnsusu-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification).

[0087] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a cap moiety at the 5'-end, the 3'-end, or both the 5' and 3' ends of the sense strand, hi other embodiments, the terminal cap moieties are inverted deoxyabasic moieties.

[0088] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

[0089] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein 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 internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides with and / or universal base modifications, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 10, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, or with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or with or without end cap molecules at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0090] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand has about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without 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 internucleotide linkages, and / or end cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0091] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both the 3'- and 5'-ends, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. In some embodiments, the antisense strand comprises about 1 to about 10, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or end cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0092] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense 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 internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5, e.g., about 1, 2, 3, 4, 5, or more, phosphorothioate internucleotide linkages, and / or end cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0093] In some embodiments, the polynucleic acid molecules described herein are chemically modified short interfering nucleic acid molecules having about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages in each strand of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand comprises a phosphate backbone modification at the 3'-end of the antisense strand. Alternatively and / or additionally, the polynucleic acid 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 embodiments, the sense strand or the antisense strand has three consecutive nucleosides linked via two phosphorothioate backbones.

[0094] In another embodiment, the polynucleic acid molecules described herein comprise a 2'-5' internucleotide linkage. In some examples, the 2'-5' internucleotide linkage is at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In additional examples, the 2'-5' internucleotide linkage is present at various other positions within one or both strands of the sequence, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of all internucleotide linkages of pyrimidine nucleotides in one or both strands of the polynucleic acid molecule comprise a 2'-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of all internucleotide linkages of purine nucleotides in one or both strands of the polynucleic acid molecule comprise a 2'-5' internucleotide linkage.

[0095] In some embodiments, the polynucleic acid molecule mediates RNAi activity in a cell or is a single-stranded polynucleic acid molecule reconstituted in an in vitro system, wherein the polynucleic acid molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, wherein a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and any purine nucleotides present in the polynucleic acid are 2'-deoxy purine nucleotides. and the terminal cap modification is optionally present at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus of the antisense sequence; the polynucleic acid molecule optionally further comprises about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxyribonucleotides at the 3'-terminus of the polynucleic acid molecule, wherein the terminal nucleotides further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages; and the polynucleic acid molecule optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0096] In some cases, one or more of the artificial nucleotide analogs described herein are more resistant to nucleases, such as ribonucleases, e.g., RNase H, deoxyribonucleases, e.g., DNase, or exonucleases, e.g., 5'-3' exonucleases and 3'-5' exonucleases, compared to naturally occurring polynucleic acid molecules. In some examples, the artificial nucleotide analogs are 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 modified 2'-O-N-methylacetamide (2'-O-N-methylacetamide). Artificial nucleotide analogs, including 2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases, such as ribonucleases, e.g., RNase H, deoxyribonucleases, e.g., DNases, or exonucleases, e.g., 5'-3' exonucleases or 3'-5' exonucleases. In some examples, the 2'-O-methyl modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some instances, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some instances, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, 2'-deoxy modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-deoxy-2'-O-fluoro modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, LNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, ENA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, HNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, morpholinos are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, PNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, methylphosphonate nucleotide modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, thiol phosphonate nucleotide modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some examples, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0097] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity for their mRNA targets compared to a comparable naturally occurring polynucleic acid molecule. One or more artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidite, have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-O-methyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-aminopropyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-deoxy modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-deoxy-2'-fluoro modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules.In some instances, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, LNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, ENA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, PNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, HNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, morpholino-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, methylphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, thiolphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, the increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0098] In some embodiments, the polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules comprising a single enantiomer. In some instances, the polynucleic acid molecules comprise L-nucleotides. In some instances, the polynucleic acid molecules comprise D-nucleotides. In some instances, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some instances, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some instances, the polynucleic acid molecules are polynucleic acid molecules described in U.S. Patent Application Publication Nos. 2014 / 194610 and 2015 / 211006; and PCT International Publication No. WO2015107425.

[0099] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer-binding moiety. In some instances, the aptamer-binding moiety is a DNA aptamer-binding moiety. In some instances, the aptamer-binding moiety is Alphamer (Centauri Therapeutics), which includes an aptamer portion that recognizes a specific cell surface target and a portion that displays a specific epitope for binding to circulating antibodies. In some instances, the polynucleic acid molecules described herein are further modified to include an aptamer-binding moiety as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0100] In additional embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some instances, the polynucleic acid molecule is modified with one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the two hydroxyl positions, such as with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, or with locked or bridged ribose structures (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule further comprises morpholino, PNA, HNA, methyl phosphonate nucleotide, thiol phosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability. In some cases, the polynucleic acid molecule is a chiral pure (or stereopure) polynucleic acid molecule. In some cases, the chiral pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications of RNA to increase the stability of delivery will be apparent to those skilled in the art.

[0101] In some cases, the polynucleic acid molecule is a double-stranded polynucleotide molecule, comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a part thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof.In some cases, the polynucleic acid molecule is assembled from two separate polynucleotides, one strand is a sense strand, and the other strand is an antisense strand, wherein the antisense strand and the sense strand are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to the nucleotide sequence of the other strand; such as when the antisense strand and the sense strand form a duplex or double-stranded structure, for example, the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a part thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. Alternatively, the polynucleic acid molecule can be assembled from a single oligonucleotide, with the self-complementary sense and antisense regions of the polynucleic acid molecule being joined by a nucleic acid-based or non-nucleic acid-based linker.

[0102] In some cases, the polynucleic acid molecule is a polynucleotide with a double, asymmetric double, hairpin, or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a part thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof.In other cases, the polynucleic acid molecule is a circular single-stranded polynucleotide with two or more loop structures and a base comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a part thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof, and the circular polynucleotide is processed in vivo or in vitro to generate an active polynucleic acid molecule that can mediate RNAi. In further cases, the polynucleic acid molecule further comprises a single-stranded polynucleotide having a nucleotide sequence complementary to that of a target nucleic acid molecule or a portion thereof (e.g., such a polynucleic acid molecule need not be present within the polynucleic acid molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-stranded polynucleotide further comprises a terminal phosphate group such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate.

[0103] In some instances, an asymmetric hairpin is a linear polynucleic acid molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region, where the sense region contains sufficient complementary nucleotides to base pair with the antisense region but fewer nucleotides than the antisense region to form a looped duplex. For example, an asymmetric hairpin polynucleic acid molecule may comprise an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in vitro system and having a loop region comprising about 4 to about 8 nucleotides, and a sense region of about 3 to about 18 nucleotides complementary to the antisense region. In some instances, the asymmetric hairpin polynucleic acid molecule further comprises a chemically modified 5'-terminal phosphate group. In further instances, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0104] In some embodiments, an asymmetric duplex is a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region has sufficient complementary nucleotides to base-pair with the antisense region but contains fewer nucleotides than the antisense region, sufficient to form a duplex. For example, an asymmetric duplex polynucleic acid molecule may comprise an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in an in vitro system, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0105] In some cases, universal base refers to the nucleotide base analogue that forms base pairs with each of the natural DNA / RNA bases that are almost indistinguishable.Non-limiting examples of universal base include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole, as known in the prior art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0106] Polynucleic acid molecule synthesis In some embodiments, the polynucleic acid molecule described herein is constructed by chemical synthesis and / or enzymatic ligation reaction using procedures known in the art.For example, polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides, or by using various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between polynucleic acid molecule and target nucleic acid.Exemplary methods include those described in the following: U.S. Patent No. 5,142,047; U.S. Patent No. 5,185,444; U.S. Patent No. 5,889,136; U.S. Patent No. 6,008,400; and U.S. Patent No. 6,111,086; PCT International Publication No. WO2009099942; or European Patent Publication No. 1579015.Additional exemplary methods include those described in: Griffey et al., "2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides," J. Med. Chem. 39(26):5100-5109 (1997); Obika, et al., "Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar puckering." Tetrahedron Letters 38(50):8735 1997; Koizumi, M. "ENA oligonucleotides as therapeutics." Current opinion in molecular therapeutics 8(2):144-149 (2006); and Abramova et al., "Novel oligonucleotide analogues based on morpholino nucleoside subunits - antisense technologies: new "Chemical Possibilities," Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, the polynucleic acid molecule can be produced biologically using an expression vector into which the polynucleic acid molecule has been subcloned in an antisense orientation (i.e., the transcribed RNA of the inserted polynucleic acid molecule will be in an antisense orientation relative to the desired target polynucleic acid molecule).

[0107] In some embodiments, the polynucleic acid molecule is synthesized by a tandem synthesis method, where both strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker, which is then cleaved to provide separate fragments or strands that hybridize to the duplex and allow for purification of the duplex.

[0108] In some instances, the polynucleic acid molecule is 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.

[0109] Further modification methods, for example, to incorporate sugar, base, and phosphate modifications, include the following: Eckstein et al., International Publication PCT No. WO 92 / 07065; Perrault et al., Nature, 1990, 344, 565-568; Pieken et al., Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al., International Publication PCT No. WO 93 / 15187; Sproat, US Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97 / 26270; Beigelman et al. al.,USPat.No.5,716,824;Usman et al.,USPat.No.5,627,053;Woolf et al.,International PCT Publication No.WO 98 / 13526;Thompson et al.,USSer.No.60 / 082,404 which was filed on Apr.20,1998;Karpeisky et al. al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. The publication describes methods and strategies for determining the location for incorporating sugar, base, and / or phosphate modifications into nucleic acid molecules without modulating catalytic activity.

[0110] In some cases, chemical modification of internucleotide bonds of polynucleic acid molecules with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate bonds improves stability, while excessive modification often causes toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide bonds is sometimes minimized. In such cases, reducing the concentration of these bonds reduces the toxicity of these molecules and increases their efficacy and specificity.

[0111] Polynucleic acid molecule conjugates In some embodiments, the polynucleic acid molecule (B) is further conjugated to a polypeptide A, which is delivered to a desired site. In some cases, the polynucleic acid molecule is conjugated to a polypeptide A, optionally with a polymer moiety. In some examples, at least one polypeptide A is conjugated to at least one B. In some examples, at least one polypeptide A is conjugated to at least one B to form an AB conjugate. In some embodiments, at least one A is conjugated to the 5' end of B, the 3' end of B, an internal site of B, or any combination thereof. In some examples, at least one polypeptide A is conjugated to at least two Bs. In some examples, at least one polypeptide A is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more Bs.

[0112] In some cases, the polynucleic acid molecule is conjugated to a polypeptide (A) and optionally a polymer moiety (C). In some embodiments, at least one polypeptide A is attached at one end of at least one B, while at least one C is attached at the opposite end of at least one B to form an ABC conjugate. In some examples, at least one polypeptide A is attached at one end of at least one B, while at least one C is attached at an internal site of at least one B. In some examples, at least one polypeptide A is directly attached to at least one C. In some examples, at least one B is indirectly attached to at least one polypeptide A via at least one C to form an ACB conjugate.

[0113] In some examples, at least one B and / or at least one C, and optionally at least one D, are linked to at least one polypeptide A. In some examples, at least one B is linked to at least one polypeptide A at a terminal end (e.g., the 5' end or the 3' end) or via an internal site. Optionally, at least one C is linked to at least one polypeptide A directly or indirectly via at least one B. Indirectly, via at least one B, at least one C is linked at the same end on B as at least one polypeptide A, at the opposite end from at least one polypeptide A, or independently at an internal site. In some examples, at least one additional polypeptide A is further linked to at least one polypeptide A, B, or C. In a further example, at least one D is optionally linked, directly or indirectly, to at least one polypeptide A, at least one B, or at least one C. When directly bound to at least one polypeptide A, at least one D is also optionally bound to at least one B to form an ADB conjugate, or optionally bound to at least one B and at least one C to form an ADBC ​​conjugate. In some examples, at least one D is directly bound to at least one polypeptide A and indirectly bound to at least one B and at least one C to form a DABC conjugate. When indirectly bound to at least one polypeptide A, at least one D is also optionally bound to at least one B to form an ABD conjugate, or optionally bound to at least one B and at least one C to form an ABDC conjugate. In some examples, at least one additional D is further bound to at least one polypeptide A, B, or C.

[0114] joining part In some embodiments, binding moiety A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some examples, the antibody or antigen-binding fragment thereof includes a humanized antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, a binding fragment having a light chain domain and a heavy chain domain, a binding fragment having two light chain domains and two heavy chain domains, a binding fragment having two or more light chain domains and heavy chain domains, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or antigen-binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

[0115] In some embodiments, binding moiety A is a bispecific antibody or antigen-binding fragment thereof. In some instances, the bispecific antibody is a trispecific antibody or a bispecific miniantibody. In some instances, the bispecific antibody is a trispecific antibody. In some instances, the trispecific antibody is a full-length monoclonal antibody that contains binding sites for two different antigens.

[0116] In some instances, the bispecific antibody is a bispecific miniantibody. In some instances, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single-chain variable fragments (scFvs), where the two scFvs target epitopes of two different antigens.

[0117] In some embodiments, binding moiety A is a bispecific miniantibody. In some instances, A is a bispecific Fab2. In some instances, A is a bispecific F(ab)'3 fragment. Optionally, A is a bispecific bis-scFv. Optionally, A is a bispecific (scFv). In some embodiments, A is a bispecific diabody. In some embodiments, A is a bispecific minibody. In some embodiments, A is a bispecific triabody. In other embodiments, A is a bispecific tetrabody. In other embodiments, A is a bispecific T cell engager (BiTE).

[0118] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody comprises a F(ab)'3 fragment or a trispecific antibody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, A is a trispecific antibody. In some embodiments, A is a trispecific antibody as described in Dimas, et al., "Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells," Mol. Pharmaceuticals, 12(9):3490-3501 (2015).

[0119] In some embodiments, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein. In some instances, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein on a muscle cell. In some instances, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein on a skeletal muscle cell.

[0120] In some embodiments, exemplary antibodies include, but are not limited to, anti-myosin antibodies, anti-transferrin receptor antibodies, and antibodies that recognize muscle-specific kinase (MuSK). In some instances, the antibody is an anti-transferrin receptor (anti-CD71) antibody.

[0121] In some embodiments, when the antibody is an anti-transferrin receptor (anti-CD71) antibody, the anti-transferrin antibody specifically binds to transferrin receptor (TfR), preferably specifically binds to transferrin receptor 1 (TfR1), or more preferably specifically binds to human transferrin receptor 1 (TfR1) (or human CD71).

[0122] In some cases, the anti-transferrin receptor antibody comprises a variable heavy (VH) region and a variable light (VL) region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q, X2 is selected from Q or E, and the HCDR3 sequence comprises SEQ ID NO: 283.

[0123] In some embodiments, the VH region of the anti-transferrin receptor antibody comprises an HCDR1, HCDR2, and HCDR3 sequence selected from Table 1.

[0124] [Table 1]

[0125] In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, 284, or 285. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, and an HCDR3 sequence comprising SEQ ID NO: 283. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising an HCDR2, a sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283.

[0126] In some embodiments, the VL region of the anti-transferrin receptor antibody comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, and if present, is F.

[0127] In some embodiments, the VL region of the anti-transferrin receptor antibody comprises an LCDR1, LCDR2, and LCDR3 sequence selected from Table 2.

[0128] [Table 2]

[0129] In some cases, the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X3 is selected from N or S.

[0130] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X4 is selected from A or G and X5 is selected from D or E.

[0131] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent and, if present, is F.

[0132] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0133] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:286, an LCDR2 sequence comprising SEQ ID NO:287, and an LCDR3 sequence comprising SEQ ID NO:288.

[0134] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:286, an LCDR2 sequence comprising SEQ ID NO:289, and an LCDR3 sequence comprising SEQ ID NO:290.

[0135] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:291, an LCDR2 sequence comprising SEQ ID NO:292, and an LCDR3 sequence comprising SEQ ID NO:290.

[0136] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0137] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX, wherein X1 is selected from N or Q, and X2 is selected from Q or E. 1 GRSNYAX2KFQG, and an HCDR3 sequence comprising SEQ ID NO: 283, wherein the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X3 is selected from N or S.

[0138] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, where X4 is selected from A or G and X5 is selected from D or E.

[0139] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and is F if present.

[0140] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0141] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO:283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:286, an LCDR2 sequence comprising SEQ ID NO:287, and an LCDR3 sequence comprising SEQ ID NO:288.

[0142] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 289, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0143] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 291, an LCDR2 sequence comprising SEQ ID NO: 292, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0144] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X3 is selected from N or S.

[0145] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X4 is selected from A or G and X5 is selected from D or E.

[0146] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 2, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and is F if present.

[0147] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:281, an HCDR2 sequence comprising SEQ ID NO:282, and an HCDR3 sequence comprising SEQ ID NO:283; the VL region comprises an LCDR1 sequence comprising SEQ ID NO:286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6; and X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0148] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 287, and an LCDR3 sequence comprising SEQ ID NO: 288.

[0149] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 9, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0150] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 282, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 291, an LCDR2 sequence comprising SEQ ID NO: 292, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0151] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X3 is selected from N or S.

[0152] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X4 is selected from A or G and X5 is selected from D or E.

[0153] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and is F if present.

[0154] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:281, an HCDR2 sequence comprising SEQ ID NO:284, and an HCDR3 sequence comprising SEQ ID NO:283; the VL region comprises an LCDR1 sequence comprising SEQ ID NO:286, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6; and X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0155] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 287, and an LCDR3 sequence comprising SEQ ID NO: 288.

[0156] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 289, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0157] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 284, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 291, an LCDR2 sequence comprising SEQ ID NO: 292, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0158] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 29, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X3 is selected from N or S.

[0159] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X4 is selected from A or G and X5 is selected from D or E.

[0160] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286 or 291, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and is F if present.

[0161] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:281, an HCDR2 sequence comprising SEQ ID NO:285, and an HCDR3 sequence comprising SEQ ID NO:283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0162] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 287, and an LCDR3 sequence comprising SEQ ID NO: 288.

[0163] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence comprising SEQ ID NO: 289, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0164] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising SEQ ID NO: 285, and an HCDR3 sequence comprising SEQ ID NO: 283, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 291, an LCDR2 sequence comprising SEQ ID NO: 292, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0165] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the sequence of the VH region comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 293-296, and the sequence of the VL region comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 298-301.

[0166] In some embodiments, the VH region comprises a sequence selected from SEQ ID NOs: 293-296 (Table 3), and the VL region comprises a sequence selected from SEQ ID NOs: 298-301 (Table 4). The underlined regions in Tables 3 and 4 represent the sequences of CDR1, CDR2, or CDR3, respectively.

[0167] [Table 3]

[0168] [Table 4]

[0169] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region as illustrated in Table 5.

[0170] [Table 5]

[0171] In some embodiments, the anti-transferrin receptor antibody described herein comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some cases, the anti-transferrin receptor antibody comprises an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the anti-transferrin receptor antibody comprises an IgG1 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2a framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2b framework. In some cases, the anti-transferrin receptor antibody comprises an IgG3 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG4 framework.

[0172] In some cases, the anti-transferrin receptor antibody contains one or more mutations in a framework region, such as the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some cases, the one or more mutations are intended to stabilize the antibody and / or extend half-life. In some cases, the one or more mutations are intended to modulate Fc receptor interaction and reduce or eliminate Fc effector function, such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional examples, the one or more mutations are intended to modulate glycosylation.

[0173] In some embodiments, one or more mutations are located in the Fc region. In some cases, the Fc region includes mutations at residue positions L234, L235, or a combination thereof. In some cases, the mutations include L234 and L235. In some cases, the mutations include L234A and L235A. In some cases, the residue positions are relative to IgG1.

[0174] In some cases, the Fc region comprises a mutation at residue positions L234, L235, D265, N297, K322, L328, or P329, or a combination thereof. In some cases, the mutation comprises L234 and L235 in combination with a mutation at residue position K322, L328, or P329. In some cases, the Fc region comprises mutations at L234, L235, and K322. In some cases, the Fc region comprises mutations at L234, L235, and L328. In some cases, the Fc region comprises mutations at L234, L235, and P329. In some cases, the Fc region comprises mutations at D265 and N297. In some cases, the residue positions are relative to IgG1.

[0175] In some cases, the Fc region comprises L234A, L235A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some cases, the Fc region comprises L234A and L235A in combination with K322G, L328R, or P329G. In some cases, the Fc region comprises L234A, L235A, and K322G. In some cases, the Fc region comprises L234A, L235A, and L328R. In some cases, the Fc region comprises L234A, L235A, and P329G. In some cases, the Fc region comprises D265A and N297G. In some cases, the residue positions are relative to IgG1.

[0176] In some cases, the Fc region comprises a mutation or combination of mutations at residue positions L235, L236, D265, N297, K322, L328, or P329. In some cases, the Fc region comprises a mutation at L235 and L236. In some cases, the Fc region comprises a mutation at L235 and L236 in combination with a mutation at residue position K322, L328, or P329. In some cases, the Fc region comprises mutations at L235, L236, and K322. In some cases, the Fc region comprises mutations at L235, L236, and L328. In some cases, the Fc region comprises mutations at L235, L236, and P329. In some cases, the Fc region comprises mutations at D265 and N297. In some cases, the residue positions are relative to IgG2b.

[0177] In some embodiments, the Fc region comprises L235A, L236A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some embodiments, the Fc region comprises L235A and L236A. In some embodiments, the Fc region comprises L235A and L236A in combination with K322G, L328R, or P329G. In some embodiments, the Fc region comprises L235A, L236A, and K322G. In some embodiments, the Fc region comprises L235A, L236A, and L328R. In some embodiments, the Fc region comprises L235A, L236A, and P329G. In some embodiments, the Fc region comprises D265A and N297G. In some embodiments, the residue positions are relative to IgG2b.

[0178] In some embodiments, the Fc region comprises a mutation at residue position L233, L234, D264, N296, K321, L327, or P328, where the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of a SEQ ID NO: 1. In some embodiments, the Fc region comprises a mutation at L233 and L234. In some embodiments, the Fc region comprises a mutation at L233 and L234 in combination with a mutation at residue position K321, L327, or P328. In some embodiments, the Fc region comprises a mutation at L233, L234, and K321. In some embodiments, the Fc region comprises a mutation at L233, L234, and L327. In some embodiments, the Fc region comprises a mutation at L233, L234, and K321. In some cases, the Fc region comprises mutations at L233, L234, and P328. In some cases, the Fc region comprises mutations at D264 and N296. In some cases, positions equivalent to residues L233, L234, D264, N296, K321, L327, or P328 in the IgG1, IgG2, IgG3, or IgG4 framework are contemplated. In some cases, mutations to residues corresponding to residues L233, L234, D264, N296, K321, L327, or P328 of SEQ ID NO: 303 in the IgG1, IgG2, or IgG4 framework are also contemplated.

[0179] In some embodiments, the Fc region comprises L233A, L234A, D264A, N296G, K321G, L327R, or P328G, residues corresponding to positions 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 303. In some embodiments, the Fc region comprises L233A and L234A. In some embodiments, the Fc region comprises L233A and L234A in combination with K321G, L327R, or P328G. In some embodiments, the Fc region comprises L233A, L234A, and K321G. In some embodiments, the Fc region comprises L233A, L234A, and L327R. In some embodiments, the Fc region comprises L233A, L234A, and K321G. In some cases, the Fc region comprises L233A, L234A, and P328G. In some cases, the Fc region comprises D264A and N296G.

[0180] In some embodiments, human IgG constant regions are used to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., Natsume et al. Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48:152-164; Alegre et al. al., 1992 J Immunol, 148:3461-3468; Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0181] In some embodiments, the anti-transferrin receptor antibodies described herein are full-length antibodies comprising a heavy chain (HC) and a light chain (LC). Optionally, the heavy chain (HC) comprises a sequence selected from Table 6. Optionally, the light chain (LC) comprises a sequence selected from Table 7. The underlined regions represent the respective CDRs.

[0182] [Table 6] JPEG2026041765000018.jpg253170 JPEG2026041765000019.jpg250170 JPEG2026041765000020.jpg247170 JPEG2026041765000021.jpg81170

[0183] [Table 7]

[0184] In some embodiments, the anti-transferrin receptor antibodies described herein have an improved serum half-life compared to a reference anti-transferrin receptor antibody. In some cases, the improved serum half-life is at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more, longer than the reference anti-transferrin receptor antibody.

[0185] In some embodiments, binding moiety A binds non-specifically to polynucleic acid molecule (B). In some instances, binding moiety A binds to polynucleic acid molecule (B) in a non-site-specific manner via a lysine or cysteine ​​residue. In some instances, binding moiety A binds to polynucleic acid molecule (B) in a non-site-specific manner via a lysine residue (e.g., a lysine residue present in binding moiety A). In some instances, binding moiety A binds to polynucleic acid molecule (B) in a non-site-specific manner via a cysteine ​​residue (e.g., a cysteine ​​residue present in binding moiety A).

[0186] In some embodiments, binding moiety A binds to polynucleic acid molecule (B) in a non-site-specific manner. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via a lysine residue, a cysteine ​​residue, at the 5'-end, at the 3'-end, at an unnatural amino acid, or at an enzyme-modified or enzyme-catalyzed residue. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via a lysine residue (e.g., a lysine residue present in binding moiety A). In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via a cysteine ​​residue (e.g., a cysteine ​​residue present in binding moiety A). In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner at the 5'-end. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner at the 3'-end. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via an unnatural amino acid. In some instances, the binding moiety A is attached to the polynucleic acid molecule (B) via an enzyme-modified or enzyme-catalyzed residue in a site-specific manner.

[0187] In some embodiments, one or more polynucleic acid molecules (B) are bound to binding moiety A. In some instances, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acid molecules are bound to one binding moiety A. In some instances, about 1 polynucleic acid molecule is bound to one binding moiety A. In some instances, about 2 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 3 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 4 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 5 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 6 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 7 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 8 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 9 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 10 polynucleic acid molecules bind to one binding moiety A. In some instances, about 11 polynucleic acid molecules bind to one binding moiety A. In some instances, about 12 polynucleic acid molecules bind to one binding moiety A. In some instances, about 13 polynucleic acid molecules bind to one binding moiety A. In some instances, about 14 polynucleic acid molecules bind to one binding moiety A. In some instances, about 15 polynucleic acid molecules bind to one binding moiety A. In some instances, about 16 polynucleic acid molecules bind to one binding moiety A. In some instances, one or more polynucleic acid molecules are the same. In other instances, one or more polynucleic acid molecules are different.

[0188] In some embodiments, the number of polynucleic acid molecules (B) bound to binding moiety A forms a ratio. In some instances, the ratio is referred to as a DAR (drug-to-antibody) ratio, and the drug as referred to herein is a polynucleic acid molecule (B). In some instances, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more. In some instances, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1 or more. In some instances, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 2 or more. In some instances, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 3 or more. In some instances, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 4 or more. In some instances, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 5 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 6 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 7 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 8 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 9 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 10 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 11 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 12 or greater.

[0189] In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 3. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 5. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 7. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 9. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 10. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 11. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 12. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 13. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 14. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 15. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 16.

[0190] In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 12.

[0191] In some instances, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety A has improved activity compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some instances, the improved activity results in an enhancement of a biologically relevant function, for example, improved stability, affinity, binding, functional activity, and efficacy in treating or preventing a disease condition. In some instances, the disease condition is the result of one or more mutated exons of a gene. In some instances, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety A results in increased exon skipping of one or more mutated exons compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some instances, exon skipping is increased by at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% in a conjugate comprising polynucleic acid molecule (B) and binding moiety A compared to a conjugate comprising polynucleic acid molecule (B) without binding moiety A.

[0192] In some embodiments, the antibody or antigen-binding fragment thereof is further modified, alone or in combination, using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, or addition, and / or by recombination and / or other modifications known in the art (e.g., post-translational and chemical modifications such as glycosylation and phosphorylation). In some examples, the modifications further include modifications to modulate interaction with Fc receptors. In some examples, one or more modifications include, for example, those described in International Publication No. WO 97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into nucleic acid sequences underlying the amino acid sequence of an antibody or antigen-binding fragment thereof are well known to those of skill in the art.

[0193] In some instances, the antigen-binding fragment further includes derivatives thereof and comprises a polypeptide sequence comprising at least one CDR.

[0194] In some instances, the term "single-chain" as used herein means that the first and second domains of the bispecific single-chain construct are covalently linked, preferably in the form of a co-linear amino acid sequence that can be encoded by a single nucleic acid molecule.

[0195] In some instances, bispecific single-chain antibody constructs relate to constructs comprising binding domains from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some instances, the scFv comprises a VH and a VL domain connected by a linker peptide. In some instances, the linker is of sufficient length and sequence to allow each of the first and second domains to retain their differential binding specificities independently of each other.

[0196] In some embodiments, as used herein, binding with or interaction by defines the binding / interaction of at least two antigen-interaction sites with each other. In some instances, an antigen-interaction site defines a polypeptide motif that exhibits a specific interaction capacity with a specific antigen or a specific group of antigens. In some instances, binding / interaction is also understood to define specific recognition. In such instances, specific recognition refers to the ability of an antibody or antigen-binding fragment thereof to specifically interact and / or bind to at least two amino acids of each of the target molecules. For example, specific recognition relates to the specificity of an antibody molecule or its ability to distinguish a specific range of target molecules. In additional examples, the specific interaction of an antigen-interaction site with its specific antigen results in the initiation of a signal, for example, by inducing a conformational change in the antigen, oligomerization of the antigen, etc. In further embodiments, binding is exemplified by the specificity of the "key-lock principle." Thus, in some instances, specific motifs in the amino acid sequence of the antigen interaction site and the antigen bind to each other as a result of their primary, secondary, or tertiary structure, as well as as a result of secondary modifications of the structure. In such cases, the specific interaction of the antigen interaction site with its specific antigen results in the site's easy binding to the antigen.

[0197] In some instances, specific interaction further refers to reduced cross-reactivity of an antibody or its antigen-binding fragment, or reduced off-target effects. For example, an antibody or its antigen-binding fragment that binds to a desired polypeptide / protein but does not or essentially does not bind to any other polypeptides is considered specific for the desired polypeptide / protein. Specificity of the antigen interaction site Examples of specific interactions with an antigen include the specificity of a ligand with its receptor, for example, the interaction of an antigenic determinant (epitope) with the antigen-binding site of an antibody.

[0198] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. In some instances, the plasma protein includes albumin. In some instances, binding moiety A is albumin. In some instances, albumin is bound to the polynucleic acid molecule by one or more of the binding chemistries described herein. In some instances, albumin is bound to the polynucleic acid molecule by native ligation chemistry. In some instances, albumin is bound to the polynucleic acid molecule by a lysine bond.

[0199] In some examples, the conjugated moiety A is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (saturated, unsaturated, substituted, or combinations thereof). In some examples, the steroid is cholesterol. In some examples, the conjugated moiety is cholesterol. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by native ligation chemistry. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by a lysine linkage.

[0200] In some instances, the binding moiety is a polymer, including but not limited to, a polynucleic acid molecule aptamer that binds to a specific surface marker on a cell. In this example, the binding moiety is a polynucleic acid that does not hybridize to the target gene or mRNA, but instead can selectively bind to the cell surface marker, similar to an antibody that binds to that specific epitope of the cell surface marker.

[0201] In some instances, the binding moiety is a peptide. In some instances, the peptide comprises about 1 to about 3 kDa. In some instances, the peptide comprises about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some instances, the peptide is a bicyclic peptide. In some instances, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the binding moiety is a bicyclic peptide (e.g., Bicycles from Bicycle Therapeutics).

[0202] In further instances, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some instances, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, where the target-binding end is capable of recognizing and interacting with a cell surface receptor. For example, in some instances, the target-binding end comprises a glutamate urea compound, which allows interaction with PSMA, thereby enhancing antibody interaction with cells expressing PSMA. In some examples, the binding moiety is a small molecule described in Zhang et al., "A remote arene-binding site on prostate-specific membrane antigen revealed by antibody-recruiting small molecules," J Am Chem Soc. 132(36):12711-12716 (2010); or McEnaney, et al., "Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease," ACS Chem Biol. 7(7):1139-1151 (2012).

[0203] Production of antibodies or antigen-binding fragments thereof In some embodiments, the polypeptides described herein (e.g., antibodies and antigen-binding fragments thereof) are produced using any method known in the art to aid in the synthesis of polypeptides (e.g., antibodies), inter alia, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0204] In some examples, antibodies or antigen-binding fragments thereof are recombinantly expressed, and nucleic acids encoding the antibodies or antigen-binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligation of the oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0205] Alternatively, nucleic acid molecules encoding antibodies are optionally produced from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0206] In some instances, the antibody or antigen binding thereof is optionally produced by immunizing an animal such as a rabbit to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of the antibody are optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) or antibody libraries (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937) for clones of Fab fragments that bind to specific antigens.

[0207] In some embodiments, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region (e.g., humanized antibodies).

[0208] In some embodiments, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are suitable for producing single-chain antibodies. Single-chain antibodies are formed by linking heavy or light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0209] In some embodiments, an expression vector containing an antibody nucleotide sequence or the antibody nucleotide sequence is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0210] In some embodiments, various host-expression vector systems are utilized to express the antibodies or antigen-binding fragments thereof described herein. Such host-expression systems not only represent vehicles in which antibody coding sequences are generated and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or antigen-binding fragments thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or antigen-binding fragment coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or antigen-binding fragment coding sequences; insect cell systems (e.g., baculovirus) infected with recombinant viral expression vectors containing the antibody or antigen-binding fragment coding sequences; and recombinant viral expression vectors (e.g., cauliflower mosaic virus). Plant cell lines infected with viruses (CaMV and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing antibody or antigen-binding fragment coding sequences; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0211] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, cells are engineered to grow in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes, grow, and form foci that are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or antigen-binding fragments thereof.

[0212] In some examples, a number of selection systems are used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes utilized in tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance has been used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy). 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods known in the art of recombinant DNA technology that can be used are generally described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0213] In some instances, antibody expression levels are increased by vector amplification (for a review, see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is related to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0214] In some examples, any method known in the art for purification or analysis of antibodies or antibody conjugates is used, for example, by chromatography (e.g., ion exchange, affinity, especially affinity to specific antigens followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and fast protein liquid chromatography.

[0215] Conjugation Chemistry In some embodiments, polynucleic acid molecule B is conjugated to a binding moiety. In some embodiments, polynucleic acid molecule B is conjugated to a binding moiety in the formula AXB, where X is a linker conjugating A and B. In some examples, the binding moiety includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and all analogs or derivatives of these classes of substances. Additional examples of binding moieties include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, steroids such as digoxigenin, and polysaccharides. In some examples, the binding moiety is an antibody or antigen-binding fragment thereof. In some examples, the polynucleic acid molecule is further conjugated to a polymer and, optionally, to an endosomolytic moiety.

[0216] In some embodiments, the polynucleic acid molecule is attached to the binding moiety by a chemical ligation process. In some instances, the polynucleic acid molecule is attached to the binding moiety by native ligation. In some examples, the conjugates are described in: Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology." Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some instances, conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleic acid molecule is site-specifically or non-specifically conjugated to a binding moiety via native ligation chemistry.

[0217] In some instances, polynucleic acid molecules are coupled to binding moieties in a site-directed manner using "traceless" coupling technology (PhiloChem). In some instances, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group on a polynucleic acid molecule that contains an aldehyde group and is then coupled to a binding moiety. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacovigilance," JACS 134(13):5887-5892 (2012)).

[0218] In some instances, polynucleic acid molecules are conjugated to binding moieties in a site-directed manner utilizing unnatural amino acids introduced into the binding moiety. In some instances, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some instances, the keto group of pAcPhe selectively binds to alkoxy-amine derived binding moieties to form oxime bonds. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40):16101-16106 (2012)).

[0219] In some instances, the polynucleic acid molecule is attached to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Catalent, Inc.). In some instances, SMARTag™ technology involves the generation of a formylglycine (FGly) residue from cysteine ​​by formylglycine generating enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent attachment of FGly to an alkylhydrazine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (See Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9):3000-3005(2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51(2013))

[0220] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some examples, the polynucleic acid molecule is conjugated to the binding moiety using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some examples, mTG is produced by Streptomyces mobaraensis. (See Strop et al., "Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates," Chemistry and Biology 20(2) 161-167 (2013)).

[0221] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by methods such as those described in PCT International Publication No. WO2014 / 140317, which utilize sequence-specific transpeptidases.

[0222] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by methods such as those described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0223] Polymer-binding moiety In some embodiments, polymer moiety C is further linked to a polynucleic acid molecule described herein, a binding moiety described herein, or a combination thereof. In some examples, polymer moiety C is a conjugated polynucleic acid molecule of the formula A-X1-B-X2-C (X1, X2 are two linkers conjugating A and B, B and C, respectively). Binds to a polynucleic acid molecule. In some cases, polymer moiety C is linked to a binding moiety. In other cases, polymer moiety C is linked to a polynucleic acid molecule binding moiety. In further cases, polymer moiety C is linked as illustrated above.

[0224] In some examples, polymer moiety C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of two- or three-dimensional monomers. In some examples, polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C includes, but is not limited to, alpha-, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (also known as poly(ethylene terephthalate), PET, PETG, or PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound, as in the context of a block copolymer. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some examples, polymer portion C includes polyalkylene oxide. In some examples, polymer portion C includes PEG. In some examples, polymer portion C includes polyethylene imide (PEI) or hydroxyethyl starch (HES).

[0225] In some instances, C is a PEG moiety. In some instances, the PEG moiety is attached at the 5' end of the polynucleic acid molecule, while the linking moiety is attached at the 3' end of the polynucleic acid molecule. In some instances, the PEG moiety is attached at the 3' end of the polynucleic acid molecule, while the linking moiety is attached at the 5' end of the polynucleic acid molecule. In some instances, the PEG moiety is attached to an internal site of the polynucleic acid molecule. In some instances, the PEG moiety, the linking moiety, or a combination thereof, is attached to an internal site of the polynucleic acid molecule. In some instances, the conjugate is a direct conjugate. In some instances, the attachment is via native ligation.

[0226] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some instances, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some instances, a monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight indicated represents the average molecular weight of the polyalkylene oxide (e.g., PEG) molecules.

[0227] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 83 700, 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.

[0228] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 830 and having a molecular weight of 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. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 850 In some instances, the molecular weight of C is about 200 Da. In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da.In some instances, the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1450 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da. In some instances, the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da.In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da.

[0229] In some embodiments, the polyalkylene oxide (e.g., PEG) comprises discrete ethylene oxide units (e.g., 4 to about 48 ethylene oxide units). In some examples, the polyalkylene oxide comprising discrete ethylene oxide units is linear. In other examples, the polyalkylene oxide comprising discrete ethylene oxide units is branched.

[0230] In some examples, polymer portion C is a polyalkylene oxide (e.g., PEG) comprising discrete ethylene oxide units. In some cases, polymer portion C comprises from about 4 to about 48 ethylene oxide units. In some cases, polymer portion C comprises 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, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units.

[0231] In some examples, polymer portion C comprises another PEG containing, for example, about 4 to about 48 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, 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, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 4 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 5 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 6 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 7 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 8 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 9 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 10 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 11 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 12 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 13 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 14 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 15 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 16 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 17 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 18 ethylene oxide units.In some cases, polymer moieties C are, for example, discrete PEGs containing about 19 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 20 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 21 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 22 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 23 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 24 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 25 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 26 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 27 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 28 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 29 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 30 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 31 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 32 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 33 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 34 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 35 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 36 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 37 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 38 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 39 ethylene oxide units.In some cases, polymer moieties C are, for example, discrete PEGs containing about 40 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 41 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 42 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 43 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 44 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 45 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 46 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 47 ethylene oxide units. In some cases, polymer moieties C are, for example, discrete PEGs containing about 48 ethylene oxide units.

[0232] In some cases, polymer moiety C is dPEG® (Quanta Biodesign Ltd).

[0233] In some embodiments, the polymer portion C comprises a cationic mucic acid-based polymer (cMAP). In some instances, the cMAP comprises one or more subunits of at least one repeating subunit, and the subunit structure is represented as formula (∨): [ka]

[0234] wherein m at each occurrence is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4-6, or 5; and n at each occurrence is independently 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0235] In some examples, cMAP is further conjugated to a PEG moiety to form a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some examples, the PEG moiety ranges from about 500 Da to about 50,000 Da. In some examples, the PEG moiety is about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 Da to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

[0236] In some instances, polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some instances, polymer moiety C is a cMAP-PEG copolymer. In other instances, polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In further instances, polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0237] In some embodiments, polymer moiety C is attached to a polynucleic acid molecule, a binding moiety, and optionally an endosomolytic moiety, as exemplified above.

[0238] Endosomolytic or cell membrane-penetrating moiety In some embodiments, the molecule of Formula (I): A-X1-B-X2-C further comprises an additional binding moiety. In some examples, the additional binding moiety is an endosomolytic moiety and / or a cell membrane-penetrating moiety. In some cases, the endosomolytic moiety is a compound capable of releasing a cellular compartment-releasing component, e.g., any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum comprising a cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer. In some cases, the cell membrane-penetrating moiety comprises a cell-penetrating peptide (CPP). In other cases, the cell membrane-penetrating moiety comprises a cell-penetrating lipid. In other cases, the cell membrane-penetrating moiety comprises a cell-penetrating small molecule.

[0239] Endosomolytic and cell membrane-penetrating polypeptides In some embodiments, the molecule of Formula (I): A-X1-B-X2-C is further linked to an endosomolytic polypeptide. Optionally, the endosomolytic polypeptide is a pH-dependent membrane-active peptide. Optionally, the endosomolytic polypeptide is an amphipathic polypeptide. In additional instances, the endosomolytic polypeptide is a peptidomimetic. In some instances, the endosomolytic polypeptide comprises INF, melittin, mucin (meucin), or their respective derivatives. In some instances, the endosomolytic polypeptide comprises INF or a derivative thereof. In other instances, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further instances, the endosomolytic polypeptide comprises mucin or a derivative thereof.

[0240] In some instances, INF7 is a 24-residue polypeptide, and these sequences include CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 331), or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 332). In some instances, INF7 or a derivative thereof includes the following sequence: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 333), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 334), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 335).

[0241] In some instances, melittin is a 26-residue polypeptide, and the sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 336), or alternatively, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 337). In some instances, melittin comprises a polypeptide sequence described in U.S. Patent No. 8,501,930.

[0242] In some instances, the mucins are antimicrobial peptides (AMPs) derived from the venom glands of the scorpion Mesobuthus eupeus. In some instances, the mucins are comprised of mucin-13 (the sequence of which comprises IFGAIAGLLKNIF-NH2 (SEQ ID NO: 338)) and mucin-18 (the sequence of which comprises FFGHLFKLATKIIPSLFQ (SEQ ID NO: 339)).

[0243] In some examples, the endosomolytic polypeptide comprises a polypeptide whose sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to INF7 or a derivative thereof, a melittin or derivative thereof, or a mucin or derivative thereof. In some examples, the endosomolytic moiety comprises INF7 or a derivative thereof, a melittin or derivative thereof, or a mucin or derivative thereof.

[0244] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 331-335. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 331. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 332-335. Optionally, the endosomolytic portion comprises SEQ ID NO: 331. Optionally, the endosomolytic portion comprises SEQ ID NOs: 332-335. Optionally, the endosomolytic portion consists of SEQ ID NOs: 331. Optionally, the endosomolytic portion consists of SEQ ID NOs: 332-335.

[0245] In some examples, the endosomolytic moiety is melittin or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 336 or 337. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 336. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 337. Optionally, the endosomolytic portion comprises SEQ ID NO: 286. Optionally, the endosomolytic portion comprises SEQ ID NO: 337. Optionally, the endosomolytic portion consists of SEQ ID NO: 336. Optionally, the endosomolytic portion consists of SEQ ID NO: 337.

[0246] In some examples, the endosomolytic moiety is a mucin or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 338 or 339. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 338. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 339. Optionally, the endosomolytic portion comprises SEQ ID NO: 338. Optionally, the endosomolytic portion comprises SEQ ID NO: 339. Optionally, the endosomolytic portion consists of SEQ ID NO: 338. Optionally, the endosomolytic portion consists of SEQ ID NO: 339.

[0247] In some instances, the endosomolytic moiety comprises a sequence as illustrated in Table 8.

[0248] [Table 8] JPEG2026041765000025.jpg107170

[0249] In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide that induces apoptosis through antagonism of inhibitory targets such as Bcl-2 and / or Bcl-xL. In some examples, the endosomolytic moiety comprises a Bak BH3 polypeptide described in Albarran, et al., "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive & Functional Polymers 71:261-265 (2011).

[0250] In some examples, the endosomolytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) such as those described in PCT International Publication No. WO2013 / 166155 or WO2015 / 069587.

[0251] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, a molecule of Formula (I): A-X1-B-X2-C is further associated with an endosomolytic lipid (e.g., a fusogenic lipid). Exemplary fusogenic lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), and N-methyl(2,2-di(9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di(9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC).

[0252] In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in PCT International Publication No. WO 09 / 126,933.

[0253] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, a molecule of Formula (I): A-X1-B-X2-C is further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), ampicillin, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, quinoline endosomolytic moieties include, but are not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline; 4-(4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino-)quinoline;7-Hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;Hydroxychloroquine phosphate;7-Chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine);7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline 7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-butylamino)quinoline;7-Fluoro ... 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-butylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-butylamino)quinoline;7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-methylbutylamino)quinoline;7-Fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-methylbutylamino)quinoline;4-(1- Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline;1-Acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-Butyryl-1,2,3,4-tetrahydroquinoline;3-chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline)(4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline; 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline)(4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline; 4-(4-hydroxy-α,α'-bis(2-methyl-1- pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde; 1,1'-pentamethylenequinolinium iodide; 8-quinolinol sulfate and amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs thereof. In some examples, the endosomolytic moiety is a small molecule described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Pat. No. 5,736,557.

[0254] Cell-penetrating polypeptides (CPPs) In some embodiments, the cell-penetrating polypeptide comprises a short, positively charged peptide having 5-30 amino acids. In some embodiments, the cell-penetrating polypeptide comprises an amino acid sequence rich in arginine or lysine. In some embodiments, the cell-penetrating polypeptide comprises any polypeptide listed in Table 9, or a combination thereof.

[0255] [Table 9]

[0256] Linker In some embodiments, the linkers described herein are cleavable or non-cleavable linkers. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker.

[0257] In some cases, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain repeating units of a monomer produced by a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In some cases, the non-polymeric linker includes a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In further cases, the non-polymeric linker does not include more than two linkers of the same type, for example, more than two homobifunctional cross-linkers or more than two peptide linkers. In further instances, the non-polymeric linker optionally includes one or more reactive functional groups.

[0258] In some instances, the non-polymeric linker does not include a polymer as described above. In some instances, the non-polymeric linker does not include a polymer encompassed by polymer moiety C. In some instances, the non-polymeric linker does not include a polyalkylene oxide (e.g., PEG). In some instances, the non-polymeric linker does not include PEG.

[0259] In some instances, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS), and the like. T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene, 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED ), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0260] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-crosslinking linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs ... Imidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidylCarbonyl-reactive and -reactive amines such as 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH) and sulfhydryl-reactive cross-linkers, amine-reactive and photoreactive cross-linkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1, 3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N -5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl7-Azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers, such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio) propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as ρ-azidophenylglyoxal (APG).

[0261] In some instances, the linker comprises a reactive functional group. In some instances, the reactive functional group comprises a nucleophilic group reactive to an electrophilic group present in the linking moiety. Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0262] In some embodiments, the linker comprises a maleimide group. In some instances, the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group further comprises caproic acid to form maleimidocaproyl (mc). In some instances, the linker comprises maleimidocaproyl (mc). In some instances, the linker is maleimidocaproyl (mc). In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), described above.

[0263] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing retro-Michael elimination. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0264] In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide moiety comprises at least 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety comprises at most 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some examples, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some examples, the peptide moiety is a non-cleavable peptide moiety. In some examples, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some examples, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

[0265] In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some examples, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some examples, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0266] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some instances, the maleimide group is maleimidocaproyl (mc). In some instances, the peptide group is val-cit. In some instances, the benzoic acid group is PABA. In some instances, the linker comprises an mc-val-cit group. In some instances, the linker comprises a val-cit-PABA group. In further instances, the linker comprises an mc-val-cit-PABA group.

[0267] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some examples, the linker includes a linker described in U.S. Pat. No. 9,089,614 or PCT Publication WO2015038426.

[0268] In some embodiments, the linker is a dendritic linker. In some instances, the dendritic linker comprises a branched multifunctional linker moiety. In some instances, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some instances, the dendritic linker comprises a PAMAM dendrimer.

[0269] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or linker group) on the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D after cleavage. Exemplary traceless linkers include, but are not limited to, a germanium linker, a silicon linker, a sulfur linker, a selenium linker, a nitrogen linker, a phosphorus linker, a boron linker, a chromium linker, or a phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497 (2013). In some examples, the linker is a traceless linker described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem. Rev. 102:2607-2024 (2002). In some examples, the linker is a traceless linker as described in US Pat. No. 6,821,783.

[0270] In some embodiments, the linker is a polymerizable compound as described in U.S. Patent Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 2013 / 0 309256; 2015 / 037360; or 2014 / 0294851; or PCT Publication Nos. WO2015057699; WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

[0271] In some embodiments, X1 and X2 are each independently a single bond or a non-polymeric linker. In some examples, X1 and X2 are each independently a single bond. In some instances, X1 and X2 are each independently a non-polymeric linker.

[0272] In some examples, X1 comprises a single bond or a non-polymeric linker. In some examples, X1 is a single bond. In some examples, X1 is a non-polymeric linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X1 is a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. When used in the context of a linker, particularly in the context of X1, alkyl refers to a saturated, straight- or branched-chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X1 comprises a homobifunctional linker or a heterobifunctional linker described above. In some cases, X1 comprises a heterobifunctional linker. In some cases, X1 comprises sMCC. In other examples, X1 comprises a heterobifunctional linker optionally bonded to a C1-C6 alkyl group. In other examples, X1 comprises sMCC optionally bonded to a C1-C6 alkyl group. In some additional examples, X1 does not comprise a homobifunctional linker or a heterobifunctional linker described above.

[0273] In some examples, X2 is a single bond or a linker. In some examples, X2 is a single bond. In other cases, X2 is a linker. In further cases, X2 is a non-polymeric linker. In some embodiments, X2 is a C1-C6 alkyl group. In some examples, X2 is a homobifunctional linker or a heterobifunctional linker described above. In some examples, X2 is a homobifunctional linker described above. In some examples, X2 is a heterobifunctional linker described above. In some examples, X2 comprises a maleimide group, such as maleimidocaproyl (mc), described above, or a self-stabilizing maleimide group. In some examples, X2 comprises a peptide moiety, such as Val-Cit. In some examples, X2 comprises a benzoic acid group, such as PABA. In further examples, X2 comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In additional examples, X2 comprises an mc group. In an additional example, X2 comprises a mc-val-cit group. In an additional example, X2 comprises a val-cit-PABA group. In an additional example, X2 comprises a mc-val-cit-PABA group.

[0274] How to use Muscle atrophy refers to the loss of muscle mass and / or the progressive weakening and degeneration of muscle. In some cases, the loss of muscle mass or the progressive weakening and degeneration of muscle is caused by a high rate of protein breakdown, a low rate of protein synthesis, or a combination of both. In some cases, the high rate of muscle protein breakdown is due to muscle protein catabolism (i.e., the breakdown of muscle protein to use amino acids as substrates for gluconeogenesis).

[0275] In one embodiment, muscle atrophy refers to a significant loss of muscle strength. By significant loss of muscle strength, we mean a decrease in the strength of a subject's diseased, damaged, or unused muscle tissue compared to the same muscle tissue in a control subject. In some embodiments, significant loss of muscle strength is a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the same muscle tissue in a control subject. In another embodiment, significant loss of muscle strength refers to a decrease in the strength of unused muscle tissue compared to the muscle strength of the same muscle tissue in the same subject prior to the period of disuse. In some embodiments, significant loss of muscle strength is a decrease of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the muscle strength of the same muscle tissue in the same subject prior to the period of disuse.

[0276] In another embodiment, muscle atrophy refers to a significant loss of muscle mass. By significant loss of muscle mass, we mean a decrease in muscle volume in a subject's diseased, damaged, or unused muscle tissue compared to the same muscle tissue in a control subject. In some embodiments, a significant loss of muscle volume is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the same muscle tissue in a control subject. In another embodiment, a significant loss of muscle mass refers to a decrease in muscle volume in unused muscle tissue compared to the muscle volume of the same muscle tissue in the same subject prior to the period of disuse. In some embodiments, a significant loss of muscle tissue is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the muscle volume of the same muscle tissue in the same subject prior to the period of disuse. Muscle volume is optionally measured by assessing muscle cross-sectional area, such as by magnetic resonance imaging (eg, by muscle volume / cross-sectional area (CSA) MRI method).

[0277] In some embodiments, methods for treating muscle atrophy in a subject are described herein, including providing a polynucleic acid molecule described herein and administering a therapeutically effective amount of a polynucleic acid molecule described herein or a polynucleic acid molecule conjugate described herein to the subject to reduce the amount of human DUX4 mRNA transcripts. In some cases, the muscle atrophy is associated with facioscapulohumeral muscular dystrophy (FSHD). The polynucleic acid moiety mediates RNA interference against human DUX4, thereby regulating muscle atrophy in the subject. In some embodiments, the expression of one or more marker genes affected by DUX4 expression is also altered or regulated (e.g., decreased) by reduced expression of human DUX4. Marker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX. In some embodiments, the expression of one or more marker genes is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated cells. In some embodiments, expression of one or more marker genes as a group or complex is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated cells.

[0278] In some embodiments, described herein is a method for treating muscle atrophy in subject, comprising providing the siRNA antibody conjugate described herein, and administering to subject a therapeutically effective amount of the siRNA antibody conjugate described herein, and reducing the level of human DUX4 mRNA transcript in said subject.In some cases, muscle atrophy is associated with FSHD.The siRNA antibody conjugate mediates the RNA interference of human DUX4 mRNA to treat muscle atrophy in subject, and comprises administering to subject a therapeutically effective amount of the siRNA antibody conjugate described herein, and reducing the level of human DUX4 mRNA transcript in said subject.

[0279] In some embodiments, the present invention provides a method for treating muscle atrophy in a subject, comprising: providing a DUX4 siRNA antibody conjugate (DUX4 siRNA conjugate or DUX4-AOC) as described herein; administering a therapeutically effective amount of the DUX4 siRNA antibody conjugate to the subject; and reducing the level of human DUX4 mRNA transcript in the subject.In some cases, the muscle atrophy is associated with FSHD.The DUX4 siRNA antibody conjugate mediates RNA interference against human DUX4 mRNA to treat muscle atrophy in a subject, comprising administering a therapeutically effective amount of the DUX4 siRNA antibody conjugate as described herein to the subject; and reducing the level of human DUX4 mRNA transcript in the subject.

[0280] In some embodiments, described herein is a method for treating FSHD in a subject, comprising providing a DUX4 siRNA antibody conjugate described herein (DUX4 siRNA conjugate or DUX4-AOC), administering a therapeutically effective amount of the DUX4 siRNA antibody conjugate described herein to the subject, and reducing the level of human DUX4 mRNA transcripts in the subject. In some cases, the FSHD is FSHD type 1 (FSHD1). In some cases, the FSHD is FSHD type 2. The DUX4 siRNA antibody conjugate mediates RNA interference against human DUX4 mRNA to treat FSHD in a subject, comprising administering a therapeutically effective amount of the DUX4 siRNA conjugate described herein to the subject and reducing the level of human DUX4 mRNA transcripts in the subject. In some embodiments, the expression levels of one or more marker genes affected by DUX4 expression are also changed or regulated by reducing the expression level of human DUX4. DUX4 biomarker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX.

[0281] In some embodiments, described herein are methods of alleviating symptoms in a subject with FSHD, comprising providing a DUX siRNA antibody conjugate described herein (DUX4-siRNA conjugate or DUX4-AOC) and administering a therapeutically effective amount of the siRNA conjugate described herein to the subject by reducing the level of human DUX4 mRNA transcript. In some cases, the FSHD is FSHD type 1 (FSHD1). In some cases, the FSHD is FSHD type 2. In another embodiment, described herein are methods of alleviating symptoms in a patient with FSHD, comprising providing an siRNA conjugate described herein and administering a therapeutically effective amount of the siRNA conjugate described herein to the patient with FSHD by reducing the level of human DUX4 mRNA transcript or reducing the level of DUX4 protein.

[0282] In some cases, FSHD symptoms affect skeletal muscles. Skeletal muscles affected by FSHD include the muscles around the eyes and mouth, shoulder muscles, upper arm muscles, lower leg muscles, abdominal muscles, and hip muscles. In some cases, FSHD symptoms also affect vision and hearing. In some cases, FSHD symptoms also affect heart and lung function. In some cases, FSHD symptoms include muscle weakness, muscle atrophy, muscular dystrophy, pain inflammation, contractures, scoliosis, lordosis, hypoventilation, retinal abnormalities, exposure to keratitis, mild hearing loss, and EMG abnormalities.

[0283] In some embodiments, described herein is a method for improving skeletal muscle function in FSHD patients by reducing the level of human DUX4 mRNA transcripts or reducing the level of DUX4 protein, comprising administering a therapeutically effective amount of the siRNA conjugate described herein to the FSHD patient. In some cases, the FSHD is type 1 FSHD (FSHD1). In some examples, the FSHD is type 2 FSHD. In some embodiments, described herein is a method for improving skeletal muscle function, vision, hearing, cardiac function, or pulmonary function in patients with FSHD, comprising administering a therapeutically effective amount of the siRNA conjugate described herein to the FSHD patient by reducing the level of human DUX4 mRNA transcripts or reducing the level of DUX4 protein.

[0284] In some embodiments, described herein is a method for treating FSHD in a subject, comprising providing an antisense oligonucleotide (ASO)-antibody conjugate (ASO conjugate) described herein, administering a therapeutically effective amount of the ASO-antibody conjugate to the subject, and reducing the level of human DUX4 mRNA transcripts in the subject. In some cases, the FSHD is type 1 FSHD (FSHD1). In some cases, the FSHD is type 2 FSHD. The ASO conjugate mediates RNA interference against human DUX4 mRNA to treat FSHD in a subject, comprising administering a therapeutically effective amount of the ASO-antibody conjugate described herein to the subject and reducing the level of human DUX4 mRNA transcripts in the subject. In some embodiments, the expression levels of one or more marker genes affected by DUX4 expression are also changed or regulated by reducing the expression level of human DUX4. DUX4 biomarker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX.

[0285] In some embodiments, a method for treating FSHD in a subject is described herein. In some cases, the FSHD subject is affected by FSHD1. In other examples, the FSHD subject is affected by FSHD. In another embodiment, the FSHD subject has muscle cells that abnormally express DUX4 protein, which is caused by genetic and epigenetic molecular changes in the D4Z4 region of the long arm of chromosome 4. The genetic molecular change in the muscle cells is a mutation that leads to a shortening of the D4Z4 region containing 1 to 10 repeats, instead of the usual 11 to 100 repeats on chromosome 4 of FSHD subjects. The epigenetic molecular change in the muscle cells is a change that leads to hypomethylation of the D4Z4 region of chromosome 4 in FSHD patients. In some cases, the muscle cells are skeletal muscle cells.

[0286] Pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal routes of administration. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet other examples, the pharmaceutical compositions described herein are formulated for nasal administration.

[0287] In some embodiments, pharmaceutical compositions include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and combined immediate and controlled release formulations.

[0288] In some instances, the pharmaceutical formulation comprises a multiparticulate formulation. In some instances, the pharmaceutical formulation comprises a nanoparticle formulation. In some instances, the nanoparticle comprises cMAP, cyclodextrin, or lipid. In some instances, the nanoparticle comprises solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those with covalently bound metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, the nanoparticles are metal nanoparticles, such as nanoparticles of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys, or oxides thereof.

[0289] In some instances, the nanoparticles comprise a core, or alternatively, a core and a shell, such as in core-shell nanoparticles.

[0290] In some examples, the nanoparticles are further coated with molecules for binding of functional elements (e.g., to one or more of the polynucleic acid molecules or binding moieties described herein). In some examples, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyldextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, or dextrin or cyclodextrin. In some examples, the nanoparticles comprise graphene-coated nanoparticles.

[0291] In some cases, the nanoparticles have at least one dimension that is less than about 500 nm, 400 nm, 300 nm, 200 nm, or even 100 nm.

[0292] In some examples, the nanoparticle formulation comprises a paramagnetic nanoparticle, a superparamagnetic nanoparticle, a metal nanoparticle, a fullerene-like material, an inorganic nanotube, a dendrimer (such as one with a covalently bound metal chelate), a nanofiber, a nanohorn, a nano-onion, a nanorod, a nanorope, or a quantum dot. In some examples, a polynucleic acid molecule or binding moiety described herein is directly or indirectly attached to the nanoparticle. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding moieties described herein are directly or indirectly attached to the nanoparticle.

[0293] In some embodiments, the pharmaceutical formulation comprises a delivery vector, e.g., a recombinant vector for delivery of a polynucleic acid molecule to a cell. In some instances, the recombinant vector is a DNA plasmid. In other instances, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. In some instances, the recombinant vector capable of expressing a polynucleic acid molecule provides stable expression in target cells. In further instances, viral vectors are used that provide transient expression of a polynucleic acid molecule.

[0294] In some embodiments, pharmaceutical formulations include a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier substances include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, humectants, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholate, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. For example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HAand Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0295] In some examples, the pharmaceutical formulation further comprises a pH adjusting or buffering agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amount necessary to maintain the pH of the composition within an acceptable range.

[0296] In some instances, the pharmaceutical formulation contains one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0297] In some cases, pharmaceutical formulations further include diluents that are used to stabilize compounds, providing a more stable environment.Salts dissolved in buffers, including but not limited to phosphate buffered saline, (which also control or maintain pH) are used as diluents in the art.In certain cases, diluents increase the size of the composition to facilitate compression or create sufficient bulk for homogeneous mixing for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; calcium hydrogen phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0298] In some cases, pharmaceutical formulations contain disintegration agents or disintegrants to promote the break-up or disintegration of materials. The term "disintegrate" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starch, such as natural starch, e.g., corn starch or potato starch, pregelatinized starch, e.g., National 1551 or Amijel®, or sodium starch glycolate, e.g., Promogel® or Explotab®, cellulose, e.g., wood products, methyl crystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Min Tia®, and Solka-Floc®), methylcellulose, croscarmellose, or crosslinked cellulose, e.g., crosslinked starch such as crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®), crosslinked carboxymethylcellulose, or crosslinked croscarmellose, crosslinked polymers such as sodium starch glycolate, crospovidone, crosslinked polyvinylpyrrolidone, alginates, e.g., alginic acid or salts of alginic acid such as sodium alginate, clays such as Veegum® HV (magnesium aluminum silicate), gums such as agar, guar, locust bean, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, natural sponge, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, etc.

[0299] In some instances, the pharmaceutical formulation comprises a filler such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0300] Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein to prevent, reduce, or inhibit adhesion or friction of materials. Typical lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil, hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), higher fatty acids, and alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol, e.g., Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium lauryl sulfate or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, starches such as corn starch, silicone oil, surfactants, and the like.

[0301] Plasticizers include compounds used to soften microencapsulation materials or film coatings, thereby reducing their brittleness. Suitable plasticizers include, for example, polyethylene glycols such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers also function as dispersing or wetting agents.

[0302] Solubilizing agents include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, docusate sodium, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide.

[0303] Stabilizers include any antioxidant, buffer, acid, preservative, and like compounds.

[0304] Suspending agents include polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having a molecular weight of from about 300 to about 6000, from about 3350 to about 4000, or from about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose stearin. Acid acetate, polysorbate 80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth, gum arabic, guar gum, xanthan including xanthan gum, sugars, cellulosics such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0305] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, poloxamers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils (e.g., polyoxyethylene (60) hydrogenated castor oil); and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., Octoxynol 10, Octoxynol 40, etc. Surfactants are often included to enhance physical stability or for other purposes.

[0306] Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0307] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, docusate sodium, sodium oleate, sodium lauryl sulfate, docusate sodium, triacetin, Tween 80, vitamin E TPGS, ammonium salts, and the like.

[0308] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic use. In some embodiments, the pharmaceutical compositions are administered once a day, twice a day, three times a day, or more. The pharmaceutical compositions are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, once every two months, once every three months, once every four months, once every five months, once every six months, or more. The pharmaceutical compositions are administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0309] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at an interval (e.g., a first administration of a first pharmaceutical composition is on day 1, followed by at least 1, 2, 3, 4, 5, or more days before administration of at least a second pharmaceutical composition).

[0310] In some embodiments, two or more different pharmaceutical compositions are administered simultaneously.In some instances, two or more different pharmaceutical compositions are administered simultaneously.In some cases, two or more different pharmaceutical compositions are administered simultaneously without any interval between administrations.In other cases, two or more different pharmaceutical compositions are administered simultaneously consecutively with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, or 2 days between administrations.

[0311] Alternatively, the dose of the administered composition may be temporarily reduced or temporarily suspended for a specified period of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday may vary between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can be 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0312] Once the patient's condition has improved, a maintenance dose is administered if necessary, after which the dosage and / or frequency of administration can be reduced, depending on the symptoms, to a level at which the improved disease, disorder, or condition is maintained.

[0313] In some embodiments, the amount of a given agent corresponding to such an amount will depend on factors such as the particular compound, the severity of the disease, the nature (e.g., body weight) of the subject or host requiring treatment, and the like, but will nevertheless be routinely determined by methods known in the art according to the particular circumstances surrounding the case, including, for example, the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dosage is conveniently presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., two, three, four or more sub-doses per day.

[0314] Because of the large number of variables associated with any particular treatment regimen, the foregoing ranges are only suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on many variables, including, but not limited to, the activity of the compound being used, the disease or condition being treated, the mode of administration, the requirements of the particular subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0315] In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage will vary within this range depending on the dosage form used and the route of administration utilized.

[0316] Kits / Products In some embodiments, the present disclosure provides kits and products that can be used with one or more compositions and methods described herein. Such kits include a carrier, packaging, or container that is partitioned to contain one or more containers, such as vials, tubes, etc., each container containing one of the individual components for using the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is made of various materials, such as glass or plastic.

[0317] The products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, jars, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0318] For example, a container contains a target nucleic acid molecule described herein. Such kits optionally include an identifying description or label or instructions for use in the methods described herein.

[0319] Kits typically include a label listing the contents and / or instructions for use, and a package insert with instructions for use. A set of instructions is also typically included.

[0320] In one embodiment, a label is on or associated with the container. In one embodiment, a label is attached to a container when letters, numbers, or other indicia forming the label are affixed to, molded into, or engraved into the container itself. A label is associated with a container when it is present in a receptacle or carrier that holds the container, for example, as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates how to use the contents, for example, in the manner described herein.

[0321] In some embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms comprising a compound provided herein. The pack may, for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accompanied by a notice attached to the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the drug form for human or animal administration. Such notice may, for example, be labeling approved by the U.S. Food and Drug Administration for prescription drugs or approved package inserts. In one embodiment, compositions comprising a compound provided herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated disease.

[0322] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. It is understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used within the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is non-limiting.

[0323] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

[0324] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0325] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. No term is limited to situations characterized by the supervision (e.g., full-time or intermittent) of a health care professional (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker).

[0326] The term "therapeutically effective amount" refers to an amount of a polynucleic acid molecule conjugate sufficient to provide a desired therapeutic effect in a mammalian subject. In some cases, this amount is a single or multiple dose administered to a patient (e.g., a human) to treat, prevent, prevent the onset of, cure, delay, reduce the severity of, or ameliorate at least one symptom of a disorder or recurrent disorder, or to prolong the patient's survival beyond that expected in the absence of such treatment. Naturally, the dosage level of a particular polynucleic acid molecule conjugate used to provide a therapeutically effective amount will vary depending on the type of injury, the subject's age, weight, and sex, the subject's medical condition, the severity of the disease, the route of administration, and the particular inhibitor used. In some examples, a therapeutically effective amount of a polynucleic acid molecule conjugate is initially assessed using cell culture and animal models, as described herein. For example, IC50 values ​​determined using cell culture methods optionally serve as a starting point in animal models, while IC50 values ​​determined in animal models are optionally used to find therapeutically effective amounts in humans.

[0327] Skeletal, or voluntary, muscles are usually anchored to bones by tendons and are commonly used to effect skeletal movement, such as during locomotion or posture. Although some control of skeletal muscles is generally maintained as an involuntary reflex (e.g., postural muscles or the diaphragm), skeletal muscles can respond to conscious control. Smooth, or involuntary, muscles are found within the walls of organs and structures such as the esophagus, stomach, intestines, uterus, urethra, and blood vessels.

[0328] Skeletal muscle is further classified into two broad types: type I (or "slow twitch") and type II (or "fast twitch"). Type I muscle fibers are densely packed with capillaries and rich in mitochondria and myoglobin, which give type I muscle tissue its characteristic red color. In some cases, type I muscle fibers carry more oxygen and use fat or carbohydrates for fuel to sustain aerobic activity. Type I muscle fibers contract for a longer period but with less force. Type II muscle fibers are further subdivided into three main subtypes (IIa, IIx, and IIb) that differ in the speed of contraction and the force generated. Type II muscle fibers contract fast and powerfully but fatigue rapidly, thus resulting in a very short, intense period of anaerobic activity before the muscle contraction becomes painful.

[0329] Unlike skeletal muscle, smooth muscle is not under conscious control.

[0330] Cardiac muscle is also an involuntary muscle, but it is structurally very similar to skeletal muscle and is found only in the heart. Cardiac and skeletal muscles are striated in that they contain sarcomeres packed into highly ordered bundles. In contrast, the myofibrils of smooth muscle cells are not arranged in sarcomeres and therefore are not striated.

[0331] Muscle cells include any cell that contributes to muscle tissue. Exemplary muscle cells include myoblasts, satellite cells, myotubes, and myofibrillar tissue.

[0332] As used herein, muscle strength is proportional to cross-sectional area (CSA), and muscle velocity is proportional to muscle fiber length. Therefore, comparison of cross-sectional area and muscle fiber size between various muscle types can provide an indication of muscle atrophy. Various methods for measuring muscle strength and muscle mass are known in the art; see, for example, "Musculoskeletal assessment: Joint range of motion and manual muscle strength" by Hazel M. Clarkson, published by Lippincott Williams & Wilkins, 2000. Computed transverse tomography (CT) for generating cross-sectional images of selected muscle tissue and sonographic evaluation are additional methods for measuring muscle strength.

[0333] The term antibody-oligonucleotide conjugate (AOC) refers to an antibody conjugated to a nucleotide.

[0334] The term siRNA conjugate or siRNA antibody conjugate refers to an antibody conjugated to an siRNA.

[0335] The term DUX4 siRNA conjugate or DUX4 siRNA antibody conjugate refers to an antibody conjugated to an siRNA that hybridizes to a target sequence of human DUX4 mRNA.

[0336] The term DUX4-AOC refers to an antibody conjugated to an siRNA that hybridizes to a target sequence of human DUX4 mRNA. [Example]

[0337] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims.

[0338] Figure 2 shows a flowchart of the in silico selection process for DUX4 siRNAs. The sequences of all siRNAs that can bind to DUX4 or a predetermined region of DUX4 are collected to generate a starting set of DUX4 siRNAs. From the starting set of DUX siRNAs, the first elimination step involves removing one or more DUX siRNAs with single nucleotide polymorphisms (SNPs) and / or MEF<-5. Next, the second elimination step involves removing DUX siRNAs with 0 and 1 mismatches (MM) in the human transcriptome (resulting in only DUX, DUX5, and DBET hits being allowed). Next, the third elimination step involves removing DUX siRNAs with 0 mismatches (MM) in human intragenic regions (resulting in only DUX1, DUX5, and DBET pseudogenes being allowed). Next, the next elimination step involves eliminating DUX siRNAs containing the human sequence of DUX4 from MM for use in the FLExDUX4FSHD mouse model. The next step is to select only or more DUX siRNAs with a predicted survival rate of 60 or higher. Next, a elimination step involves removing one or more DUX siRNAs that match the seed region of known miRNAs 1-1000. This is followed by a elimination step that removes DUX siRNA molecules with a GC% content of 75 or higher. The final selection process then involves selecting 70 candidate DUX siRNAs with no more than 8 predicted off-target hits in 2 mm, except for the region 295-1132, where a maximum of 12 hits are allowed. Using this series of selection steps, we were able to select the final 70 candidate DUX siRNAs from the starting set of 1694 DUX siRNAs. Figure 3 shows the location and number of DUX4 siRNAs so selected in the DUX4 mRNA transcript (NM_001306068).

[0339] The identified siRNA candidates share common features in their sequences, as shown in Table 10 below. The identified siRNAs mostly have 2'-O-Me modifications, with 2'-F located only at positions 7, 8, and 9 of the sense strand and only at positions 1, 2, 6, 14, and 16 of the antisense strand. The identified siRNAs also contain four thioate modifications in each strand, located at the last two linkages at the 5' and 3' ends. The identified siRNAs further contain "Uf" at the first position of the 5' end of the antisense strand (with "a" attached at the last position of the 3' end of the sense strand), regardless of the actual target mRNA sequence. The identified siRNAs further contain a "uu" overhang only at the 3' end of the antisense strand, and no overhang at the 3' end of the sense strand. Optimization of the identified siRNAs may include phosphonate vinyl nucleotides, inverted abasic moieties, or amine linkers to the passenger or guide strand.

[0340] [Table 10]

[0341] Tables 11, 12, and 13 illustrate the identification of siRNA candidates for the regulation of human DUX4.

[0342] [Table 11] JPEG2026041765000029.jpg243170

[0343] [Table 12] JPEG2026041765000031.jpg249160 JPEG2026041765000032.jpg165170

[0344] [Table 13] JPEG2026041765000034.jpg35156

[0345] Example 2. siRNA Sequence and Synthesis All siRNA single strands were fully assembled on a solid phase using standard phosphoramidite chemistry and purified by HPLC. The purified single strands were then duplexed to obtain double-stranded siRNAs. All siRNA passenger strands contain conjugation handles in various formats, such as C6-NH2 and / or C6-SH, one at each end of the strand. The conjugation handle or handles are connected to the siRNA passenger strand or siRNA guide strand via reverse abasic phosphodiester or phosphorothioate. Representative structures of the formats used in in vivo experiments are shown below. [ka]

[0346] The typical structure of an siRNA comprises a C6-NH2 conjugation handle at the 5' end of the passenger or guide strand and a C6-SH at the 3' end. [ka]

[0347] A typical structure of an siRNA passenger or guide strand has a C6-NH2 conjugation handle at the 5' end and a C6-S-PEG at the 3' end. [ka]

[0348] The typical structure of an siRNA passenger or guide strand has a C6-NH2 conjugation handle at the 5' end and a C6-S-NEM at the 3' end. [ka]

[0349] A typical structure of an siRNA passenger or guide strand has a C6-N-SMCC conjugation handle at the 5' end and a C6-S-NEM at the 3' end. [ka]

[0350] A typical structure of an siRNA passenger strand or guide strand has PEG at the 5' end and C6-SH at the 3' end. [ka]

[0351] A typical structure of an siRNA passenger or guide strand has a C6-S-NEM at the 5' end and a C6-NH2 conjugation handle at the 3' end.

[0352] Example 3. Conjugate synthesis The following structures illustrate exemplary A-X1-B-X2-Y (Formula I) structures described herein: [ka]

[0353] Structure-1: Antibody-Cys-SMCC-S-5'-passenger chain. This conjugate was generated by interchain cysteine ​​conjugation of the antibody to the maleimide (SMCC) at the 5' end of the passenger chain. [ka]

[0354] Structure-2: Antibody-Cys-SMCC-S-3'-passenger chain. This conjugate was generated by interchain cysteine ​​conjugation of the antibody to the maleimide (SMCC) at the 3' end of the passenger chain. [ka]

[0355] ASC Structure-3: Antibody-Cys-bisMal-3'-passenger chain. This conjugate was generated by interchain cysteine ​​conjugation of the antibody to a bismaleimide (bisMal) linker at the 3' end of the passenger chain. [ka]

[0356] ASC Structure-4: Model structure of Fab-Cys-bisMal-3'-passenger chain. This conjugate was generated by interchain cysteine ​​conjugation of Fab to a bismaleimide (bisMal) linker at the 3' end of the passenger chain. [ka]

[0357] ASC Structure-5: Model structure of an antibody-siRNA conjugate with two different siRNAs bound to one antibody molecule. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNAs to the reduced mAb interchain cysteines relative to the bismaleimide (bisMal) linker at the 3' end of the passenger strand of each siRNA. [ka]

[0358] ASC Structure-6: Model structure of an antibody-siRNA conjugate with two different siRNAs attached. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNAs to the mAb interchain cysteine ​​reduced to maleimide (SMCC) linkers at the 3' end of the passenger strand of each siRNA.

[0359] Example 3.1 Antibody-siRNA Conjugate Synthesis Using SMCC Linker [ka] Step 1: Antibody interchain disulfide reduction with TCEP The antibody was buffer-exchanged into borax buffer (pH 8) to a concentration of up to 10 mg / ml. Two equivalents of TCEP in water were added to this solution and rotated at room temperature for 2 hours. The resulting reaction mixture was buffer-exchanged into PBS, pH 7.4, containing 5 mM EDTA and added to a solution of SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-X kDa (2 equivalents) (X = 0.5 kDa to 10 kDa) in PBS, pH 7.4, containing 5 mM EDTA at room temperature and rotated overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed antibody-siRNA conjugates along with unreacted antibody and siRNA.

[0360] Step 2: Purification The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1 as described in Example 3.4. Fractions containing DAR1 and DAR>2 antibody-siRNA-PEG conjugates were isolated, concentrated, and buffer-exchanged into PBS, pH 7.4.

[0361] Step 3: Analysis of purified conjugates The isolated conjugates were characterized by SEC, SAX chromatography, and SDS-PAGE. The purity of the conjugates was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3. Both methods are described in Example 3.4. The isolated DAR1 conjugates typically eluted at 9.0±0.3 minutes on the analytical SAX method and were greater than 90% pure. A typical DAR>2 cysteine ​​conjugate contains greater than 85% DAR2 and less than 15% DAR3.

[0362] Example 3.2. Antibody-siRNA Conjugate Synthesis Using a Bismaleimide (BisMal) Linker [ka] Step 1: Reduction of antibody with TCEP The antibody was buffer-exchanged into borax buffer (pH 8) to a maximum concentration of 5 mg / ml. Two equivalents of TCEP in water were added to this solution and rotated at room temperature for 2 hours. The resulting reaction mixture was exchanged with PBS containing 5 mM EDTA, pH 7.4, and added to a solution of BisMal-C6-siRNA-C6-S-NEM (2 equivalents) in PBS containing 5 mM EDTA, pH 7.4, at room temperature and maintained at 40°C overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed the presence of antibody-siRNA conjugates along with unreacted antibody and siRNA.

[0363] Step 2: Purification The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR2 antibody-siRNA conjugates were isolated, concentrated, and buffer-exchanged into PBS, pH 7.4.

[0364] Step 3: Analysis of purified conjugates The isolated conjugates were characterized by either mass spectrometry or SDS-PAGE. The purity of the conjugates was assessed by analytical HPLC using either anion exchange chromatography method-2 or size exclusion chromatography method-1.

[0365] Example 3.3. Fab' Generation from mAb and Conjugation to siRNA [ka]

[0366] Step 1: Antibody digestion with pepsin The antibody was buffer exchanged into 20 mM sodium acetate / acetic acid buffer, pH 4.0, to a concentration of up to 5 mg / ml. Immobilized pepsin (Thermo Scientific, Product #20343) was added and incubated at 37°C for 3 hours. The reaction mixture was filtered using a 30 kDa MWCO Amicon spin filter and PBS, pH 7.4. The retentate was collected and purified using size exclusion chromatography to isolate F(ab')2. The collected F(ab')2 was then reduced with 10 equivalents of TCEP and conjugated with SMCC-C6-siRNA-PEG5 in PBS, pH 7.4, at room temperature. Analysis of the reaction mixture on SAX chromatography revealed the Fab-siRNA conjugate along with unreacted Fab and siRNA-PEG.

[0367] Step 2: Purification The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. The fractions containing DAR1 and DAR2 Fab-siRNA conjugates were isolated, concentrated, and buffer exchanged into PBS pH 7.4.

[0368] Step 3: Analysis of purified conjugates The characterization and purity of the isolated conjugates were assessed by analytical HPLC using anion exchange chromatography methods-2 or 3 as well as SEC method-1.

[0369] Example 3.4. Purification and analytical methods Anion Exchange Chromatography Method (SAX)-1. 1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5mm ID x 15cm, 13um 2. Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 ​​mM TRIS, 1.5 M NaCl, pH 8.0; Flow rate: 6.0 mL / min 3. Gradient: a. %A %B Column volume b.100 0 1.00 c.60 40 18.00 d.40 60 2.00 e.40 60 5.00 f.0 100 2.00 g.100 0 2.00

[0370] Anion Exchange Chromatography (SAX) Method-2 1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4 x 250 mm 2. Solvent A: 80%, 10 mM TRIS, pH 8, 20% ethanol; Solvent B: 80%, 10 mM TRIS, pH 8, 20% ethanol, 1.5 M NaCl; Flow rate: 0.75 mL / min 3. Gradient: a.Time %A %B b.0.0 90 10 c.3.00 90 10 d.11.00 40 60 e.13.00 40 60 f.15.00 90 10 g.20.00 90 10

[0371] Anion Exchange Chromatography (SAX) Method-3 1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4 x 250 mm 2. Solvent A: 80%, 10 mM TRIS, pH 8, 20% ethanol; Solvent B: 80%, 10 mM TRIS, pH 8, 20% ethanol, 1.5 M NaCl; 3.Flow rate: 0.75mL / min 4. Gradient: a.Time %A %B b.0.0 90 10 c.3.00 90 10 d.11.00 40 60 e.23.00 40 60 f.25.00 90 10 g.30.00 90 10

[0372] Size Exclusion Chromatography (SEC) Method-1 1. Column: TOSOH Biosciences, TSKgel G3000SW XL, 7.8 x 300 mm, 5 μM 2. Mobile phase: 150 mM phosphate buffer 3. Flow rate: 1.0 ml / min for 15 minutes

[0373] Example 3.5. Antibody-siRNA Conjugate Synthesis Using a Bismaleimide (BisMal) Linker Step 1: Reduction of antibody with TCEP The antibody was buffer exchanged into 25 mM borate buffer (pH 8) containing 1 mM DTPA to a maximum concentration of 10 mg / ml. To this solution, 4 equivalents of TCEP in the same borate buffer were added and incubated at 37°C for 2 hours. The resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer, pH 6.0, at room temperature and maintained at 4°C overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate along with unreacted antibody and siRNA. To cap any remaining free cysteine ​​residues, the reaction mixture was treated with 10 eq of N-ethylmaleimide (10 mg / mL in DMSO).

[0374] Step 2: Purification The crude reaction mixture was purified by AKTA Pure FPLC using anion exchange chromatography (SAX) method-1. Fractions containing DAR1 and DAR2 antibody-siRNA conjugates were isolated, concentrated, and buffer-exchanged with PBS, pH 7.4.

[0375] Anion Exchange Chromatography Method (SAX)-1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5mm ID x 15cm, 13um Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 ​​mM TRIS, 1.5 M NaCl, pH 8.0; Flow rate: 6.0 ml / min gradient: a. %A %B Column volume b.100 0 1 c.81 19 0.5 d.50 50 13 e.40 60 0.5 f.0 100 0.5 g.100 0 2

[0376] Anion Exchange Chromatography (SAX) Method-2 Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4 x 250 mm Solvent A: 80%, 10 mM TRIS, pH 8, 20% ethanol; Solvent B: 80%, 10 mM TRIS, pH 8, 20% ethanol, 1.5 M NaCl; Flow rate: 0.75 ml / min gradient: a.Time %A %B b.0.0 90 10 c.3.00 90 10 d.11.00 40 60 e.14.00 40 60 f.15.00 20 80 g.16.00 90 10 h.20.00 90 10

[0377] Example 4. Expression profile of DUX4 in myonuclei Myotubes derived from healthy individuals and FSHD patients were evaluated for DUX4 expression. Myotubes were immunostained to detect DUX4 expression, as shown in Figure 4. The nuclei and cytoplasm of muscle cells were immunostained with DAPI (4',6-diamidino-2-phenylindole) and troponin T. As shown, DUX4 expression was detected in less than 1% of myonuclei, indicating that direct detection of DUX4 expression from cells may be difficult in determining the effects of DUX4 siRNA activity.

[0378] Example 5. Expression profile of DUX4-dependent marker genes in FSHD donor muscle cells treated with DUX4 siRNA Two DUX4 siRNAs (siDUX4-1 and siDUX4-4, disclosed in Geng LN et al., Dev. Cell, 2012) were used to treat diseased myocytes (FSHD donor myocytes), and the RNA expression levels of five DUX4-dependent biomarker genes were quantified, as shown in the bar graph in Figure 5. As shown, both siDUX4-1 and siDUX4-4 significantly reduced the expression of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, and LEUTX compared to baseline (100%). More specifically, siDUX4-4 reduced the expression of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, and LEUTX by at least 75% compared to baseline (100%). DUX4 target genes as biomarkers were sensitive for measuring siRNA-mediated DUX4 downregulation.

[0379] Example 6. DUX4 siRNA-mediated reduction of five DUX4 target biomarker gene expression and the FSHD complex of DUX4 target biomarker genes in cultured FSHD primary myotubes DUX4 siRNA activity was assessed at dose-dependent concentrations using two published DUX4 siRNAs, siDUX4- and siDUX4-4 (Geng LN et al., Dev. Cell, 2012), using primary myoblasts (MB06) derived from an FSHD1 patient. FSHD primary myoblasts (MB06(FSHD1)) were cultured in the recommended medium. Prior to seeding, 96-well tissue culture plates (Costar) were coated with 50 μL of 1% Matrigel per well for at least 2 hours at 37°C and washed twice with PBS. After coating, myoblasts were seeded in quadruplicate at 4000 cells / well without antibiotics and maintained for 24 hours prior to transfection. On the day of transfection, DUX4-4 siRNA was formulated using the commercially available transfection reagents Lipofectamine RNAiMAX (Life Technologies) and OptiMEM (Life Technologies) according to the manufacturer's instructions for "forward transfection." DUX4-4 siRNA was synthesized by Integrated DNA Technologies (IDT). Myoblasts were transfected with a high concentration of 25 nM DUX4-4 siRNA in a 9-fold serial dilution. 24 h after transfection, myogenic differentiation was induced in differentiation medium containing 15% KOSR. Myotubes were collected with Trizol 4 days after induction of differentiation and stored at -80°C until processing. RNA isolation was performed using the Direct-zol-96 RNA Isolation Kit (Zymo) according to the manufacturer's instructions. 100–500 ng of purified RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using a SimpliAmp Thermal Cycler (Applied Biosystems).cDNA was analyzed by qPCR using TaqMan Fast Universal Master Mix II (Thermo Fisher) and TaqMan probes (Thermo Fisher) in duplicate on a QuantStudio 6 or 7 Flex Real-Time PCR instrument (Applied Biosystems). Data were analyzed using QuantStudio™ Real-Time PCR Software v1.3 (Applied Biosystems). Expression levels of five DUX4 target genes, MBD3L2, ZSCAN4, LEUTX, KHDC1L, and TRIM43, were assessed. Expression of DUX4 target genes was normalized to a complex of two reference genes, AHSA1 and RPL27. Percentages of target mRNA expression were calculated using a 2. -ΔΔCt The expression of five DUX4 target biomarker genes was determined compared to mock-treated cells using the Livak and Schmittgen, Methods 2001 method. Figures 6A-B show DUX4 siRNA-mediated reduction of five DUX4 target biomarker gene expression in cultured FSHD primary myotubes. DUX4 siRNA reduced the expression of five individual DUX4 target biomarker genes (MBD3L2, ZSCAN4, LEUTX, KHDC1L, and TRIM43) (Figure 6A) or the FSHD complex of five DUX4 target biomarker genes (Figure 6B) in FSHD primary myotubes.

[0380] Example 7. DUX4 siRNA Library Screening—First Round at Two Concentrations of MB02 and MB06 (10 and 0.5 nM) In this example, 70 DUX4 siRNAs were screened for their activity in two FSHD primary myoblast cell lines (MB02 (FSHD1) and MB06 (FSHD1)) to identify more desirable siRNA candidates. Cells were seeded at a density of 4,000 cells / well (MW96) and then transfected in quadruplicate with DUX4 siRNA and a control siRNA. Transfection was performed 24 hours after plating at 10 nM. Myogenic differentiation was induced 2 days (24 hours after transfection) after plating in 15% KOSR (in DMEM / F-12) medium. Depending on the cell line, samples were harvested 3 or 4 days after induction of differentiation. DUX4 downstream target gene expression was assessed by RT-qPCR (normalized to the combined AHSA1 and RPL27 housekeeping gene expression values). Data presented in this example are expressed as the mean FSHD combined − / + SEM. N=4.

[0381] FIG. 7 shows a bar graph of the assessment of myotube differentiation and viability at 10 nM concentration of siDUX4 by monitoring ACTA1 expression in MB02(FSHD1) and MB06(FSHD1) cell lines.

[0382] Figure 8 shows a bar graph of the screening of 70 DUX4 siRNAs for activity at 10 nM concentration by measuring FSHD complex expression (FSHD complex calculation: Dct = (average ct4DUX4 target gene) - (average ct2HKG), DDct). = Dct(siDUX4) - Dct(mock), Complex = 2^-DDct * 100(%)).

[0383] Figure 9 shows a bar graph of the activity screening of 70 DUX4 siRNAs at a concentration of 0.5 nM by measuring FSHD complex expression. As used herein, the FSHD complex is made up of four DUX4 target genes: MBD3L2, ZSCAN4, LEUTX, and KHDC1L. As shown in Tables 14-15, the downregulation of FSHD complex expression correlated well with the downregulation of individual genes in the complex when normalized to the HKG complex (AHSA1, RPL27) (n=4), indicating that effective downregulation of FSHD complex expression is a good indicator of DUX4 siRNA efficacy.

[0384] [Table 14]

[0385] [Table 15]

[0386] As shown in Figure 10, from a DUX4 siRNA library containing 70 siRNA candidates, 37 candidates with KDs of less than 70% in at least one cell line at 10 nM were eliminated. Next, three additional siRNA candidates that reduced ACTA1 expression to less than 70% in both cell lines were eliminated. Next, two additional siRNA candidates with null activity (<10% KD) at 0.5 nM in both cell lines were eliminated, leaving a total of 28 siRNA candidates for the next screening process. Table 16 lists the 28 DUX4 siRNAs selected in this way and the downregulation of FSHD complex expression in two cell lines at concentrations of 10 nM and 0.5 nM for the selected 28 DUX4 siRNAs (also shown as bar graphs in Figures 11A and 11B).

[0387] [Table 16]

[0388] Human polymorphism databases are unreliable in their iterations and may miss polymorphic positions. Therefore, in this round of selection, we excluded siRNAs that performed poorly in one of the two myoblast cell lines, with the goal of verifying that the selected DUX4 siRNAs were active in myoblasts derived from various FSHD patients. Figure 12 shows the distribution of 70 siRNAs by their potency in downregulating the FSHD complex in the two myoblast cell lines. KD correlation analysis identified approximately 10% of DUX4 siRNAs that functioned only in one of the two FSHD primary myoblast cell lines used in the full library screening.

[0389] Example 8. DUX4 siRNA Library Screening - Second Round at 10 nM in Four Different Patient-Derived Primary Myoblast Cell Lines In this example, a total of nine high-quality FSHD patient-derived primary myoblast cell lines (six FSHD1 and three FSHD2) were used. Conditions enabling reliable detection of DUX4 target gene expression in FSHD myotubes were established in-house by culturing cells in differentiation medium (15% KOSR in DMEM / F-12). Time points were specifically selected for each cell line. All nine FSHD cell lines showed a concentration-dependent response to the tool DUX4 siRNA.

[0390] Figure 13 shows a bar graph of ACTA1 expression levels in four different patient-derived primary myoblast cell lines (MB01, MB05, MB11, and MB12). While most of the top 28 DUX4 siRNAs did not affect ACTA1 expression levels in these cell lines, several siRNAs showed a greater than 30% reduction in four additional FSHD primary cell lines tested (8 in MB11, 3 in MB05, 3 in MB12, and none in MB01). Furthermore, as shown in Figure 14, the top 28 DUX4 siRNAs were active in all four additional FSHD primary cell lines. Several siRNAs showed greater than 75% KD in three cell lines: MB05, MB11, and MB12. KD levels were generally lower in MB01. Figure 15 shows the activity of the top 28 DUX4 siRNAs at 10 nM in all FSHD cell lines (MB01, MB02, MB05, MB06, MB11, and MB12).

[0391] From the top 28 DUX4 siRNAs, we selected the top 14 DUX4 siRNAs. These top 14 DUX4 siRNAs were selected based on i) siRNAs that showed no or minimal cytotoxicity (visual identification and assessment of ACTA1 expression levels) and ii) siRNAs that showed the highest activity at both 10 nM and 0.5 nM concentrations. Table 17 lists the top 14 DUX4 siRNAs and their downregulation of FSHD complex expression in six primary FSHD cell lines (MB01, MB02, MB05, MB06, MB11, and MB12) at both 10 nM and 0.5 nM concentrations, and their downregulation of ACTA1 expression in six primary FSHD cell lines at 10 nM. Table 18 lists the top 14 DUX4 siRNAs and their downregulation of FSHD complex expression in all nine FSHD primary myotubes.

[0392] [Table 17]

[0393] [Table 18]

[0394] Example 9. Top 14 DUX4 siRNAs evaluated for concentration-response efficacy in MB02, MB05, MB06 The objective of this experiment was to select eight siRNAs with the best Emax and potency for off-target analysis. Three FSHD primary myoblast cell lines (MB02, MB05, and MB06) were used. Cells were seeded at a density of 4,000 cells / well (MW96) and transfected in quadruplicate with 14 selected DUX4 siRNAs. Transfection was performed 24 hours after plating. Myogenic differentiation was induced 2 days (24 hours after transfection) after plating in 15% KOSR (in DMEM / F-12) medium. Depending on the cell line, samples were harvested 3 or 4 days after induction of differentiation. DUX4 downstream target gene expression was assessed by RT-qPCR (normalized to the combined AHSA1 and RPL27 housekeeping gene expression values). Data are presented as mean - / + SEM. N=4.

[0395] Figures 16A-C show the concentration response of 14 selected DUX4 siRNAs in three FSHD patient-derived primary myotubes, MB02, MB05, and MB06, respectively. DUX-4 target gene expression was reduced by more than 75% for most of the top 14 DUX4 siRNAs in three FSHD patient-derived primary myoblast cell lines. Differences in potency between siRNAs were also observed, ranging from 60- to 100-fold depending on the cell line. Tables 19-22 show the potency of DUX4 siRNAs evaluated in three FSHD patient-derived primary myotubes based on the FSHD complex.

[0396] [Table 19]

[0397] [Table 20]

[0398] [Table 21]

[0399] [Table 22]

[0400] Example 11. Antibody-DUX4 siRNA conjugate (DUX4-AOC) mediated reduction of FSHD complex (complex of DUX4 target biomarker genes) expression in cultured FSHD primary myotubes The in vitro concentration-response potency and maximal efficacy of 16 DUX4-AOCs (8 vpUq AOCs or 8 non-VP AOCs) were evaluated in primary myotubes (MB06) derived from an FSHD1 patient. The guide strand of the DUX4 siRNA in the AOC either contained a vinyl phosphonate at the 5' end of the strand (vpUq) or did not contain a vinyl phosphonate at the 5' end of the strand (non-VP).

[0401] A human IgG1 antibody against human TfR1 was expressed in a CHO stable pool created by transfecting the CHOK1SV GS-KO host cell line with a dual gene vector. The antibody was captured from the cell culture supernatant using protein A affinity chromatography. The resulting antibody was further purified using hydrophobic interaction chromatography (to reduce aggregates) and anion exchange chromatography (to reduce host cell DNA and endotoxins). The final antibody was buffer-exchanged into either PBS or 50 mM sodium citrate buffer, pH 6.5, at a concentration of 20 mg / mL. Antibody purity was assessed by size exclusion chromatography.

[0402] The guide and perfectly complementary RNA passenger strands were assembled on a solid phase using standard phosphamidite chemistry and purified by HPLC. The purified single strands were then double-stranded to yield double-stranded siRNA. The guide strand was generated with a vinylphosphonate-modified nucleotide structure at the 5' end. The passenger strand had a conjugation handle at the 5' end via a phosphorothioate-inverted abasic phosphodiester linker.

[0403] Antibody-oligonucleotide conjugates (AOCs) were generated using the random cysteine ​​conjugation method. The interchain disulfide bonds of the antibody were partially reduced with TCEP before conjugation with maleimide linker-siRNA. The reaction mixture was purified using strong anion exchange chromatography to ensure a drug-antibody ratio (DAR) of 1 (i.e., one siRNA molecule per antibody molecule). The collected AOC fractions were concentrated, buffer exchanged into PBS, and sterile filtered using a 0.2 μm filter. The purity of the AOCs was assessed using strong anion exchange chromatography, size exclusion chromatography, and SDS-PAGE.

[0404] FSHD primary myoblasts (MB06(FSHD1)) were grown in the recommended medium. Prior to seeding, 96-well tissue culture plates (Costar) were coated with 50 μL of 1% Matrigel per well for at least 2 hours at 37°C and washed twice with PBS. After coating, myoblasts were seeded at 4,000 cells / well in quadruplicate. Two days after plating, myogenic differentiation was induced with differentiation medium containing 15% KOSR. 24 hours after differentiation induction, siDUX4-AOC was added to the medium at a high concentration of 100 nM in 10-fold serial dilutions. Untreated cells were maintained as a control. After 3 days of incubation with DUX4-AOC, myotubes were collected in Trizol and stored at -80°C until processing. RNA isolation was performed using the Direct-zol-96 RNA Isolation Kit (Zymo) according to the manufacturer's instructions. 100–500 ng of purified RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a SimpliAmp Thermal Cycler (Applied Biosystems). cDNA was analyzed by qPCR using TaqMan Fast Universal Master Mix II (Thermo Fisher) and TaqMan probes (Thermo Fisher) in duplicate on a QuantStudio 6 or 7 Flex Real-Time PCR instrument (Applied Biosystems). Data were analyzed using QuantStudio™ Real-Time PCR Software v1.3 (Applied Biosystems). Expression levels of DUX4 target genes were assessed by calculating an FSHD composite score, which integrated the expression levels of four DUX4 target genes (MBD3L2, ZSCAN4, LEUTX, and KHDC1L) normalized to two reference genes (AHSA1 and RPL27).

[0405] Figures 18A-B show dose-response curves for DUX4-AOC-mediated reduction of DUX4 target biomarker gene expression in cultured FSHD primary myotubes. Most DUX4-AOCs containing vinyl phosphonate (Figure 18A) and some DUX4-AOCs without vinyl phosphonate (Figure 18B) reduced the expression of the FSHD complex of four DUX4 target biomarker genes (MBD3L2, ZSCAN4, LEUTX, and KHDC1L) in primary myotubes from FSHD1 patients. Overall, DUX4-AOC reduced the expression of DUX4 target biomarker gene expression, and the presence of vinyl phosphonate in DUX4 siRNA in AOCs enhanced the reduction of DUX4 target biomarker gene expression.

[0406] Example 12. Malat1-siRNA AOC mediated in vivo reduction of nuclear-localized Inc-RNA Malat1 mRNA levels in three different mouse skeletal muscles Wild-type female CD-1 mice (approximately 6–8 weeks old) were administered a single IV bolus injection of either Malat1 or Scramble AOC into the tail vein at 5 mL / kg body weight, where siRNA was conjugated to a mouse anti-transferrin receptor (mTfR1) antibody at doses of 0.3, 1, 3, or 6 mg / kg body weight (siRNA amount). Two weeks after treatment, muscle tissue samples were collected in tubes containing ceramic beads, snap-frozen in liquid nitrogen, and then homogenized in 1 mL of chilled Trizol using a FastPrep-24 (MP Biomedicals). The homogenate supernatant was used for RNA isolation using the Direct-zol-96 RNA Isolation Kit (Zymo) according to the manufacturer's instructions. 100–500 ng of purified RNA was converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using a SimpliAmp Thermal Cycler (Applied Biosystems). cDNA was analyzed by qPCR using TaqMan Fast Universal Master Mix II (Thermo Fisher) and TaqMan probes (Thermo Fisher) in duplicate using a QuantStudio 6 or 7 Flex Real-Time PCR instrument (Applied Biosystems). Data were analyzed using QuantStudio™ Real-Time PCR Software v1.3 (Applied Biosystems). Target gene expression was normalized to the reference gene Ppib. -ΔΔCt The percentage of target mRNA expression in treated samples compared to the control treatment was determined using the . Data are expressed as % of the PBS control (mean ± SEM; N = 4 for siMalat1 and siScramble AOC, N = 5 for the PBS group).

[0407] Figure 19 shows Malat1 siRNA-AOC-mediated in vivo reduction of nuclear-localized lnc-RNA Malat1 levels in mouse skeletal muscle. A single dose of up to 6 mg / kg of Malat1 siRNA-AOC (siRNA dose) in mice reduced nuclear Malat1 expression in skeletal muscle by up to 80% two weeks after administration. The reduction in nuclear Malat1 mRNA expression levels demonstrates the in vivo ability of AOC to target nuclear RNA for degradation.

[0408] Example 13. Sustained AOC-mediated in vivo reduction of SSB mRNA levels in mouse skeletal muscle by a single dose of 3 mg / kg siRNA AOC over an 8-week period Wild-type male C57BL / 6 mice (approximately 12–16 weeks old) were administered a single IV bolus injection of 5 mL / kg body weight of Ssb siRNA conjugated to a mouse anti-TfR1 (mTfR1) antibody at a dose of 3 mg / kg body weight (siRNA amount) either without vinyl phosphonate (non-VP) or with vinyl phosphonate (vpUq) via the tail vein. Gastrocnemius muscles were collected at the following time points after administration: days 1, 7, 14, 28, 43, and 57. Muscles were placed in tubes containing ceramic beads, flash-frozen in liquid nitrogen, and then homogenized in 1 mL of cold Trizol using FastPrep-24 (MP Biomedicals). The homogenate supernatant was used for RNA isolation using the Direct-zol-96 RNA Isolation Kit (Zymo) according to the manufacturer's instructions. 100–500 ng of purified RNA was converted to cDNA using the High-Capacity DNA Reverse Transcription Kit (Applied Biosystems) in a SimpliAmp Thermal Cycler (Applied Biosystems). cDNA was analyzed by qPCR using TaqMan Fast Universal Master Mix II (Thermo Fisher) and TaqMan probes (Thermo Fisher) in duplicate using a QuantStudio 6 or 7 Flex Real-Time PCR instrument (Applied Biosystems). Data were analyzed using QuantStudio™ Real-Time PCR Software v1.3 (Applied Biosystems). Ssb gene expression was normalized to the reference gene Ppib. -ΔΔCt The percentage of target mRNA expression in treated samples compared to the control treatment (PBS) was determined using the . Data are expressed as % of the PBS control (mean ± SEM; N = 4 for siSsb-AOCs, N = 3-5 for PBS group).

[0409] Figure 20 shows SSB siRNA-AOC-mediated reduction of in vivo SSB mRNA levels in mouse skeletal muscle for 8 weeks by a single administration of 3 mg / kg siRNA. After a single administration of 3 mg / kg (siRNA dose) of SSB siRNA-AOC both without vinylphosphonate (non-VP) and with vinylphosphonate (vpUq), a sustained AOC-mediated in vivo reduction of SSB mRNA expression levels was achieved in mouse skeletal muscle.

[0410] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the disclosure described herein may be utilized in practicing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of the claims and their equivalents be covered thereby.

Claims

1. A polynucleic acid molecule conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DUX4, and mediates RNA interference against DUX4.

2. 2. The polynucleic acid molecule conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a non-human antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or antigen-binding fragment thereof.

3. The polynucleic acid molecule conjugate of any one of claims 1-2, wherein the antibody or antigen-binding fragment thereof is an anti-transferrin receptor antibody or antigen-binding fragment thereof.

4. 4. The polynucleic acid molecule conjugate of any one of claims 1 to 3, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and the sense strand and / or the antisense strand each independently comprise at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

5. 5. The polynucleic acid molecule conjugate of claim 1, wherein the polynucleotide hybridizes to at least 8 consecutive bases of a target sequence of DUX4.

6. The polynucleic acid molecule conjugate of any one of claims 1 to 5, wherein the polynucleotide is from about 8 to about 50 nucleotides in length, or from about 10 to about 30 nucleotides in length.

7. 7. The polynucleic acid molecule conjugate of any one of claims 1 to 6, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and the sense strand comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 1-70 or 141-210.

8. 8. The polynucleic acid molecule conjugate of any one of claims 1 to 7, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and the antisense strand comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 71-140 or 211-280.

9. the polynucleotide comprises at least one 2'-modified nucleotide, and the 2'-modified nucleotide is 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-N-methylacetamide (2'-O-NMA) modified nucleotides; comprising a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA), or including combinations of these A polynucleic acid molecule conjugate according to any one of claims 1 to 8.

10. 10. The polynucleic acid molecule conjugate of claim 1, wherein at least one modified internucleotide linkage comprises a phosphorothioate or phosphorodithioate linkage.

11. The polynucleic acid molecule conjugate of any one of claims 1 to 10, wherein the polynucleic acid molecule comprises three or more 2'-modified nucleotides selected from 2'-O-methyl and 2'-deoxy-2'-fluoro.

12. The polynucleic acid molecule conjugate of any one of claims 1 to 11, wherein the polynucleic acid molecule comprises a 5'-terminal vinylphosphonate modified nucleotide.

13. 13. The polynucleic acid molecule conjugate of any one of claims 1 to 12, wherein the 2'-modified nucleotide is a 2'-O-methyl modified nucleotide, and the 2'-O-methyl modified nucleotide is at the 5' end of the sense strand and / or the antisense strand.

14. The polynucleic acid molecule conjugate of claim 13, wherein the 2'-O-methyl modified nucleotide is a purine nucleotide.

15. The polynucleic acid molecule conjugate of claim 13, wherein the 2'-O-methyl modified nucleotide is a pyridine nucleotide.

16. The polynucleic acid molecule conjugate of any one of claims 13 to 15, wherein the sense strand and / or the antisense strand comprises at least two, three, or four consecutive 2'-O-methyl modified nucleotides at the 5' end.

17. 17. The polynucleic acid molecule conjugate of any one of claims 1 to 16, comprising a linker connecting said antibody or antigen-binding fragment thereof to said polynucleic acid molecule.

18. The polynucleic acid molecule conjugate of claim 17, wherein the linker is a C1-C6 alkyl linker.

19. 18. The polynucleic acid molecule conjugate of claim 17, wherein the linker is a homobifunctional linker or a heterobifunctional linker and comprises a maleimide group, a dipeptide moiety, a benzoic acid group, or a derivative thereof.

20. 18. The polynucleic acid molecule conjugate of claim 17, wherein the linker is a cleavable or non-cleavable linker.

21. 21. The polynucleic acid molecule conjugate of any one of claims 1-20, wherein the ratio between said polynucleic acid molecule and said antibody or antigen-binding fragment thereof is about 1:1, 2:1, 3:1, or 4:

1.

22. 22. The polynucleic acid molecule conjugate of any one of claims 1-21, wherein the polynucleic acid molecule mediates RNA interference against human DUX4 and regulates muscle atrophy in a subject.

23. The polynucleic acid molecule conjugate of claim 22, wherein the RNA interference comprises reducing the expression of the mRNA transcript of the DUX4 gene by at least 50%, at least 60%, or at least 70% or more compared to the amount of the mRNA transcript of the DUX4 gene in an untreated cell.

24. The polynucleic acid molecule conjugate of any one of claims 22-23, wherein the RNA interference comprises affecting the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX in a cell.

25. 25. The polynucleic acid molecule conjugate of claim 24, wherein affecting the expression of the marker gene reduces the expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or more.

26. The polynucleic acid molecule conjugate of any one of claims 22 to 25, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

27. Formula (I) A-X-B (Formula I) wherein: A is the antibody or antigen-binding fragment thereof; B is a polynucleic acid molecule that hybridizes to a target sequence of DUX4; X is a bond or a non-polymeric linker; wherein X is attached to a cysteine ​​residue of A; A polynucleic acid molecule conjugate according to any one of claims 1 to 26.

28. A polynucleic acid molecule conjugate according to claims 1-27, and pharmaceutically acceptable excipients A pharmaceutical composition comprising:

29. 29. The pharmaceutical composition of claim 28, formulated as a nanoparticle formulation.

30. 30. The pharmaceutical composition of any one of claims 28-29, formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intrathecal administration.

31. 1. A method for treating muscular dystrophy in a subject in need thereof, comprising: Providing a polynucleic acid conjugate according to any one of claims 1 to 30; and administering a polynucleic acid conjugate to a subject in need thereof to treat muscular dystrophy, wherein said polynucleic acid conjugate reduces the amount of human DUX4 mRNA transcript; A method comprising:

32. 32. The method of claim 31, wherein the polynucleic acid portion mediates RNA interference against human DUX4 and regulates muscle atrophy in a subject.

33. 33. The method of claim 32, wherein the RNA interference affects the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX in cells affected by muscular dystrophy.

34. 34. The method of any one of claims 31-33, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

35. Use of a polynucleic acid molecule conjugate according to any one of claims 1 to 27 or a pharmaceutical composition according to any one of claims 28 to 30 for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

36. Use of a polynucleic acid molecule conjugate according to any one of claims 1 to 27 or a pharmaceutical composition according to any one of claims 28 to 30 for the manufacture of a medicament for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

37. A kit comprising a polynucleic acid molecule conjugate according to any one of claims 1-27 or a pharmaceutical composition according to any one of claims 28-30.

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