Compositions and methods for treating fascioscapulohumeral muscular dystrophy

Abstract

Polynucleic acid molecules, pharmaceutical compositions, and methods for treating facioscapulohumeral muscular dystrophy (FSHD) are provided.SOLUTION: Provided are polynucleic acid molecules conjugates comprising antibodies or antigen-binding fragments thereof conjugated to polynucleic acid molecules that hybridize to a target sequence of DUX4, wherein the polynucleic acid molecules comprise specific sequences and comprise 2 ' - F modified nucleotides at positions 2, 6, 14, and 16, and wherein the polynucleic acid molecules mediate RNA interferences against the DUX4.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 245,123, filed September 16, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Muscle atrophy is the loss of muscle mass or the progressive weakening and degeneration of muscles, including skeletal and voluntary muscles that control movement, cardiac muscles, and smooth muscles. Various pathophysiological conditions, including disuse, starvation, cancer, diabetes, and renal failure, or treatment with glucocorticoids, can result in muscle atrophy and loss of strength. The phenotypic effects of muscle atrophy are mediated 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.

[0003] FSHD is a rare, progressive, disabling disease for which there is no approved treatment. It is one of the most common forms of muscular dystrophy, affecting both sexes equally and typically occurring in adolescents and young adults. FSHD is characterized by progressive skeletal muscle loss, initially causing weakness in the face, shoulders, arms, and trunk, and progressing to weakness in the lower limbs and pelvic girdle. Skeletal muscle weakness leads to significant physical limitations, including progressive loss of facial muscles, which can make it difficult to smile or communicate, perform daily activities using the arms, and stand up. Many patients ultimately become wheelchair-dependent for daily activities. Many FSHD patients also report the development of chronic pain, anxiety, and depression.

[0004] FSHD is caused by abnormal expression of the gene DUX4 in skeletal muscle, resulting in inappropriate expression of the DUX4 protein. Gene silencing by RNA-guided gene silencing can result in several levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) produces long-lasting effects that span multiple cell divisions. Therefore, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease treatment.

[0005] Incorporation by Reference 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

[0006]

[0010] In some aspects, described herein 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 comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 5%, or 100% identical to a sequence selected from SEQ ID NOs: 72, 76, 126, or 131-136, wherein the polynucleic acid molecule comprises 2'-F modified nucleotides at positions 2, 6, 14, and 16, and wherein the polynucleic acid molecule mediates RNA interference against DUX4. In some 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 some embodiments, the antibody or antigen-binding fragment thereof is an anti-transferrin receptor antibody or antigen-binding fragment thereof. In some embodiments, the polynucleic acid molecule is about 16 to about 30 nucleotides in length. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least one of the nucleic acid sequences UfsNfsnnnNfnnnnnnnNfnNfnnnsusu, usNfsnnnNfnnnnnnnNfnNfnnnsusu, or vpNsNfsnnNfnnnnnnnnNfnnnnsus, where vpN = vinylphosphonate VpUq, lowercase (n) = 2'-O-Me modification, Nf = 2'-F modification, and s = phosphorothioate backbone modification. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438.In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 2, 6, 56, or 61-66, and comprises at least two or at least three consecutive 2'-F modified nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 2, 6, 56, or 61-66. In some embodiments, the polynucleic acid molecule comprises phosphorothioate or phosphorodithioate linkages. In some embodiments, the polynucleic acid molecule comprises six or more 2'-modified nucleotides selected from 2'-O-methyl and 2'-deoxy-2'-fluoro. In some embodiments, the polynucleic acid molecule comprises a 5'-terminal vinylphosphonate modified nucleotide. In some embodiments, the 5'-terminal vinylphosphonate modified nucleotide is:

[0007] [ka] wherein B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group connecting adjacent nucleotides of the polynucleic acid molecule. In some embodiments, the sense strand and / or antisense strand comprises at least two, at least three, or at least four consecutive 2'-O-methyl modified nucleotides at the 5'- or 3'-end. In some embodiments, the polynucleic acid molecule conjugate comprises a linker connecting the antibody or antigen-binding fragment thereof to the polynucleic acid molecule via a cysteine ​​or lysine residue on the antibody or antigen-binding fragment. In some embodiments, the linker is a C1-C6 alkyl linker. In some embodiments, the linker is a homobifunctional or heterobifunctional linker and comprises a maleimide group, a dipeptide moiety, a benzoic acid group, or derivatives thereof. In some embodiments, the linker is a cleavable or non-cleavable linker. In some embodiments, the polynucleic acid molecule conjugate has a ratio of polynucleic acid molecule to antibody or antigen-binding fragment thereof of about 1:1, 2:1, 3:1, or 4:1. In some embodiments, the polynucleic acid molecule mediates RNA interference against human DUX4 and regulates muscle atrophy in a subject. In some embodiments, the RNA interference comprises reducing the expression of mRNA transcription of the DUX4 gene by at least 50%, at least 60%, or at least 70% or more compared to the amount of mRNA transcription of the DUX4 gene in untreated cells. In some embodiments, the RNA interference comprises affecting the expression of a marker gene in the cell selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX. In some embodiments, the RNA interference comprises affecting the expression of a marker gene in the cell selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD. In some embodiments, affecting expression of the marker gene comprises reducing expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more, hi some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0008] In some aspects herein, a pharmaceutical composition is described, comprising the 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, intravenous, subcutaneous, or intrathecal administration.

[0009] Some aspects of the present specification describe a method for treating muscular dystrophy in a subject in need thereof, the method comprising: providing a polynucleic acid conjugate described herein; and administering the polynucleic acid conjugate to the subject to treat the muscular dystrophy, wherein the polynucleic acid conjugate reduces the amount of mRNA transcription of human DUX4. In some embodiments, the polynucleic acid conjugate mediates RNA interference against human DUX4 and regulates muscular dystrophy in the subject. In some embodiments, the RNA interference comprises affecting the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD in cells affected by muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0010] Some aspects herein describe the use of a polynucleic acid molecule conjugate described herein or a pharmaceutical composition described herein for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, some aspects herein describe the use of a polynucleic acid molecule conjugate described herein or a pharmaceutical composition described herein in the manufacture of a medicament for treating a subject diagnosed with or suspected of having FSHD. Some aspects herein describe kits comprising a polynucleic acid molecule conjugate described herein or a pharmaceutical composition described herein.

[0011] In some embodiments, described herein are polynucleic acid molecules that mediate RNA interference against DUX4, the polynucleic acid molecules comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438.

[0012] In some embodiments, described herein is a double-stranded polynucleic acid molecule that mediates RNA interference against DUX4, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438, and the sense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

[0013]

[0010] In some embodiments, the present specification describes a double-stranded polynucleic acid molecule that mediates RNA interference against DUX4, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence comprising at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from a sequence selected from SEQ ID NOs: 412-420 or 430-438, and the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

[0014] In certain embodiments, disclosed herein are polynucleic acid molecules and pharmaceutical compositions for regulating genes associated with muscle wasting, particularly facioscapulohumeral muscular dystrophy (FSHD). Also described herein in some embodiments are methods for treating muscle wasting, particularly FSHD, with the polynucleic acid molecules or polynucleic acid molecule conjugates disclosed herein.

[0015] In certain embodiments, disclosed herein is a polynucleic acid molecule conjugate comprising an antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DUX4, the polynucleic acid molecule conjugate mediating RNA interference against DUX4. In certain embodiments, the antibody or its antigen-binding fragment comprises a non-human antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a monoclonal antibody or its antigen-binding fragment, 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 its antigen-binding fragment. In certain embodiments, the antibody or its antigen-binding fragment is an anti-transferrin receptor antibody or its antigen-binding fragment.

[0016] 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 eight contiguous 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 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. Alternatively and / or additionally, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand 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. Alternatively and / or additionally, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand 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: 142, 146, 196, 201-206, 412-420, or 430-438. In some aspects, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand 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: 412-420 or 430-438. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand is identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438.In some aspects, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand 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: 142, 146, 196, or 201-206. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand is identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

[0017] In certain embodiments, the polynucleotide comprises at least one 2'-modified nucleotide, further comprising a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modified nucleotide, or a locked nucleic acid (LNA) or ethylene nucleic acid (ENA), or a combination thereof. In certain embodiments, at least one modified internucleotide linkage comprises a phosphorothioate or phosphorodithioate bond. 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.

[0018] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0019] [ka] wherein B is a heterocyclic base moiety.

[0020] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0021] [ka] is selected from wherein B is a heterocyclic base moiety, R1, R2, and R3 are independently selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0022] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0023] [ka] is selected from wherein B is a heterocyclic base moiety, R4 and R5 are independently selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0024] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0025] [ka] is selected from wherein B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0026] Another embodiment provides a polynucleic acid molecule of a polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

[0027] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0028] [ka] is selected from A polynucleic acid molecule is provided wherein B is a heterocyclic base moiety and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0029] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0030] [ka] wherein B is a heterocyclic base moiety and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0031] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0032] [ka] is selected from wherein B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0033] Another embodiment is a polynucleic acid molecule of the polynucleic acid molecule conjugate, wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is

[0034] [ka] The present invention provides a polynucleic acid molecule selected from:

[0035] 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 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.

[0036] In certain embodiments, the polynucleic acid molecule contains 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 a homobifunctional or heterobifunctional linker, including 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 of the polynucleic acid moiety to the target cell binding moiety is about 1:1, 2:1, 3:1, or 4:1.

[0037] In certain embodiments, the polynucleic acid portion mediates RNA interference against human DUX4 and regulates the symptoms of muscular dystrophy or muscular atrophy in a subject. In some embodiments, the RNA interference comprises reducing the expression of mRNA transcription of DUX4 gene by at least 50%, at least 60%, or at least 70% or more compared to the amount of mRNA transcription of 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 or including 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). Alternatively and / or additionally, the RNA interference comprises affecting the expression of a marker gene in the cell, including or selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD. 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).

[0038] 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 conjugated to a cysteine ​​residue of A.

[0039] In certain aspects herein, disclosed is a pharmaceutical composition comprising the polynucleic acid molecule conjugate described herein and a pharmaceutically acceptable excipient.In some aspects, the pharmaceutical composition is formulated as a nanoparticle formulation.In some aspects, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, or intravenous, subcutaneous, or intrathecal administration.

[0040] Symptoms of FSHD include effects on skeletal muscles. FSHD affects muscles around the eyes and mouth, shoulder muscles, upper arm muscles, lower leg muscles, abdominal muscles, and gluteal muscles. In some cases, FSHD symptoms also affect vision and hearing. In some cases, FSHD symptoms also affect cardiac or pulmonary function. In some instances, FSHD symptoms include muscle weakness, muscle atrophy, muscular dystrophy, pain, inflammation, spasticity, 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 muscle-related effects of FSHD.

[0041] In certain embodiments herein, a method is disclosed 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 to treat the muscular dystrophy. The polynucleic acid conjugate reduces the amount of mRNA transcription of human DUX4. In some embodiments, the polynucleic acid portion mediates RNA interference against human DUX4, thereby regulating muscular atrophy in the subject. In certain embodiments, the RNA interference comprises affecting the expression of a marker gene for DUX4 in cells affected by muscular dystrophy, the marker gene being selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX. In certain embodiments, the RNA interference comprises affecting the expression of a marker gene for DUX4 in cells affected by muscular dystrophy, the marker gene being selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD.

[0042] Preferably, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0043] In certain embodiments disclosed herein 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 described in certain embodiments herein is the use of a polynucleic acid molecule conjugate or pharmaceutical composition described herein in the manufacture of a medicament for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

[0044] In certain aspects herein, disclosed are kits comprising the polynucleic acid molecule conjugates or pharmaceutical compositions described herein. [Brief explanation of the drawings]

[0045] Various aspects of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood 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, in which:

[0046] [Figure 1] Illustrate a diagram of FSHD pathology. [Figure 2] A flowchart diagram of in silico selection of DUX4 siRNA is shown. [Figure 3] The location and number of selected DUX4 siRNAs in the DUX4 mRNA transcript are illustrated. [Figure 4A] FIG. 1 shows a graph of in vivo downregulation of DUX4 target genes in skeletal muscle of a mouse model of FSHD. [Figure 4B] FIG. 1 shows a graph of in vivo muscle tissue concentration of DUX-4 siRNA. [Figure 5A]A representative structure of an siRNA is depicted, with a C6-NH2 conjugation handle at the 5' end of the passenger or guide strand and a C6-SH at the 3' end. [Figure 5B] A representative structure of an siRNA passenger or guide strand is depicted, having a C6-NH2 conjugation handle at the 5' end and a C6-S-PEG at the 3' end. [Figure 5C] A representative structure of an siRNA passenger or guide strand is depicted, having a C6-NH2 conjugation handle at the 5' end and a C6-S-NEM at the 3' end. [Figure 5D] A representative structure of an siRNA passenger strand is depicted, with a C6-N-SMCC conjugation handle at the 5' end and a C6-S-NEM at the 3' end. [Figure 5E] A representative structure of an siRNA passenger or guide strand is depicted, having PEG at the 5' end and C6-SH at the 3' end. [Figure 5F] A representative structure of an siRNA passenger or guide strand is depicted, with a C6-S-NEM at the 5' end and a C6-NH2 conjugation handle at the 3' end. [Figure 6A] Illustrates antibody-Cys-SMCC-5'-passenger strand (Architecture 1). This conjugate was generated by antibody interchain cysteine ​​conjugation to maleimide (SMCC) at the 5' end of the passenger strand. [Figure 6B] Illustrates antibody-Cys-SMCC-3'-passenger chain (Architecture 2). This conjugate was generated by antibody interchain cysteine ​​conjugation to maleimide (SMCC) at the 3' end of the passenger chain. [Figure 6C] Illustrates antibody-Cys-bisMal-3'-passenger chain (ASC Architecture 3). This conjugate was generated by antibody interchain cysteine ​​conjugation to a bismaleimide (bisMal) linker at the 3' end of the passenger chain. [Figure 6D] A model structure of Fab-Cys-bisMal-3'-passenger strand (ASC architecture 4) is shown. This conjugate was generated by inter-Fab chain cysteine ​​conjugation to a bismaleimide (bisMal) linker at the 3' end of the passenger strand. [Figure 6E] A model structure of an antibody-siRNA conjugate (ASC Architecture 5) is shown, in which two different siRNAs are attached to one antibody molecule. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNA to a reduced mAb interchain cysteine ​​to bismaleimide (bisMal) linker at the 3' end of the passenger strand of each siRNA. [Figure 6F] A model structure of an antibody-siRNA conjugate (ASC Architecture 6) with two different siRNAs attached was generated by conjugating a mixture of SSB and HPRT siRNA to a reduced mAb interchain cysteine ​​to maleimide (SMCC) linker at the 3' end of the passenger strand of each siRNA. [Figure 7A] FIG. 1 illustrates an exemplary synthetic scheme (Synthetic Scheme 1) for antibody-Cys-SMCC-siRNA-PEG conjugates via antibody-cysteine ​​conjugation. [Figure 7B] 1 illustrates an exemplary synthetic scheme for antibody-Cys-BisMal-siRNA-PEG conjugates (Synthetic Scheme 2). [Figure 7C] A typical synthetic scheme for producing Fab-siRNA conjugates (Synthetic Scheme 3) is illustrated. DETAILED DESCRIPTION OF THE INVENTION

[0047] 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 lead to muscle pathology. Normally, DUX4-driven gene expression is restricted to the germline and early stem cell development. In FSHD patients, 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 signature that distinguishes FSHD-affected muscle tissue from healthy muscle. Aberrant DUX4 expression in FSHD results in muscle death and replacement with fat, resulting in skeletal muscle weakness and progressive disability. Data suggest that reduced expression of the DUX4 gene and its downstream transcriptional program could provide a disease-modifying therapeutic approach for the treatment of FSHD, which is the underlying cause of the condition.

[0048] There are two ways that the DUX4 gene can be silenced or derepressed. FSHD1, which comprises approximately 95% of FSHD patients, has a mutation that shortens an array of DNA 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, encompassing between 1 and 10 repeats instead of the normal 11 to 100 repeats. This contraction results in hypomethylation of the D4Z4 region and derepression of DUX4. FSHD2 patients do not have significant D4Z4 repeat contractions but have mutations in a regulatory gene known as SMCHD1, which normally contributes to DUX4 gene repression through DNA methylation. When this repression is lost due to a mutation in the SMCHD1 gene that causes hypomethylation of the D4Z4 region, DUX4 is inappropriately expressed, leading to the disease state. Figure 1 shows an exemplary diagram of FSHD pathology.

[0049] Nucleic acid (for example, RNAi) therapy is a highly selective and specific targeted therapy.However, in some cases, nucleic acid therapy is also hindered by poor intracellular uptake, limited blood stability, and non-specific immune stimulation.To address these problems, various modifications of nucleic acid compositions have been explored, such as new linkers for good stabilization and / or low toxicity, optimization of binding moieties for increasing target specificity and / or target delivery, and nucleic acid polymer modifications for increasing stability and / or reducing off-target effects.

[0050] In some embodiments, the arrangement or order of the various components comprising a nucleic acid composition further affects intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, when a nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (i.e., a polynucleotide), the order or arrangement of the binding moiety, polymer, and / or polynucleic acid molecule (i.e., polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule) further affects intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0051] In some embodiments, described herein are polynucleic acid molecules and polynucleic acid molecule conjugates for the treatment of facioscapulohumeral muscular dystrophy (FSHD), particularly muscular dystrophy and / or muscular atrophy associated therewith. In some examples, the polynucleic acid molecule conjugates described herein have improved intracellular uptake, stability, and / or efficacy. Optionally, the polynucleic acid molecule conjugate comprises an antibody or antigen-binding fragment thereof conjugated to the polynucleic acid molecule. Optionally, a polynucleic acid molecule that hybridizes to a target sequence of DUX4, preferably human DUX4. Optionally, a nucleic acid molecule that hybridizes to a target sequence of human DUX4 having accession number NM_001306068. Optionally, a nucleic acid molecule that hybridizes to a target sequence of human DUX4 having SEQ ID NO: 439.

[0052] 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.

[0053] Polynucleic acid molecule In certain embodiments, the polynucleic acid molecule hybridizes to a target sequence of the Double homeobox 4 (DUX4) gene. In some examples, the polynucleic acid molecules described herein hybridize to a target sequence of the human DUX4 gene (DUX4) and reduce DUX4 mRNA in muscle cells.

[0054] In some embodiments, the polynucleic acid molecule 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 embodiments, the polynucleic acid molecule 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 embodiments, the polynucleic acid molecule 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 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.

[0055] In some embodiments, the polynucleic acid molecule 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: 142, 146, 196, 201-206, 412-420, or 430-438.

[0056] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some examples, 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. Optionally, 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. Optionally, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some examples, 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. Optionally, 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.

[0057] In some examples, 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: 142, 146, 196, or 201-206. Optionally, 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: 412-420 or 430-438.

[0058] In some embodiments, the polynucleic acid molecule comprises a sense strand (e.g., a passenger strand) and an antisense strand (e.g., a guide strand). In some examples, the sense strand (e.g., the 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 examples, the antisense strand (e.g., the 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., a passenger strand) and an antisense strand (e.g., a guide strand). In some examples, the sense strand (e.g., the 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 examples, the antisense strand (e.g., the 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. In some examples, 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: 142, 146, 196, or 201-206. In some examples, 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: 412-420 or 430-438.

[0059] In some examples, 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 NO: 1, 2, 3, 6, 14, 36, 52, 56, 61, 62, 63, 65, or 66. In some examples, 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, or 136. In some examples, the siRNA comprises the sense strand and antisense strand shown in Table 11.

[0060] In some examples, 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, or 206. In some examples, 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, or 276. In some examples, the siRNA comprises the sense strand and antisense strand shown in Table 12.

[0061] In some examples, 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: 142, 146, 196, or 201-206 in Table 14 and Table 15.

[0062] In some examples, 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: 412-420 or 430-438 in Table 14 and Table 15.

[0063] In some aspects, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand 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: 412-420 or 430-438. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand is identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438. In some aspects, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand 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: 142, 146, 196, or 201-206. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand is identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

[0064] In some embodiments, the polynucleic acid molecule has at least 14, 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides, no more than 2 nucleotides, or no more than 1 nucleotide from any one of SEQ ID NOs: 142, 146, 196, or 201-206, or SEQ ID NOs: 412-420, or 430-438. In some embodiments, the polynucleic acid molecule is single-stranded. In some embodiments, the polynucleic acid molecule is double-stranded.

[0065] In some embodiments, the polynucleic acid molecules described herein comprise RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some cases, the RNA comprises small interfering RNA (siRNA), short hairpin RNA (shRNA), μRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some cases, the RNA comprises shRNA. In some cases, the RNA comprises miRNA. In some cases, the RNA comprises dsRNA. In some cases, the RNA comprises tRNA. In some cases, the RNA comprises rRNA. In some cases, the RNA comprises hnRNA. In some cases, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO), which is a short single-stranded oligonucleotide analogue constructed with a backbone of morpholine rings connected by phosphorodithioate linkages. In some cases, the RNA comprises siRNA. In some cases, the polynucleic acid molecule comprises siRNA.

[0066] In some embodiments, the polynucleic acid molecule is about 8 to about 50 nucleotides in length. In some embodiments, the polynucleic acid molecule is about 10 to about 50 nucleotides in length. In some examples, 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.

[0067] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some examples, the polynucleic acid molecule is about 45 nucleotides in length. In some examples, the polynucleic acid molecule is about 40 nucleotides in length. In some examples, the polynucleic acid molecule is about 35 nucleotides in length. In some examples, the polynucleic acid molecule is about 30 nucleotides in length. In some examples, the polynucleic acid molecule is about 25 nucleotides in length. In some examples, the polynucleic acid molecule is about 20 nucleotides in length. In some examples, the polynucleic acid molecule is about 19 nucleotides in length. In some examples, the polynucleic acid molecule is about 18 nucleotides in length. In some examples, the polynucleic acid molecule is about 17 nucleotides in length. In some examples, the polynucleic acid molecule is about 16 nucleotides in length. In some examples, the polynucleic acid molecule is about 15 nucleotides in length. In some examples, the polynucleic acid molecule is about 14 nucleotides in length. In some examples, the polynucleic acid molecule is about 13 nucleotides in length. In some examples, the polynucleic acid molecule is about 12 nucleotides in length. In some examples, the polynucleic acid molecule is about 11 nucleotides in length. In some examples, the polynucleic acid molecule is about 10 nucleotides in length. In some examples, the polynucleic acid molecule is about 8 nucleotides in length. In some examples, the polynucleic acid molecule is between about 8 and about 50 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 50 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 45 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 40 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 35 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 30 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 25 nucleotides in length. In some examples, the polynucleic acid molecule is between about 10 and about 20 nucleotides in length. In some examples, the polynucleic acid molecule is between about 15 and about 25 nucleotides in length. In some examples, the polynucleic acid molecule is between about 15 and about 30 nucleotides in length. In some instances, the polynucleic acid molecule is between about 12 and about 30 nucleotides in length.

[0068] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide. In some examples, the polynucleic acid molecule comprises a second polynucleotide. In some examples, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide is a sense strand or a passenger strand. In some examples, the second polynucleotide is an antisense strand or a guide strand.

[0069] 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. In some embodiments, the first polynucleotide is about 10 to about 50 nucleotides in length. In some examples, 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.

[0070] In some examples, the first polynucleotide is about 50 nucleotides in length. In some examples, the first polynucleotide is about 45 nucleotides in length. In some examples, the first polynucleotide is about 40 nucleotides in length. In some examples, the first polynucleotide is about 35 nucleotides in length. In some examples, the first polynucleotide is about 30 nucleotides in length. In some examples, the first polynucleotide is about 25 nucleotides in length. In some examples, the first polynucleotide is about 20 nucleotides in length. In some examples, the first polynucleotide is about 19 nucleotides in length. In some examples, the first polynucleotide is about 18 nucleotides in length. In some examples, the first polynucleotide is about 17 nucleotides in length. In some examples, the first polynucleotide is about 16 nucleotides in length. In some examples, the first polynucleotide is about 15 nucleotides in length. In some examples, 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 between about 8 and about 50 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 50 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 45 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 40 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 35 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 30 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 25 nucleotides in length. In some examples, the first polynucleotide is between about 10 and about 20 nucleotides in length. In some examples, the first polynucleotide is between about 15 and about 25 nucleotides in length.In some examples, the first polynucleotide is between about 15 and about 30 nucleotides in length. In some examples, the first polynucleotide is between about 12 and about 30 nucleotides in length.

[0071] 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 examples, 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.

[0072] In some examples, the second polynucleotide is about 50 nucleotides in length. In some examples, the second polynucleotide is about 45 nucleotides in length. In some examples, the second polynucleotide is about 40 nucleotides in length. In some examples, the second polynucleotide is about 35 nucleotides in length. In some examples, the second polynucleotide is about 30 nucleotides in length. In some examples, the second polynucleotide is about 25 nucleotides in length. In some examples, the second polynucleotide is about 20 nucleotides in length. In some examples, the second polynucleotide is about 19 nucleotides in length. In some examples, the second polynucleotide is about 18 nucleotides in length. In some examples, the second polynucleotide is about 17 nucleotides in length. In some examples, the second polynucleotide is about 16 nucleotides in length. In some examples, the second polynucleotide is about 15 nucleotides in length. In some examples, the second polynucleotide is about 14 nucleotides in length. In some examples, the second polynucleotide is about 13 nucleotides in length. In some examples, the second polynucleotide is about 12 nucleotides in length. In some examples, the second polynucleotide is about 11 nucleotides in length. In some examples, the second polynucleotide is about 10 nucleotides in length. In some examples, the second polynucleotide is about 8 nucleotides in length. In some examples, the second polynucleotide is between about 8 and about 50 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 50 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 45 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 40 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 35 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 30 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 25 nucleotides in length. In some examples, the second polynucleotide is between about 10 and about 20 nucleotides in length. In some examples, the second polynucleotide is between about 15 and about 25 nucleotides in length.In some examples, the second polynucleotide is between about 15 and about 30 nucleotides in length. In some examples, the second polynucleotide is between about 12 and about 30 nucleotides in length.

[0073] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the polynucleic acid molecule further comprises a blunt end, an overhang, or a combination thereof. In some examples, 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, 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 four 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-paired nucleotides at the 3' end as an overhang, while the sense strand has no overhang. Optionally, in such embodiments, the non-base-paired 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.

[0074] 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 of about 10-50 base pairs, about 15-50 base pairs, 15-40 base pairs, 15-30 base pairs, or 15-25 base pairs in length in DUX4, wherein the first nucleotide of the target sequence starts at any nucleotide in the DUX4 mRNA transcript, in the coding region or in the 5' or 3' untranslated region (UTR). For example, the first nucleotide of the target sequence can be selected to start at nucleic acid position (nal, the number starting from the 5'-end of the full length of the DUX mRNA, e.g., the first nucleotide at the 5'-end is nal.1) 1, nal2, nal3, nal4, nal5, nal6, nal7, nal8, nal9, nal10, nal11, nal12, nal13, nal14, nal15, nal15, nal16, nal17, or any other nucleic acid position in the coding or non-coding portion (5' or 3' untranslated region) of the DUX mRNA.In some embodiments, the first nucleotide of the target sequence is nal10 to nal15, nal10 to nal20, nal50 to nal60, nal55 to nal65, nal75 to nal85, nal95 to nal105, nal135 to nal145, nal155 to nal165, nal225 to nal235, nal265 to nal275, nal275 to nal285, nal285 to nal295, nal325 to nal335, nal335 to nal345, al345, nal385~nal395, nal515~nal525, nal665~nal675, nal675~nal685, nal695~nal705, nal705~nal715, nal875~nal88 5, nal885~nal895, nal895~nal905, nal1035~nal1045, nal1045~nal1055, nal1125~nal1135, nal1135~nal1145, nal1145~ nal1155, nal1155~nal1165, nal1125~nal1135, nal1155~nal1165, nal1225~nal1235, nal1235~nal1245, nal1275~nal12 85, nal1285~nal1295, nal1305~nal1315, nal1125~nal1135, nal1155~nal1165, nal1225~nal1235, nal1235~nal1245, nal You can choose to start at a location within or between 1275-nal1285, nal1285-nal1295, nal1305-nal1315, nal1315-nal1325, nal1335-nal1345, nal1345-nal1355, nal1525-nal1535, nal1535-nal1545, nal1605-nal1615, nal1615-c.1625, nal1625-nal1635.

[0075] In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to the target sequence described herein. In some examples, the sequence of the polynucleic acid molecule is 100% complementary to the target sequence described herein.

[0076] In some embodiments, the sequence of the polynucleic acid molecule has five or fewer mismatches to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule has four or fewer mismatches to the target sequence described herein. In some examples, the sequence of the polynucleic acid molecule has three or fewer mismatches to the target sequence described herein. Optionally, the sequence of the polynucleic acid molecule has two or fewer mismatches to the target sequence described herein. Optionally, the sequence of the polynucleic acid molecule has one or fewer mismatches to the target sequence described herein.

[0077] In some embodiments, a group of polynucleic acid molecules in all polynucleic acid molecules potentially binds to the DUX4 target sequence selected to generate the polynucleic acid molecule library. In certain embodiments, such a selection process is performed in silico by one or more steps of eliminating 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 MEFs below -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 (such that the only hits allowed 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 (such that the only hits allowed are DUX1, DUX5, and DBET pseudogenes). Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules with a MM relative to the DUX4 human sequence 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 viability of less than 60. Alternatively and / or additionally, such a selection process includes carrying forward one or more polynucleic acid molecules with a predicted viability of 60 or greater. Alternatively and / or additionally, in some embodiments, the selection process includes eliminating one or more polynucleic acid molecules with a match to the seed region of known miRNAs 1-1000. Alternatively and / or additionally, in some embodiments, the selection process includes eliminating 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 eliminating eight or fewer predicted off-target hits with two MMs. In some embodiments, for the region 295-1132 (nal295-1132), no more than 12 predicted off-target hits with two MMs are allowed.

[0078] In some embodiments, the selection process is carried out in silico by one or more successive steps to remove less desirable polynucleic acid molecules from the candidates. For example, in some embodiments, the selection process begins with collecting candidate polynucleic acid molecules to generate a library. From this library, the first removal step involves removing one or more polynucleic acid molecules with single nucleotide polymorphisms (SNPs) and / or MEFs below -5. Then, the second removal step involves removing one or more polynucleic acid molecules with MMs of 0 and 1 within the human transcriptome (where the only allowed hits are DUX, DUX5, and DBET). Then, the third removal step involves removing one or more polynucleic acid molecules with MMs of 0 within human intragenic regions (where the only allowed hits are DUX1, DUX5, and DBET pseudogenes). Then, the next removal step involves removing one or more polynucleic acid molecules with MMs relative to the DUX4 human sequence used in the FLExDUX4 FSHD mouse model. The next step is then to carry forward only one or more polynucleic acid molecules with a predicted viability of 60 or greater. Next, a elimination step involves eliminating one or more polynucleic acid molecules with matches to the seed region of known miRNAs 1-1000. The elimination step then continues by eliminating one or more polynucleic acid molecules with a GC content% of 75 or greater. The final selection process then excludes the region 295-1132 and contains no more than eight predicted off-target hits with two MMs, where a maximum of 12 hits is allowed.

[0079] In some embodiments, the specificity of a polynucleic acid molecule hybridizing 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 examples, hybridization is under highly stringent hybridization conditions.

[0080] In some embodiments, polynucleic acid molecule has less off-target effect.In some cases, " off-target " or " off-target effect " refers to all cases where polynucleic acid polymer directed to a given target causes unintended effect by direct or indirect interaction with other mRNA sequence, DNA sequence, or cellular protein or other parts.In some cases, " off-target effect " occurs when other transcripts are simultaneously degraded due to the partial homology or complementarity between other transcripts and the sense strand and / or antisense strand of polynucleic acid molecule.

[0081] In some embodiments, polynucleic acid molecules contain natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules contain DNA, RNA, and / or a combination of nucleotide analogs. In some examples, synthetic or artificial nucleotide analogs or bases contain modifications to one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[0082] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid containing a modification at the 2' hydroxyl group of the ribose moiety. In some examples, the modification includes H, OR, R, halo, SH, SR, NH2, NHR, NR2, 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 a heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In some examples, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0083] In some instances, the modification of the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some instances, 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 an adenosine molecule and a 2'-O-methyl modification and a uridine 2'-O-methoxyethyl modification are illustrated below.

[0084] [ka]

[0085] In some cases, 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 cases, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving cellular uptake properties due to its zwitterionic properties. A typical chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is shown below.

[0086] [ka]

[0087] In some instances, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid, or LNA) in which an oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer. A typical representation of the chemical structure of an LNA is illustrated below. The representation on the left highlights the chemical connections of the LNA monomer. The representation on the right highlights the locked 3'-endo (3E) conformation of the furanose ring of the LNA monomer.

[0088] [ka]

[0089] In some cases, the modification at the 2' hydroxyl group includes, for example, ethylene nucleic acid (ENA), such as 2'-4'-ethylene-bridged nucleic acid, which locks the sugar conformation into a C3'-endo sugar puckering conformation. ENA is part of the bridged nucleic acid class of modified nucleic acids, which further includes LNA. Typical chemical structures of ENA and bridged nucleic acid are illustrated below.

[0090] [ka]

[0091] 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).

[0092] In some embodiments, the 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-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine. Modified bases include 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl, and substituted naphthyl groups, all O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, phenyl and modified phenyl groups such as 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, aminophenol, or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified on the sugar moiety, as well as nucleotides having non-ribosyl sugars or analogs thereof. For example, the sugar moiety in some cases is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what is known in the art as a universal base.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0093] In some embodiments, the nucleotide analogue 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 phosphoramidite morpholino oligo (PMO) comprises a synthetic molecule whose structure mimics the natural nucleic acid structure by deviating from the normal sugar and phosphate structure. 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, the ribose monomer is linked by a phosphoramidate group instead of a phosphate group. In such cases, the backbone modification removes all positive and negative charges, making the morpholino neutral molecule capable of crossing cell membranes without the aid of a cellular delivery agent, such as those used by charged oligonucleotides.

[0094] [ka]

[0095] In some embodiments, peptide nucleic acids (PNAs) contain no sugar backbone rings or phosphate linkages; the bases are joined and appropriately spaced by oligoglycine-like molecules, thus eliminating backbone charge.

[0096] [ka]

[0097] In some embodiments, one or more modifications are optionally made in the internucleotide linkage.In some examples, the modified internucleotide linkage is phosphorothioate, phosphorodithioate, methyl phosphonate, 5'-alkylene phosphonate, 5'-methyl phosphonate, 3'-alkylene phosphonate, boron trifluoride, 3'-5'-linked or 2'-5'-linked boranophosphate ester and selenophosphate, phosphotriester, alkyl phosphotriester of thio, hydrogen phosphonate linkage, alkyl phosphonate, alkyl phosphonothioate, aryl phosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkyl phosphoramidate, amino alkyl phosphoramidate, thionophosphoramidate, phosphoropiperazidate, phosphoroanilothioate, phosphoroanilidate. Phosphorothioate antisense oligonucleotides (PS ASOs) include, but are not limited to, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazides, methylenedimethylhydrazides, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamidates, linkages with riboacetyl groups, aminoethylglycines, silyl or siloxane linkages, saturated or unsaturated, substituted, and / or heteroatom-containing alkyl or cycloalkyl linkages of 1 to 10 carbon atoms with or without heteroatoms, linkages with morpholino structures, amides, polyamides in which the bases are directly or indirectly bound to the aza nitrogens of the backbone, and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate linkages. A typical PS ASO is illustrated below.

[0098] [ka]

[0099] 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.

[0100] [ka]

[0101] In some instances, the modified nucleotides include:

[0102] [ka] Examples of suitable phosphoramidites include, but are not limited to, 2'-fluoro N3-P5'-phosphoramidites exemplified by:

[0103] In some instances, the modified nucleotides include:

[0104] [ka] and 5'-vinylphosphonate modified non-natural nucleotides selected from the following: wherein B is a heterocyclic base moiety.

[0105] In some instances, the modified nucleotides include:

[0106] [ka] wherein B is a heterocyclic base moiety; R1, R2, and R3 are independently selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that links adjacent nucleotides in a polynucleotide.

[0107] In some instances, the modified nucleotides include:

[0108] [ka] wherein B is a heterocyclic base moiety; R4 and R5 are independently selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that links adjacent nucleotides in a polynucleotide.

[0109] In some instances, the modified nucleotides include:

[0110] [ka] wherein B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that links adjacent nucleotides in a polynucleotide.

[0111] In some examples, the modified nucleotides include, but are not limited to, one 5'-vinylphosphonate modified non-natural nucleotide selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA).

[0112] In some instances, the modified nucleotides include:

[0113] [ka] wherein B is a heterocyclic base moiety and J is an internucleotide linking group that links adjacent nucleotides in a polynucleotide.

[0114] In some instances, the modified nucleotides include:

[0115] [ka] wherein B is a heterocyclic base moiety and J is an internucleotide linking group that links adjacent nucleotides in a polynucleotide.

[0116] In some instances, the modified nucleotides include:

[0117] [ka] wherein B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that links adjacent nucleotides in a polynucleotide.

[0118] In some instances, the modified nucleotides include:

[0119] [ka] Examples of suitable 5'-vinylphosphonate modified non-natural nucleotides include, but are not limited to, one 5'-vinylphosphonate modified non-natural nucleotide,

[0120] In some instances, the modified nucleotides include:

[0121] [ka] Examples of hexitol nucleic acids include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)) exemplified by:

[0122] In some embodiments, the one or more modifications optionally further include modifications of the ribose moiety, phosphate backbone, and nucleoside at the 3' or 5' end, or modifications of the nucleotide analog. For example, the 3' end optionally includes a 3' cationic group, or the nucleoside is reversed at the 3' end by a 3'-3' linkage. In another alternative, the 3' end is optionally conjugated with an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In a further alternative, the 3' end is optionally conjugated with an abasic site, such as an apurinic or apyrimidinic site. In some examples, the 5' end is conjugated with an aminoalkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5' end is conjugated with an abasic site, such as an apurinic or apyrimidinic site.

[0123] In some embodiments, polynucleic acid molecules contain one or more of the artificial nucleotide analogs described herein. In some examples, polynucleic acid molecules contain one or more of the 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 examples, the polynucleic acid molecule is selected from the group consisting of 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), and 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 selected from 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof. In some examples, the 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 examples, the 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 of 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, the 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 of thiol phosphonate nucleotides.

[0124] In some examples, the polynucleic acid molecule comprises at least one of about 5% to about 100% modifications, about 10% to about 100% modifications, about 20% to about 100% modifications, about 30% to about 100% modifications, about 40% to about 100% modifications, about 50% to about 100% modifications, about 60% to about 100% modifications, about 70% to about 100% modifications, about 80% to about 100% modifications, and about 90% to about 100% modifications.

[0125] In some cases, the polynucleic acid molecule comprises at least one of about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications.

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

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

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

[0129] Optionally, the polynucleic acid molecule comprises at least one of about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications.

[0130] Optionally, the polynucleic acid molecule comprises at least one of about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0131] Optionally, the polynucleic acid molecule comprises at least one of about 10% to about 30% modifications and about 20% to about 30% modifications.

[0132] Optionally, the polynucleic acid molecule contains about 10% to about 20% modifications.

[0133] Optionally, 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.

[0134] In further cases, the polynucleic acid molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0135] 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, or about 22 or more modifications.

[0136] 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, or about 22 or more modified nucleotides.

[0137] 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 cases, about 35% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 40% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 45% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 50% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 55% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 60% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 65% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 70% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 75% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 80% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, about 85% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some cases, 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, 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.

[0138] In some embodiments, the polynucleic acid molecule comprises from about 1 to about 25 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 1 modification, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 2 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 3 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 4 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 5 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 6 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 7 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 8 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 9 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 10 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 15 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 16 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 17 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 18 modifications, including an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 19 modifications, including an artificial nucleotide analog described herein.In some embodiments, the polynucleic acid molecule comprises about 20 modifications, including artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 21 modifications, including artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 22 modifications, including artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 23 modifications, including artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 24 modifications, including artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 25 modifications, including artificial nucleotide analogs described herein.

[0139] In some embodiments, the polynucleic acid molecule is assembled from two separate polynucleotides, one polynucleotide comprising the sense strand and another polynucleotide comprising the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is connected to the antisense strand via a linker molecule, which in some cases is a polynucleotide linker or a non-nucleotide linker.

[0140] 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.

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

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

[0143] 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 four, at least five, or at least six consecutive 2'-O-methyl modified nucleotides at its 5'-end and / or 3'-end, or both. Optionally, in such embodiments, the sense strand of the 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 3'-terminus of at least four, at least five, or at least six consecutive 2'-O-methyl modified nucleotides at the 5'-terminus of the polynucleotide, or at the 5'-terminus of at least four, at least five, or at least six consecutive 2'-O-methyl modified nucleotides at the 3'-terminus of the polynucleotide. Further optionally, the at least two, at least three, or at least four 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides.

[0144] 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.

[0145] 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 the 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 the 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.

[0146] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid of 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, wherein the antisense strand comprises a nucleic acid of 5'-UfsNfsnnnNfnnnnnnnNfnNfnnnsusu-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 a nucleic acid of 5'-nsnsnnnnNfNfNfnnnnnnnnsnsa-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification) and the antisense strand comprises a nucleic acid of 5'-UfsNfsnnnNfnnnnnnnnNfnNfnnnsusu-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification).

[0147] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand comprises a terminal cap moiety at the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the sense strand. In other embodiments, the terminal cap moiety is an inverted deoxyabasic moiety.

[0148] 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.

[0149] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally an end cap molecule at the 3'-end, the 5'-end, or both the 3'-end and the 5'-end of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro, if present on the same or different strands, or if absent, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus.

[0150] In some embodiments, the 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 about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the sense strand. and the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally an end cap molecule at the 3'-end, the 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro, if present on the same or different strands, or if absent, from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus.

[0151] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides at the 3'-end, the 5'-end, or both the 3'- and 5'-ends of the sense strand and / or antisense strand, and optionally an end cap molecule at the 3'-end, the 5'-end, or both the 3'- and 5'-ends of the sense strand. In some embodiments, the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both the 3'-end and the 5'-end of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro, if present on the same or different strands, or if absent, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus.

[0152] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'-terminus and the 5'-terminus of the sense strand. Both contain terminal cap molecules, and the antisense strand contains from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro, if present on the same or different strands, or if absent, from about 1 to about 5, e.g., about 1, 2, 3, 4, 5 or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus.

[0153] 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 examples, 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.

[0154] In another embodiment, the polynucleic acid molecules described herein comprise 2'-5' internucleotide linkages. In some examples, the 2'-5' internucleotide linkages are at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In further examples, the 2'-5' internucleotide linkages are present at various other positions within one or both strands of the sequence, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more internucleotide linkages, including all internucleotide linkages of pyrimidine nucleotides in one or both strands of the polynucleic acid molecule, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more internucleotide linkages, including all internucleotide linkages of purine nucleotides in one or both strands of the polynucleic acid molecule.

[0155] In some embodiments, the polynucleic acid molecule is a single-stranded polynucleic acid molecule that mediates RNAi activity in a cellular system or a reconstituted in vitro system, the polynucleic acid molecule comprising a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and all purine nucleotides present in the polynucleic acid are 2'-deoxy purine nucleotides (e.g., all purine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides). and a terminal cap modification optionally present at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of the antisense strand, wherein the nucleotide is a 2'-deoxypurine nucleotide, or alternatively the plurality of purine nucleotides are 2'-deoxypurine nucleotides, and 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 internucleoside linkages, and the polynucleic acid molecule optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0156] In some cases, one or more of the artificial nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases, compared to naturally occurring polynucleic acid molecules. In some examples, 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'-O-N-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or combinations thereof, e.g., RNase inhibitors. The modified polynucleic acid molecules are resistant to nucleases, such as ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases or 3'-5' exonucleases. In some examples, 2'-O-methyl modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases). In some examples, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases). In some examples, 2'-O-aminopropyl modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, 2'-deoxy modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some examples, 2'-deoxy-2'-fluoro modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, LNA-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, ENA-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, HNA-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some examples, morpholinos exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, PNA-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, methylphosphonate nucleotide-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, thiolphosphonate nucleotide-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some examples, polynucleic acid molecules comprising 2'-fluoroN3-P5'-phosphoramidites exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). 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.

[0157] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity for mRNA compared to an equivalent naturally occurring polynucleic acid molecule. One or more of the 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 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, or 2'-fluoro N3-P5'-phosphoramidite, have increased binding affinity for mRNA compared to the equivalent naturally occurring polynucleic acid molecule. In some instances, 2'-O-methyl modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-O-aminopropyl modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-deoxy modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-deoxy-2'-fluoro modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules.In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, LNA modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, ENA modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, PNA modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, HNA modified polynucleic acid molecules have increased binding affinity for mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, morpholino-modified polynucleic acid molecules have increased binding affinity to mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, methylphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity to mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, thiolphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity to mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity to mRNA compared to equivalent naturally occurring polynucleic acid molecules. In some cases, increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0158] In some embodiments, the polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules containing a single enantiomer. In some examples, the polynucleic acid molecules contain L-nucleotides. In some examples, the polynucleic acid molecules contain D-nucleotides. In some examples, the polynucleic acid molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of its mirror image enantiomer. In some cases, the polynucleic acid molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of a racemic mixture. In some examples, the polynucleic acid molecule is a polynucleic acid molecule described in U.S. Patent Application Publication Nos. 2014 / 194610 and 2015 / 211006 and International Publication No. WO2015107425.

[0159] In some embodiments, the polynucleic acid molecules described herein are further modified to include aptamer conjugation moieties.In some examples, the aptamer conjugation moieties are DNA aptamer conjugation moieties.In some examples, the aptamer conjugation moieties are Alphamer (Centauri Therapeutics), which include an aptamer moiety that recognizes specific cell surface targets and a moiety that presents specific epitopes for attachment to circulating antibodies.In some examples, the polynucleic acid molecules described herein are further modified to include aptamer conjugation moieties as described in U.S. Patent Nos. 8,604,184, 8,591,910 and 7,850,975.

[0160] In a further aspect, 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 examples, the polynucleic acid molecule is modified by one or more of the above-mentioned modifications to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2'-hydroxyl position by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, or by a locked or bridged ribose conformation (e.g., LNA or ENA). Optionally, the polynucleic acid molecule is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. Optionally, the polynucleic acid molecule also contains morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability. In some examples, the polynucleic acid molecule is a chiral pure (or stereopure) polynucleic acid molecule. In some examples, the chiral pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications to RNA to increase stability for delivery will be apparent to those skilled in the art.

[0161] In some examples, 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 a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. In some examples, the polynucleic acid molecule is assembled from two separate polynucleotides, wherein one strand is a sense strand and the other is an antisense strand, and the antisense strand and the sense strand are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to a nucleotide sequence in the other strand, e.g., the antisense strand and the sense strand form a duplex or double-stranded structure, e.g., the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs), wherein the antisense strand comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. Alternatively, the polynucleic acid molecule can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the polynucleic acid molecule are joined by a nucleic acid-based or non-nucleic acid-based linker.

[0162] In some cases, polynucleic acid molecule is a polynucleotide that comprises a double, asymmetric double, hairpin, or asymmetric hairpin secondary structure, with self-complementary sense region and antisense region, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence in a separate target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence that corresponds to the target nucleic acid sequence or a part thereof.In other cases, polynucleic acid molecule is a circular single-stranded polynucleotide that has a stem that comprises two or more loop structures and self-complementary sense region and antisense region, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence in a target nucleic acid molecule or a part thereof, and sense strand has the nucleotide sequence that corresponds 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, a polynucleic acid molecule also includes a single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., 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 includes a terminal phosphate group, such as a 5'-phosphate or a 5',3'-diphosphate.

[0163] 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 comprising fewer nucleotides than the antisense region, but sufficient complementary nucleotides to base pair with the antisense region and form a duplex with the loop. For example, an asymmetric hairpin polynucleic acid molecule comprises an antisense region of sufficient length (e.g., about 19 to about 22 nucleotides) to mediate RNAi in a cellular or in vitro system, 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 also comprises a chemically modified 5'-terminal phosphate group. In further instances, the loop region of the asymmetric hairpin polynucleic acid molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0164] In some embodiments, an asymmetric duplex is a polynucleic acid molecule having two separate strands, including a sense region and an antisense region, where the sense region contains fewer nucleotides than the antisense region, but has sufficient complementary nucleotides to base-pair with the antisense region and form a duplex with the loop. For example, an asymmetric duplex polynucleic acid molecule includes an antisense region having a length sufficient to mediate RNAi in a cellular or in vitro system (e.g., about 19 to about 22 nucleotides), and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0165] In some cases, universal base refers to nucleotide base analogues that form base pairs with each of the natural DNA / RNA bases, with little distinction between them.Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives known in the art, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole.

[0166] Synthesis of polynucleic acid molecules In some embodiments, the polynucleic acid molecule described herein is constructed by chemical synthesis and / or enzyme ligation reaction using procedures known in the art.For example, polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between polynucleic acid molecule and target nucleic acid.Typical methods include those described in 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 Publication WO2009099942 or European Patent Application Publication No. 1579015.Further exemplary methods include 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., RNA transcribed from the inserted polynucleic acid molecule is in an antisense orientation relative to the target polynucleic acid molecule of interest).

[0167] In some embodiments, the polynucleic acid molecule is synthesized by tandem synthesis, where both strands are synthesized as one continuous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize the duplex and allow for its purification.

[0168] In some instances, the polynucleic acid molecule is also assembled from two separate nucleic acid strands or fragments, one fragment comprising the sense region and another fragment comprising the antisense region of the molecule.

[0169] For example, further modification methods for incorporating sugar, base, and phosphate modifications include those described in Eckstein et al., International Publication 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, pp. 334-339; International Publication WO 93 / 15187 by Usman et al.; U.S. Pat. No. 5,334,711 by Sproat; 1995, J. Biol. Chem., 270, pp. 25702 by Beigelman et al.; International Publication WO 97 / 26270 by Beigelman et al.; U.S. Pat. No. 5,716,824 by Beigelman et al.; U.S. Pat. No. 5,627,053 by Usman et al.; International Publication WO 98 / 13526 by Woolf et al.; U.S. Provisional Patent Application No. 60 / 082,404 filed April 20, 1998 by Thompson et al.; 1998, Tetrahedron Lett., 39, pp. 1131 by Karpeisky et al.; 1998, Earnshaw and Gait Biopolymers (Nucleic Acid Sciences), 48, pp. 39-55, Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, pp. 99-134, and Burlina et al., 1997, Bioorg. Med. Chem., 5, pp. 1999-2010. These publications describe general methods and strategies for determining the location of sugar, base, and / or phosphate modifications, etc., into nucleic acid molecules without the need for catalytic regulation.

[0170] In some cases, chemical modification of internucleotide linkages of polynucleic acid molecules with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate improves stability, but excessive modification can cause toxicity or reduced activity.Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages is sometimes minimized.In such cases, reducing the concentration of these linkages reduces toxicity, increases the effectiveness of these molecules, and improves specificity.

[0171] Polynucleic acid molecule conjugates In some embodiments, the polynucleic acid molecule (B) is further conjugated to the polypeptide (A) for delivery to a site of interest. 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.

[0172] 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 conjugated to one end of at least one B, while at least one C is conjugated to the opposite end of at least one B to form an ABC conjugate. In some examples, at least one polypeptide A is conjugated to one end of at least one B, while at least one C is conjugated at an internal site of at least one B. In some examples, at least one polypeptide A is directly conjugated to at least one C. In some examples, at least one B is indirectly conjugated to at least one polypeptide A via at least one C to form an ACB conjugate.

[0173] In some examples, at least one B and / or at least one C, and optionally at least one D, are conjugated to at least one polypeptide A. In some examples, at least one B is conjugated to at least one polypeptide A at an end (e.g., the 5'-end or 3'-end) or via an internal site. Optionally, at least one C is conjugated to at least one polypeptide A directly or indirectly via at least one B. When conjugated indirectly via at least one B, at least one C is conjugated at the same end as at least one polypeptide A on B, at the opposite end of at least one polypeptide A, or independently at an internal site. In some examples, at least one additional polypeptide A is further conjugated to at least one polypeptide A, B, or C. In a further example, at least one D is optionally conjugated directly or indirectly to at least one polypeptide A, at least one B, or at least one C. When performed directly on at least one polypeptide A, at least one D is also optionally conjugated to at least one B to form an ADB conjugate, or optionally conjugated to at least one B and at least one C to form an ADBC ​​conjugate. In some examples, at least one D is directly conjugated to at least one polypeptide A and indirectly conjugated to at least one B and at least one C to form a DABC conjugate. When performed indirectly on at least one polypeptide A, at least one D is also optionally conjugated to at least one B to form an ABD conjugate, or optionally conjugated 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 conjugated to at least one polypeptide A, B, or C.

[0174] joining part In some embodiments, binding moiety A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. Optionally, the fragment is an antigen-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.

[0175] In some embodiments, binding moiety A is a bispecific antibody or an antigen-binding fragment thereof. In some examples, the bispecific antibody is a trispecific antibody or a bispecific miniantibody. Optionally, the bispecific antibody is a trispecific antibody. In some examples, the trispecific antibody is a full-length monoclonal antibody that contains binding sites for two different antigens.

[0176] In some cases, the bispecific antibody is a bispecific miniantibody. In some examples, 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 containing two single-chain variable fragments (scFv), where the two scFvs target epitopes of two different antigens.

[0177] In some embodiments, binding moiety A is a bispecific miniantibody. In some examples, A is a bispecific Fab2. In some examples, A is a bispecific F(ab)'3 fragment. Optionally, A is a bispecific bis-scFv. Optionally, A is a bispecific (scFv)2. 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).

[0178] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody comprises a F(ab)'3 fragment or a triabody. In some examples, A is a trispecific F(ab)'3 fragment. Optionally, A is a triabody. 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).

[0179] In some embodiments, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein. In some examples, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein on a muscle cell. Optionally, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein on a skeletal muscle cell.

[0180] 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 examples, the antibody is an anti-transferrin receptor (anti-CD71) antibody.

[0181] In some embodiments where 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).

[0182] In some examples, the anti-transferrin receptor antibody comprises a variable heavy chain (VH) region and a variable light chain (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.

[0183] In some embodiments, the VH region of the anti-transferrin receptor antibody comprises an HCDR1, HCDR2, and HCDR3 sequence selected from Table 1.

[0184] [Table 1]

[0185] 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, and an HCDR3 sequence comprising SEQ ID NO: 283. In some examples, 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 examples, 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 examples, 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.

[0186] 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.

[0187] In some embodiments, the VL region of the anti-transferrin receptor antibody comprises an LCDR1, LCDR2, and LCDR3 sequence selected from Table 2.

[0188] [Table 2]

[0189] In some examples, 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.

[0190] In some examples, 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.

[0191] In some examples, 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, wherein X6 is present or absent and, if present, is F.

[0192] In some examples, 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.

[0193] In some examples, 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.

[0194] In some examples, 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.

[0195] In some examples, 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.

[0196] 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 of 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, or if present, is F.

[0197] In some examples, 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 of 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 RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence comprising SEQ ID NO: 288 or 290, where X3 is selected from N or S.

[0198] In some examples, 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 of SEQ ID NO: 282, wherein the HCDR2 sequence is 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.

[0199] In some examples, 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 of SEQ ID NO: 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 if present, is F.

[0200] In some examples, 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 of SEQ ID NO: 282, a HCDR2 sequence of SEQ ID NO: 283, and a HCDR3 sequence of SEQ ID NO: 283; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, a LCDR2 sequence of SEQ ID NO: 283, and a LCDR3 sequence of SEQ ID NO: 286, a LCDR2 sequence of SEQ ID NO: 284, and a LCDR3 sequence of SEQ ID NO: 285, wherein X5 is selected from D or E, and X6 is present or absent, and if present, is F.

[0201] In some examples, 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 of 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, an LCDR2 sequence comprising SEQ ID NO: 287, and an LCDR3 sequence comprising SEQ ID NO: 288.

[0202] In some examples, 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 of 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, an LCDR2 sequence comprising SEQ ID NO: 289, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0203] In some examples, 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 of 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: 291, an LCDR2 sequence comprising SEQ ID NO: 292, and an LCDR3 sequence comprising SEQ ID NO: 290.

[0204] In some examples, 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.

[0205] In some examples, 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.

[0206] In some examples, 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, wherein X6 is present or absent, and, if present, is F.

[0207] In some examples, 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, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6; X5 is selected from D or E; and X6 is present or absent, and if present, is F.

[0208] In some examples, 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.

[0209] In some examples, 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.

[0210] In some examples, 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.

[0211] In some examples, 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.

[0212] In some examples, 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.

[0213] In some examples, 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, wherein X6 is present or absent, and, if present, is F.

[0214] In some examples, 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; X5 is selected from D or E; and X6 is present or absent, and if present, is F.

[0215] In some examples, 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.

[0216] In some examples, 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.

[0217] In some examples, 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.

[0218] In some examples, 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.

[0219] In some examples, 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.

[0220] In some examples, 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, wherein X6 is present or absent, and, if present, is F.

[0221] In some examples, 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; the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 286, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6; X5 is selected from D or E; and X6 is present or absent, and if present, is F.

[0222] In some examples, 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.

[0223] In some examples, 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.

[0224] In some examples, 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.

[0225] 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.

[0226] 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 corresponding CDR1, CDR2, or CDR3 sequences.

[0227] [Table 3]

[0228] [Table 4]

[0229] In some embodiments, the anti-transferrin receptor antibody comprises the VH and VL regions illustrated in Table 5.

[0230] [Table 5]

[0231] In some embodiments, the anti-transferrin receptor antibodies described herein comprise an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some examples, the anti-transferrin receptor antibody comprises an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). Optionally, the anti-transferrin receptor antibody comprises an IgG1 framework. Optionally, the anti-transferrin receptor antibody comprises an IgG2 (e.g., IgG2a or IgG2b) framework. Optionally, the anti-transferrin receptor antibody comprises an IgG2a framework. Optionally, the anti-transferrin receptor antibody comprises an IgG2b framework. Optionally, the anti-transferrin receptor antibody comprises an IgG3 framework. Optionally, the anti-transferrin receptor antibody comprises an IgG4 framework.

[0232] 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 examples, the one or more mutations stabilize the antibody and / or increase half-life. In some examples, the one or more mutations modulate Fc receptor interaction to reduce or eliminate Fc effector function, such as FcyR, antibody-dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In a further example, the one or more mutations modulate glycosylation.

[0233] In some embodiments, the one or more mutations are located in the Fc region. In some examples, the Fc region includes a mutation at residue position L234, L235, or a combination thereof. In some examples, the mutation includes L234 and L235. In some examples, the mutation includes L234A and L235A. Optionally, the residue positions are relative to IgG1.

[0234] In some examples, the Fc region comprises mutations at residue positions L234, L235, D265, N297, K322, L328, or P329, or a combination thereof. In some examples, the mutations comprise L234 and L235 in combination with mutations at residue positions K322, L328, or P329. Optionally, the Fc region comprises mutations at L234, L235, and K322. Optionally, the Fc region comprises mutations at L234, L235, and L328. Optionally, the Fc region comprises mutations at L234, L235, and P329. Optionally, the Fc region comprises mutations at D265 and N297. Optionally, the residue positions are relative to IgG1.

[0235] In some examples, the Fc region comprises L234A, L235A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some examples, the Fc region comprises L234A and L235A in combination with K322G, L328R, or P329G. Optionally, the Fc region comprises L234A, L235A, and K322G. Optionally, the Fc region comprises L234A, L235A, and L328R. Optionally, the Fc region comprises L234A, L235A, and P329G. Optionally, the Fc region comprises D265A and N297G. Optionally, the residue positions are relative to IgG1.

[0236] In some examples, the Fc region comprises mutations at residue positions L235, L236, D265, N297, K322, L328, or P329, or a combination of these mutations. In some examples, the Fc region comprises mutations at L235 and L236. In some examples, the Fc region comprises mutations at L235 and L236 in combination with mutations at residue positions K322, L328, or P329. Optionally, the Fc region comprises mutations at L235, L236, and K322. Optionally, the Fc region comprises mutations at L235, L236, and L328. Optionally, the Fc region comprises mutations at L235, L236, and P329. Optionally, the Fc region comprises mutations at D265 and N297. Optionally, the residue positions are associated with IgG2.

[0237] In some embodiments, the Fc region comprises L235A, L236A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some examples, the Fc region comprises L235A and L236A. In some examples, the Fc region comprises L235A and L236A in combination with K322G, L328R, or P329G. Optionally, the Fc region comprises L235A, L236A, and K322G. Optionally, the Fc region comprises L235A, L236A, and L328R. Optionally, the Fc region comprises L235A, L236A, and P329G. Optionally, the Fc region comprises D265A and N297G. Optionally, the residue positions are relative to IgG2.

[0238] In some embodiments, the Fc region comprises a mutation at residue positions L233, L234, D264, N296, K321, L327, or P328, where the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 303. In some examples, the Fc region comprises a mutation at L233 and L234. In some examples, the Fc region comprises a mutation at L233 and L234 in combination with a mutation at residue position K321, L327, or P328. Optionally, the Fc region comprises a mutation at L233, L234, and K321. Optionally, the Fc region comprises a mutation at L233, L234, and L327. Optionally, the Fc region comprises a mutation at L233, L234, and K321. Optionally, the Fc region comprises mutations at L233, L234, and P328. In some examples, the Fc region comprises mutations at D264 and N296. Optionally, equivalent positions to residues L233, L234, D264, N296, K321, L327, or P328 in an IgG1, IgG2, IgG3, or IgG4 framework are contemplated. Optionally, mutations to residues corresponding to residues L233, L234, D264, N296, K321, L327, or P328 of SEQ ID NO: 23 in an IgG1, IgG2, or IgG4 framework are also contemplated.

[0239] In some embodiments, the Fc region comprises residue positions L233A, L234A, D264A, N296G, K321G, L327R, or P328G, which correspond to residues 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 303. In some examples, the Fc region comprises L233A and L234A. In some examples, the Fc region comprises L233A and L234A in combination with K321G, L327R, or P328G. Optionally, the Fc region comprises L233A, L234A, and K321G. Optionally, the Fc region comprises L233A, L234A, and L327R. Optionally, the Fc region comprises L233A, L234A, and K321G. Optionally, the Fc region comprises L233A, L234A, and P328G. In some examples, the Fc region comprises mutations at D264A and N296G.

[0240] In some embodiments, the human IgG constant region can be selected from the group consisting of those described, for example, in Natsume et al., 2008 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., 1992 J The amino acid modifications described in Immunol, 148: 3461-3468, reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11 are modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0241] 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 corresponding CDRs.

[0242] [Table 6-1]

[0243] [Table 6-2]

[0244] [Table 6-3]

[0245] [Table 6-4]

[0246] [Table 6-5]

[0247] [Table 6-6]

[0248] [Table 7]

[0249] In some embodiments, the anti-transferrin receptor antibodies described herein have improved serum half-lives compared to a reference anti-transferrin receptor antibody. In some examples, 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.

[0250] In some embodiments, binding moiety A is non-specifically conjugated to polynucleic acid molecule (B). In some examples, binding moiety A is non-site-specifically conjugated to polynucleic acid molecule (B) via a lysine residue or a cysteine ​​residue. In some examples, binding moiety A is non-site-specifically conjugated to polynucleic acid molecule (B) via a lysine residue (e.g., a lysine residue present in binding moiety A). In some cases, binding moiety A is non-site-specifically conjugated to polynucleic acid molecule (B) via a cysteine ​​residue (e.g., a cysteine ​​residue present in binding moiety A).

[0251] In some embodiments, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner through a lysine residue, through a cysteine ​​residue, at the 5'-end, at the 3'-end, through an unnatural amino acid, or through an enzyme-modified or enzyme-catalyzed residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner through a lysine residue (e.g., a lysine residue present in binding moiety A). In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner through a cysteine ​​residue (e.g., a cysteine ​​residue present in binding moiety A). In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner at the 5'-end. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner at the 3'-end. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner through an unnatural amino acid. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner through an enzyme modification or enzyme catalytic residue.

[0252] In some embodiments, one or more polynucleic acid molecules (B) are conjugated to a binding moiety A. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 1 polynucleic acid molecule is conjugated to one binding moiety A. In some examples, about 2 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 3 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 4 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 5 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 6 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 7 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 8 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 9 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 10 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 11 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 12 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 13 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 14 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 15 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 16 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, the one or more polynucleic acid molecules are the same. In other cases, the one or more polynucleic acid molecules are different.

[0253] In some embodiments, the number of polynucleic acid molecules (B) conjugated to binding moiety A forms a ratio. In some examples, the ratio is referred to as the DAR (drug-to-antibody) ratio, and the drug referred to herein is the polynucleic acid molecule (B). In some examples, the DAR ratio between the polynucleic acid molecule (B) and the 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 examples, the DAR ratio between the polynucleic acid molecule (B) and the binding moiety A is about 1 or more. In some examples, the DAR ratio between the polynucleic acid molecule (B) and the binding moiety A is about 2 or more. In some examples, the DAR ratio between the polynucleic acid molecule (B) and the binding moiety A is about 3 or more. In some examples, the DAR ratio between the polynucleic acid molecule (B) and the binding moiety A is about 4 or more. In some examples, the DAR ratio between the polynucleic acid molecule (B) and the binding moiety A is about 5 or more. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 6 or greater. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 7 or greater. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 8 or greater. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 9 or greater. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 10 or greater. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 11 or greater. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 12 or greater.

[0254] In some examples, the DAR ratio between polynucleic acid molecule (B) and 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 between polynucleic acid molecule (B) and binding moiety A is about 1. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 2. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 3. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 4. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 5. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 6. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 7. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 8. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 9. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 10. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 11. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 12. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 13. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 14. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 15. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is about 16.

[0255] In some examples, the DAR ratio between polynucleic acid molecule (B) and 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 between polynucleic acid molecule (B) and binding moiety A is 1. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is 2. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is 4. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is 6. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is 8. In some examples, the DAR ratio between polynucleic acid molecule (B) and binding moiety A is 12.

[0256] In some examples, 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) but not a binding moiety A. In some examples, the improved activity results in improved biologically relevant function in the treatment or prevention of a disease state, for example, improved stability, affinity, binding, functional activity, and efficacy. In some examples, the disease state is the result of one or more mutated exons of a gene. In some examples, 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) but not a binding moiety A. In some examples, 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 a polynucleic acid molecule (B) and a binding moiety A compared to a conjugate comprising a polynucleic acid molecule (B) but not a binding moiety A.

[0257] In some embodiments, the antibody or antigen-binding fragment is further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, and / or recombination, and / or by any other modification known in the art, alone or in combination (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, the one or more modifications include, for example, those described in International Publication WO 97 / 34631, which discloses that amino acid residues are involved in the interaction of the Fc domain with the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence underlying the amino acid sequence of an antibody or antigen-binding fragment are well known to those of skill in the art.

[0258] In some examples, an antigen-binding fragment, including further derivatives thereof, comprises a polypeptide sequence containing at least one CDR.

[0259] 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 colinear amino acid sequence that can be encoded by a single nucleic acid molecule.

[0260] In some instances, bispecific single-chain antibody constructs relate to constructs comprising binding domains derived from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some instances, the scFv contains a VH domain and a VL domain connected by a linker peptide. In some instances, the linker is of sufficient length and sequence to ensure that each of the first and second domains can retain their unique binding specificity independently of each other.

[0261] In some embodiments, the term "binding" or "interaction" as used herein defines the binding / interaction between at least two antigen-interaction sites. In some examples, the antigen-interaction site defines a polypeptide motif that exhibits the ability to specifically interact with a specific antigen or a group of specific antigens. In some cases, the binding / interaction is also understood to define specific recognition. In such cases, specific recognition refers to the ability of an antibody or its antigen-binding fragment to specifically interact with and / or bind to at least two amino acids of each of the target molecules. For example, specific recognition is related to the specificity of the antibody molecule or its ability to discriminate specific regions of the target molecule. In further examples, the specific interaction between the antigen-interaction site and its specific antigen results in the initiation of a signal, for example, due to the induction of changes in the conformation of the antigen, oligomerization of the antigen, etc. In further embodiments, the binding is exemplified by the specificity of the "key-lock principle." Thus, in some examples, the specific motifs in the amino acid sequences 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 secondary modifications of that structure. In such cases, the specific interaction of the antigen-interaction site with its specific antigen similarly results in simple binding of the site to the antigen.

[0262] In some cases, specific interaction also refers to the reduction of cross-reactivity of antibody or antigen-binding fragment or the reduction of off-target effect.For example, antibody or antigen-binding fragment that binds to polypeptide / protein of interest but does not bind or essentially does not bind to any other polypeptide is considered to be specific for polypeptide / protein of interest.Examples of the specific interaction between antigen interaction site and specific antigen include the specificity of ligand to its receptor, for example, the interaction between antigenic determinant (epitope) and antigenic binding site of antibody.

[0263] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. In some examples, the plasma protein includes albumin. In some examples, binding moiety A is albumin. In some examples, the albumin is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some examples, the albumin is conjugated to the polynucleic acid molecule by native ligation chemistry. In some examples, the albumin is conjugated to the polynucleic acid molecule by lysine conjugation.

[0264] In some examples, the binding moiety is a steroid. Typical steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, and carbohydrates, which may be saturated, unsaturated, substituted, or a combination thereof. In some examples, the steroid is cholesterol. In some examples, the binding 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 lysine conjugation.

[0265] 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 is capable of selectively binding to the cell surface marker in a manner similar to an antibody binding to its specific epitope on the cell surface marker.

[0266] Optionally, the binding moiety is a peptide. Optionally, the peptide comprises between about 1 and about 3 kDa. Optionally, the peptide comprises between about 1.2 and about 2.8 kDa, between about 1.5 and about 2.5 kDa, or between about 1.5 and 2 kDa. In some examples, the peptide is a bicyclic peptide. Optionally, the bicyclic peptide is a constrained bicyclic peptide. In some examples, the binding moiety is a bicyclic peptide (e.g., Bicycle Therapeutics bicycles).

[0267] In further instances, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. Optionally, the antibody-recruiting small molecule includes 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 includes a glutamate urea compound, allowing interaction with PSMA, thereby enhancing antibody interaction with PSMA-expressing cells. In some instances, 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).

[0268] Production of antibodies or antigen-binding fragments thereof In some aspects, the polypeptides (e.g., antibodies and antigen-binding fragments) described herein are produced by any method known in the art to be useful for synthesizing polypeptides (e.g., antibodies), specifically chemical synthesis or recombinant expression, preferably by recombinant expression techniques.

[0269] In some cases, 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., those described in Kutmeier et al., 1994, BioTechniques 17:242), which require the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligation of those oligonucleotides, and subsequent PCR amplification of the ligated oligonucleotides.

[0270] Alternatively, nucleic acid molecules encoding antibodies are optionally generated 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 hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0271] In some examples, the antibody or antigen-binding fragment thereof is optionally produced by immunizing an animal such as a rabbit to generate polyclonal antibodies, or more preferably, by generating monoclonal antibodies as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), by Kozbor et al. (1983, Immunology Today 4:72), or by 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, e.g., 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 a specific antigen.

[0272] In some embodiments, techniques developed to produce "chimeric antibodies" by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity (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. 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.

[0273] 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 the heavy and light chain fragments of the Fv portion 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).

[0274] 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 promoter, an inducible promoter, or a tissue-specific promoter.

[0275] In some aspects, 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 for producing and subsequently purifying the antibody coding sequence, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, express the antibody or antigen-binding fragment thereof in situ. These include 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 sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or antigen-binding fragment coding sequence; Pichia); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence for the antibody or antigen-binding fragment thereof; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence for the antibody or antigen-binding fragment thereof; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0276] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, 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 regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells are grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to 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.

[0277] In some instances, a number of selection systems are used, including, but not limited to, the genes for herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817), which are employed in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance is also mediated by 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 and 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 for Cancer Research 12:101-102). 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 generally known in the field of recombinant DNA technology that can be used are 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; Chapters 12 and 13 of Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0278] In some cases, 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 site will be linked to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0279] 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, particularly by affinity for 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 constrained protein liquid chromatography.

[0280] 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 of the formula AXB, where X is a linker that conjugates A and B. In some examples, binding moieties include amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, as well as all analogs or derivatives of these classes of substances. Additional examples of binding moieties also 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, an endosomolytic moiety.

[0281] In some aspects, the polynucleic acid molecule is conjugated to the binding moiety by a chemical ligation process. In some examples, the polynucleic acid molecule is conjugated to the binding moiety by native ligation. In some examples, the conjugation is as described in Dawson et al., "Synthesis of proteins by native chemical ligation," Science 1994, 266, pp. 776-779; Dawson et al., "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, pp. 4325-4329; Hackeng et al., "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology." Proc. Natl. Acad. Sci. USA 1999, 96, pp. 10068-10073; or Wu et al., "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, pp. 4116-4125. In some examples, the 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.

[0282] In some instances, polynucleic acid molecules are conjugated to binding moieties by a site-specific method utilizing "traceless" coupling technology (Philochem). In some instances, the "traceless" coupling technology conjugates to an N-terminal 1,2-aminothiol group on a binding moiety conjugated with a polynucleic acid molecule containing an aldehyde group (see Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacovigilance," JACS 134(13):5887-5892 (2012)).

[0283] In some examples, polynucleic acid molecules are conjugated to binding moieties by site-specific methods that utilize unnatural amino acids incorporated into the binding moiety. In some examples, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some examples, the keto group of pAcPhe is selectively attached to an alkoxy-amine derived conjugation moiety to form an oxime bond (see Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40):16101-16106 (2012)).

[0284] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a site-specific method that utilizes an enzyme-catalyzed process. In some instances, the site-specific method utilizes SMARTag™ technology (Catalent, Inc.). In some examples, SMARTag™ technology involves the generation of formylglycine (FGly) residues from cysteines by formylglycine generating enzyme (FGE) through an oxidation process in the presence of an aldehyde tag and subsequent conjugation of FGly to alkylhydrazine-functionalized polynucleic acids by 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)).

[0285] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). Optionally, a polynucleic acid molecule is conjugated to a binding moiety using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes covalent bond formation 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)).

[0286] In some examples, the polynucleic acid molecule is conjugated to the binding moiety by the method described in International Publication WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0287] In some examples, the polynucleic acid molecule is conjugated to a binding moiety by the methods described in U.S. Patent Application Nos. 2015 / 0105539 and 2015 / 0105540.

[0288] Polymer conjugation moiety In some embodiments, the polymer moiety C is further conjugated to a polynucleic acid molecule described herein, a binding moiety described herein, or a combination thereof. In some examples, the polymer moiety C is conjugated to the polynucleic acid molecule according to the formula A-X1-B-X2-C (X1 and X2 are two linkers that conjugate A and B, and B and C, respectively). In some cases, the polymer moiety C is conjugated to a binding moiety. In other cases, the polymer moiety C is conjugated to a polynucleic acid molecule-binding moiety molecule. In further cases, the polymer moiety C is conjugated as described above.

[0289] In some examples, polymer moiety C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or two- or three-dimensional cross-linked networks of monomers. In some examples, polymer moiety C comprises a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some examples, at least one polymer moiety C includes, but is not limited to, alpha- or omega-dihydroxypolyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (also known as poly(ethylene terephthalate), PET, PETG, or PETE), polytetramethylene glycol (PTG), or polyurethane, as well as mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound, as well 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 polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0290] In some examples, C is a PEG moiety. In some examples, the PEG moiety is conjugated at the 5' end of the polynucleic acid molecule, while the binding moiety is conjugated at the 3' end of the polynucleic acid molecule. In some examples, the PEG moiety is conjugated at the 3' end of the polynucleic acid molecule, while the binding moiety is conjugated at the 5' end of the polynucleic acid molecule. In some examples, the PEG moiety is conjugated to an internal site of the polynucleic acid molecule. In some examples, the PEG moiety, the binding moiety, or a combination thereof is conjugated to an internal site of the polynucleic acid molecule. In some examples, the conjugation is direct conjugation. In some examples, the conjugation is via native ligation.

[0291] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, polydisperse materials contain a dispersed distribution of materials of different molecular weights, characterized by average weight (weight-average) size and dispersity. In some cases, monodisperse PEG contains 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 molecules of the polyalkylene oxide, e.g., PEG.

[0292] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, , 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0293] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 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, and having a molecular weight of 800, 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, The C has a molecular weight of 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 examples, the molecular weight of C is about 200 Da. In some examples, the molecular weight of C is about 300 Da. In some examples, the molecular weight of C is about 400 Da. In some examples, the molecular weight of C is about 500 Da. In some examples, the molecular weight of C is about 600 Da. In some examples, the molecular weight of C is about 700 Da. In some examples, the molecular weight of C is about 800 Da. In some examples, the molecular weight of C is about 900 Da. In some examples, the molecular weight of C is about 1000 Da. In some examples, the molecular weight of C is about 1100 Da. In some examples, the molecular weight of C is about 1200 Da. In some examples, the molecular weight of C is about 1300 Da. In some examples, the molecular weight of C is about 1400 Da. In some examples, the molecular weight of C is about 1450 Da.In some examples, the molecular weight of C is about 1500 Da. In some examples, the molecular weight of C is about 1600 Da. In some examples, the molecular weight of C is about 1700 Da. In some examples, the molecular weight of C is about 1800 Da. In some examples, the molecular weight of C is about 1900 Da. In some examples, the molecular weight of C is about 2000 Da. In some examples, the molecular weight of C is about 2100 Da. In some examples, the molecular weight of C is about 2200 Da. In some examples, the molecular weight of C is about 2300 Da. In some examples, the molecular weight of C is about 2400 Da. In some examples, the molecular weight of C is about 2500 Da. In some examples, the molecular weight of C is about 2600 Da. In some examples, the molecular weight of C is about 2700 Da. In some examples, the molecular weight of C is about 2800 Da. In some examples, the molecular weight of C is about 2900 Da. In some examples, the molecular weight of C is about 3000 Da. In some examples, the molecular weight of C is about 3250 Da. In some examples, the molecular weight of C is about 3350 Da. In some examples, the molecular weight of C is about 3500 Da. In some examples, the molecular weight of C is about 3750 Da. In some examples, the molecular weight of C is about 4000 Da. In some examples, the molecular weight of C is about 4250 Da. In some examples, the molecular weight of C is about 4500 Da. In some examples, the molecular weight of C is about 4600 Da. In some examples, the molecular weight of C is about 4750 Da. In some examples, the molecular weight of C is about 5000 Da. In some examples, the molecular weight of C is about 5500 Da. In some examples, the molecular weight of C is about 6000 Da. In some examples, the molecular weight of C is about 6500 Da. In some examples, the molecular weight of C is about 7000 Da. In some examples, the molecular weight of C is about 7500 Da. In some examples, the molecular weight of C is about 8000 Da. In some examples, the molecular weight of C is about 10,000 Da. In some examples, the molecular weight of C is about 12,000 Da. In some examples, the molecular weight of C is about 20,000 Da. In some examples, the molecular weight of C is about 35,000 Da. In some examples, the molecular weight of C is about 40,000 Da. In some examples, the molecular weight of C is about 50,000 Da. In some examples, the molecular weight of C is about 60,000 Da.In some instances, the molecular weight of C is about 100,000 Da.

[0294] 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 instances, the polyalkylene oxide comprising discrete ethylene oxide units is linear. In other instances, the polyalkylene oxide comprising discrete ethylene oxide units is branched.

[0295] In some instances, polymer portion C is a polyalkylene oxide (e.g., PEG) comprising discrete ethylene oxide units. Optionally, polymer portion C comprises between about 4 and about 48 ethylene oxide units. Optionally, 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.

[0296] In some examples, polymer moiety C is a discrete PEG comprising, for example, between about 4 and about 48 ethylene oxide units. Optionally, polymer moiety C is a discrete PEG comprising, 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 discrete PEG containing, for example, about 4 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 5 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 6 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 7 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 8 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 9 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 10 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 11 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 12 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 13 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 14 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 15 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 16 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 17 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 18 ethylene oxide units.In some cases, polymer portion C is a discrete PEG containing, for example, about 19 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 20 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 21 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 22 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 23 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 24 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 25 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 26 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 27 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 28 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 29 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 30 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 31 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 32 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 33 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 34 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 35 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 36 ethylene oxide units. Optionally, polymer portion C is a discrete PEG, for example, comprising about 37 ethylene oxide units. Optionally, polymer portion C is a discrete PEG, for example, comprising about 38 ethylene oxide units.Optionally, polymer portion C is a discrete PEG containing, for example, about 39 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 40 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 41 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 42 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 43 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 44 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 45 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 46 ethylene oxide units. Optionally, polymer portion C is a discrete PEG containing, for example, about 47 ethylene oxide units. Optionally, polymer moiety C is a discrete PEG containing, for example, about 48 ethylene oxide units.

[0297] Optionally, polymer moiety C is dPEG® (Quanta Biodesign Ltd).

[0298] In some embodiments, the polymer portion C comprises a cationic mucic acid-based polymer (cMAP). In some examples, the cMAP comprises one or more subunits of at least one repeating subunit, the subunit structure being represented by formula (V):

[0299] [ka] It is expressed as:

[0300] wherein m, independently at each occurrence, is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 to 6 or 5, and n, independently at each occurrence, is 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0301] In some examples, cMAP is 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 is in the range of about 500 Da to about 50,000 Da. In some examples, the PEG moiety is in the range of 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.

[0302] In some instances, polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. Optionally, 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.

[0303] In some embodiments, the polymer moiety C is conjugated to a polynucleic acid molecule, a binding moiety, and optionally an endosomolytic moiety as described above.

[0304] Endosomolytic or cell membrane-penetrating moiety In some embodiments, the molecule of formula (I): A-X1-B-X2-C further comprises an additional conjugation moiety. In some examples, the additional conjugation moiety is an endosomolytic moiety and / or a cell membrane-penetrating moiety. Optionally, the endosomolytic moiety is a cellular compartment release component, such as a compound releasable from any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum, including cells. Optionally, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. Optionally, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer. Optionally, 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.

[0305] Endosomolytic and cell membrane-penetrating polypeptides In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further conjugated with an endosomolytic polypeptide. Optionally, the endosomolytic polypeptide is a pH-dependent membrane active peptide. Optionally, the endosomolytic polypeptide is an amphipathic polypeptide. In further cases, the endosomolytic polypeptide is a peptidomimetic. In some examples, the endosomolytic polypeptide comprises INF, melittin, meucin, or a corresponding derivative thereof. In some examples, the endosomolytic polypeptide comprises INF or a derivative thereof. In other cases, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further cases, the endosomolytic polypeptide comprises melittin or a derivative thereof.

[0306] In some examples, INF7 is a 24-residue polypeptide, the sequence of which includes CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 331), or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 332). In some examples, 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).

[0307] In some instances, melittin is a 26-residue polypeptide, the sequence of which comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 336), or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 337). In some instances, melittin comprises a polypeptide sequence described in U.S. Patent No. 8,501,930.

[0308] In some instances, meucin is an antimicrobial peptide (AMP) derived from the venom gland of the scorpion Mesobuthus eupeus. In some instances, meucin is comprised of meucin-13, which has the sequence IFGAIAGLLKNIF-NH2 (SEQ ID NO: 338), and meucin-18, which has the sequence FFGHLFKLATKIIPSLFQ (SEQ ID NO: 339).

[0309] In some examples, the endosomolytic polypeptide comprises a polypeptide having a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to INF7 or a derivative thereof, melittin or a derivative thereof, or meucin or a derivative thereof. In some examples, the endosomolytic moiety comprises INF7 or a derivative thereof, melittin or a derivative thereof, or meucin or a derivative thereof.

[0310] In some examples, the endosomolytic moiety is INF or a derivative thereof. Optionally, 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. Optionally, 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. Optionally, 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.

[0311] In some examples, the endosomolytic moiety is melittin or a derivative thereof. Optionally, 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. Optionally, 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. Optionally, 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.

[0312] In some examples, the endosomolytic portion is meucin or a derivative thereof. Optionally, 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: 338 or 339. Optionally, 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: 338. Optionally, 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. In some examples, the endosomolytic portion comprises a sequence exemplified in Table 8.

[0313] [Table 8-1]

[0314] [Table 8-2]

[0315] Optionally, the endosomolytic moiety comprises Bcl-2 and / or Bcl- XLIn some examples, the endosomolytic moiety comprises a Bak BH3 polypeptide described in "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier" by Albarran et al., Reactive & Functional Polymers 71:261-265 (2011).

[0316] In some instances, the endosomolytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) described in PCT Publication WO2013 / 166155 or WO2015 / 069587.

[0317] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further conjugated with an endosomolytic lipid (e.g., a fusogenic lipid). Exemplary fusion 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), 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).

[0318] In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in International Publication WO 09 / 126,933.

[0319] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further conjugated with an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), amopyroquines, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, the quinoline endosomolytic moiety may be 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 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-hydroxy-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, 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-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline, 3-fluoro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline, 4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline, Examples of suitable endosomolytic moieties include, but are not limited to, [1-(2H)-butyl-amino]-6-methoxyquinoline, 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde, 1,1'-pentamethylenediquinoline diiodide, 8-quinolinol sulfate, and their amino, aldehyde, carboxyl, 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. Patent No. 5,736,557.

[0320] 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 arginine- or lysine-rich amino acid sequence. In some embodiments, the cell-penetrating polypeptide comprises any polypeptide listed in Table 9, or a combination thereof.

[0321] [Table 9]

[0322] Linker In some aspects, the linkers described herein are cleavable linkers or non-cleavable linkers. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker.

[0323] 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. Typical non-polymeric linkers include, but are not limited to, a C1-C6 alkyl group (e.g., C5, C4, C3, C2, or C1 alkyl group), a homobifunctional crosslinker, a heterobifunctional crosslinker, 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., C5, C4, C3, C2, or C1 alkyl group), a homobifunctional crosslinker, a heterobifunctional crosslinker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In other cases, the non-polymeric linker does not include more than two of the same type of linker, for example, more than two homobifunctional crosslinkers or more than two peptide linkers. In further cases, the non-polymeric linker optionally includes one or more reactive functional groups.

[0324] In some cases, the non-polymeric linker does not include a polymer as described above. In some cases, the non-polymeric linker does not include a polymer encompassed by the polymer moiety C. In some cases, the non-polymeric linker does not include a polyalkylene oxide (e.g., PEG). In some cases, the non-polymeric linker does not include PEG.

[0325] In some examples, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tetratate (DST), disulfosuccinimidyl tetratate (sulfoDST), ethylene glycol dithiobis(succinimidyl) tetratate (dithiobis(sulfosuccinimidyl) tetratate) ... Lycobis (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)propionamide) Examples of suitable aryl ethers include, but are not limited to, hexane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB) such as 1,5-difluoro-2,4-dinitrobenzene, 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0326] In some examples, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include amine-reactive and sulfhydryl crosslinkers, 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)toluene]-, and sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluene]-. -(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), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl (4-iodoacetyl) )aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide esters (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexa noate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, e.g.,4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (MCH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate ( sulfo-NHs-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-s ANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), 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 Cinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-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 crosslinkers, such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB),These crosslinkers include, but are not limited to, N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive crosslinkers, such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as ρ-azidophenylglyoxal (APG).

[0327] In some instances, the linker comprises a reactive functional group. In some instances, the reactive functional group comprises a nucleophilic group reactive with an electrophilic group present on the binding moiety. Typical 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. Typical nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0328] In some examples, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further comprises caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, 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.

[0329] 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, providing an intramolecular catalyst for thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing retro-Michael elimination. In some examples, the self-stabilizing maleimide is the 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.

[0330] In some examples, 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 up to 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 (SEQ ID NO: 294223), 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 (SEQ ID NO: 294224), or Gly-Phe-Leu-Gly (SEQ ID NO: 294225). In some examples, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 294223), 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 (SEQ ID NO: 294224), or Gly-Phe-Leu-Gly (SEQ ID NO: 294225). Optionally, the linker comprises Val-Cit. Optionally, the linker is Val-Cit.

[0331] 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).

[0332] 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 examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises a mc-val-cit-PABA group.

[0333] In some embodiments, linker is a self-immolative linker or a self-eliminating linker.In some cases, linker is a self-immolative linker.In other cases, linker is a self-eliminating linker (for example, a cyclized self-eliminating linker).In some examples, linker comprises the linker described in U.S. Patent No. 9,089,614 or PCT Publication WO2015038426.

[0334] In some examples, the linker is a dendritic linker. In some examples, the dendritic linker comprises a branched multifunctional linker moiety. In some examples, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some examples, the dendritic linker comprises a PAMAM dendrimer.

[0335] In some embodiments, the linker is a traceless linker, or a linker that does not leave a linker moiety (e.g., an atomic group or a linker group) after cleavage to the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D. 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, as described by 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 as 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 U.S. Patent No. 6,821,783.

[0336] In some examples, the linker may be a polymerizable compound such as those described in U.S. Pat. Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688; U.S. Patent Application Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 28697 0, 2013 / 0309256, 2015 / 037360, or 2014 / 0294851, or PCT Publications WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0337] In some embodiments, X1 and X2 are each independently a bond or a non-polymeric linker. In some examples, X1 and X2 are each independently a bond. In some cases, X1 and X2 are each independently a non-polymeric linker.

[0338] In some examples, X1 is a bond or a non-polymeric linker. In some examples, X1 is a bond. In some examples, X1 is a non-polymeric linker. In some examples, the linker is a C1-C6 alkyl group. Optionally, X1 is a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. Optionally, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. As used in the context of linkers, specifically in the context of X1, alkyl means a saturated, straight- or branched-chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X1 comprises a homobifunctional linker or heterobifunctional linker as described above. Optionally, X1 comprises a heterobifunctional linker. Optionally, X1 comprises sMCC. In other examples, X1 comprises a heterobifunctional linker optionally conjugated to a C1-C6 alkyl group. In other examples, X1 comprises sMCC optionally conjugated to a C1-C6 alkyl group. In a further example, X1 does not comprise a homobifunctional linker or a heterobifunctional linker as described above.

[0339] In some examples, X2 is a bond or a non-polymeric linker. In some examples, X2 is a bond. In other cases, X2 is a linker. In further cases, X2 is a non-polymeric linker. In some embodiments, X1 is a C1-C6 alkyl group. In some examples, X2 comprises a homobifunctional linker or a heterobifunctional linker as described above. In some examples, X2 is a homobifunctional linker as described above. In some examples, X2 is a heterobifunctional linker as described above. In some examples, X2 comprises a maleimide group, such as the maleimidocaproyl (mc) or self-stabilizing maleimide group described above. 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 a further example, X2 comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In a further example, X2 comprises an mc group. In a further example, X2 comprises an mc-val-cit group. In a further example, X2 comprises a val-cit-PABA group. In a further example, X2 comprises a mc-val-cit-PABA group.

[0340] 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 and / or the progressive weakening and degeneration of muscle is caused by a high rate of protein degradation, a low rate of protein synthesis, or a combination of both. In some cases, the high rate of muscle protein degradation is due to muscle protein catabolism (i.e., the breakdown of muscle protein to use amino acids as substrates for gluconeogenesis).

[0341] In one embodiment, muscle atrophy refers to a substantial loss of muscle strength. Substantial loss of muscle strength refers to a decrease in the strength of a subject's diseased, injured, or unused muscle tissue compared to the same muscle tissue in a control subject. In one embodiment, a substantial loss of muscle strength refers to 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%, or at least 50% or more compared to the same muscle tissue in a control subject. In another embodiment, a substantial loss of muscle strength refers to a decrease in strength of unused muscle tissue compared to the strength of the same muscle tissue in the same subject before the period of disuse. In one embodiment, a substantial loss of muscle strength refers to 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%, or at least 50% or more compared to the strength of the same muscle tissue in the same subject before the period of disuse.

[0342] In another embodiment, muscle atrophy refers to a substantial loss of muscle mass. Substantial loss of muscle mass refers to a decrease in muscle mass in a subject's diseased, damaged, or unused muscle tissue compared to the same muscle tissue in a control subject. In one embodiment, substantial loss of muscle mass refers to 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%, or at least 50% or more compared to the same muscle tissue in a control subject. In another embodiment, substantial loss of muscle mass refers to a decrease in muscle mass in unused muscle tissue compared to the muscle mass of the same muscle tissue in the same subject before the period of disuse. In one embodiment, substantial loss of muscle tissue refers to 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%, or at least 50% or more compared to the muscle mass of the same muscle tissue in the same subject before the period of disuse. Muscle mass is optionally measured by assessing muscle cross-sectional area, such as by magnetic resonance imaging (eg, by the muscle mass / cross-sectional area (CSA) MRI method).

[0343] In some aspects, described herein are methods of treating muscle wasting in a subject, the method comprising obtaining a polynucleic acid molecule described herein and administering to the subject a therapeutically effective amount of a polynucleic acid molecule described herein or a polynucleic acid molecule conjugate described herein to reduce the amount of human DUX4 mRNA transcript. In some aspects, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand 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: 412-420 or 430-438. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the antisense strand is identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438. In some aspects, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand 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: 142, 146, 196, or 201-206. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, wherein the sense strand is identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

[0344] In some examples, muscle atrophy is associated with facioscapulohumeral muscular dystrophy (FSHD). The polynucleic acid portion mediates RNA interference against human DUX4, so as to regulate muscle atrophy in the subject. In some embodiments, the expression of one or more marker genes affected by DUX4 expression is also modified or regulated (e.g., reduced) by reducing the expression of human DUX4. Marker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD. 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.

[0345] In some embodiments, the present specification describes a method for treating muscle atrophy in a subject, the method comprising: obtaining the siRNA-antibody conjugate described herein; administering the siRNA-antibody conjugate described herein to the subject in a therapeutically effective amount; and reducing the level of the mRNA transcript of human DUX4 in the subject.In some examples, the muscle atrophy is associated with FSHD.The siRNA-antibody conjugate mediates RNA interference against human DUX4 mRNA, so as to treat the muscle atrophy in the subject, the method comprises administering the siRNA-antibody conjugate described herein to the subject in a therapeutically effective amount; and reducing the level of the mRNA transcript of human DUX4 in the subject.

[0346] In some embodiments, the present disclosure describes a method for treating muscle atrophy in a subject, the method comprising: obtaining a DUX4 siRNA-antibody conjugate (DUX4 siRNA-conjugate or DUX4-AOC) described herein; 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 examples, the muscle atrophy is associated with FSHD. The DUX4 siRNA-antibody conjugate mediates RNA interference against human DUX4 mRNA to treat muscle atrophy in the subject, and includes 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.

[0347] In some embodiments, the present specification describes a method of treating FSHD in a subject, the method comprising obtaining 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 examples, the FSHD is FSHD type 1 (FSHD1). In some examples, the FSHD is FSHD type 2 (FSHD2). The DUX4 siRNA-antibody conjugate mediates RNA interference against human DUX4 mRNA to treat FSHD in a subject, and includes administering a therapeutically effective amount of a DUX4 siRNA-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 modified or regulated (e.g., reduced) by reducing the expression level of human DUX4. DUX4 biomarker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD.

[0348] In some aspects, described herein are methods of alleviating symptoms in a subject with FSHD, comprising obtaining a DUX4 siRNA-antibody conjugate (DUX4-siRNA-conjugate or DUX4-AOC) described herein and administering to the subject a therapeutically effective amount of the siRNA conjugate described herein, thereby reducing the level of human DUX4 mRNA transcripts in the subject. In some examples, the FSHD is FSHD type 1 (FSHD1). In some examples, the FSHD is FSHD type 2 (FSHD2). In another aspect, described herein are methods of alleviating symptoms in a patient with FSHD, comprising obtaining a siRNA conjugate described herein and administering to the patient with FSHD a therapeutically effective amount of the siRNA conjugate described herein, thereby reducing the level of human DUX4 mRNA transcripts or reducing the level of DUX4 protein.

[0349] In some instances, FSHD symptoms affect skeletal muscles, including the muscles around the eyes and mouth, shoulder muscles, upper arm muscles, lower leg muscles, abdominal muscles, and gluteal muscles. In some instances, FSHD symptoms also affect vision and hearing. In some instances, FSHD symptoms also affect cardiac or pulmonary function. In some instances, FSHD symptoms include muscle weakness, muscle atrophy, muscular dystrophy, pain, inflammation, spasticity, scoliosis, lordosis, hypoventilation, retinal abnormalities, exposure to keratitis, mild hearing loss, and EMG abnormalities.

[0350] In some embodiments, described herein is a method for improving skeletal muscle function in a patient with FSHD, comprising administering to the patient with FSHD a therapeutically effective amount of a siRNA conjugate described herein, by reducing the level of human DUX4 mRNA transcripts or reducing the level of DUX4 protein. In some examples, the FSHD is FSHD type 1 (FSHD1). In some examples, it is FSHD type 2. In some embodiments, described herein is a method for improving skeletal muscle function, vision, tension, cardiac function, or pulmonary function in a patient with FSHD, comprising administering to the patient with FSHD a therapeutically effective amount of a siRNA conjugate described herein, by reducing the level of human DUX4 mRNA transcripts or reducing the level of DUX4 protein.

[0351] In some embodiments, the present specification describes a method for treating FSHD in a subject, the method comprising obtaining an antisense oligonucleotide (ASO)-antibody conjugate described herein, 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 examples, the FSHD is FSHD type 1 (FSHD1). In some examples, it is FSHD type 2. The ASO-antibody 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 modified or regulated by reducing the expression level of human DUX4. DUX4 biomarker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD.

[0352] In some aspects, the present specification describes methods for treating FSHD in a subject. In some examples, the FSHD subject has FSHD1. In other examples, the FSHD subject has FSHD2. In another embodiment, the FSHD subject has muscle cells that abnormally express the DUX4 protein, 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 results in a reduction of the D4Z4 region site on chromosome 4 in the FSHD subject, which contains 1-10 repeats instead of the usual 11-100 repeats. The epigenetic molecular change in the muscle cells is a change that results in hypomethylation of the D4Z4 region site on chromosome 4 in the FSHD subject. In some examples, the muscle cells are skeletal muscle cells.

[0353] 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, 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 intranasal administration.

[0354] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid 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, sustained release formulations, pulsed release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and combined immediate and controlled release formulations.

[0355] In some cases, the pharmaceutical formulation comprises a multiparticulate formulation. In some cases, the pharmaceutical formulation comprises a nanoparticle formulation. In some cases, the nanoparticle comprises cMAP, cyclodextrin, or lipid. In some cases, the nanoparticle comprises a solid lipid nanoparticle, a polymeric nanoparticle, a self-emulsifying nanoparticle, a liposome, a microemulsion, or a micellar solution. 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 attached metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, the nanoparticles are metal nanoparticles, such as 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.

[0356] In some cases, the nanoparticles comprise a core or a core and a shell, such as core-shell nanoparticles.

[0357] In some examples, the nanoparticles are further coated with molecules for binding 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.

[0358] Optionally, the nanoparticles have at least one dimension less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

[0359] In some embodiments, 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 having a covalently attached metal chelate), a nanofiber, a nanohorn, a nanoonion, a nanorod, a nanorope, or a quantum dot. In some examples, the polynucleic acid molecule or binding moiety described herein is directly or indirectly conjugated 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 conjugated to the nanoparticle.

[0360] In some embodiments, the pharmaceutical preparation comprises a delivery vector, for example, a recombinant vector, that is, the delivery of a polynucleic acid molecule to cells. In some examples, the recombinant vector is a DNA plasmid. In other examples, the recombinant vector is a viral vector. Typical viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some examples, the recombinant vector capable of expressing a polynucleic acid molecule provides stable expression in target cells. In another example, a viral vector is used that provides transient expression of a polynucleic acid molecule.

[0361] In some aspects, pharmaceutical formulations include a carrier or carrier material selected based on compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Typical carrier materials include, for example, binders, suspending agents, active agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, dextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1975; Liberman, H.A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999).

[0362] In some cases, the pharmaceutical preparation 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 tris-hydroxymethylaminomethane; 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.

[0363] In some cases, the pharmaceutical formulation contains one or more salts in an amount necessary to make the osmolality of the composition acceptable. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium sulfite, and ammonium sulfate.

[0364] In some cases, pharmaceutical formulations further comprise diluents, which provide a more stable environment and are used to stabilize the compound. Salts dissolved in buffer solutions (which also control or maintain pH) are utilized as diluents in the art, including, but not limited to, phosphate buffered saline. In certain cases, diluents increase the volume of the composition to facilitate compression or create a sufficient volume for homogeneous mixing for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch such as Di-Pac® (Amstar), compressible sugar; 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.

[0365] In some cases, pharmaceutical formulations contain disintegrants or disintegrants to facilitate the disintegration or breakdown of materials. The term "disintegrate" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches such as corn starch and potato starch, pregelatinized starches such as National 1551 and Amijel®, or sodium starch glycolates such as Promogel® and Explotab®, celluloses such as wood products, methylcrystalline cellulose, such as Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming crosslinked celluloses such as Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®), crosslinked carboxymethylcellulose, or crosslinked croscarmellose; crosslinked starches such as sodium starch glycolate; crosslinked polymers such as crospovidone; crosslinked polyvinylpyrrolidone; alginates such as alginic acid or alginates such as sodium alginate; clays such as Veegum® HV (magnesium aluminum silicate); gums such as agar, guar gum, 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 starch blends.

[0366] In some examples, the pharmaceutical formulation comprises a filler such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0367] 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, or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts, for example, aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (for example, PEG-4000) or methoxypolyethylene glycol such as Carbowax®, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid®, starch such as Cab-O-Sil®, corn starch, silicone oil, surfactants, etc.

[0368] Plasticizers include compounds used to soften microencapsulation materials or film coatings so they do not become too brittle. Suitable plasticizers include polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers also function as dispersing or wetting agents.

[0369] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, 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.

[0370] Stabilizers include any antioxidant, buffer, acid, preservative, or other compound.

[0371] Suspending agents include polyvinylpyrrolidone, for example, polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol, for example, polyethylene glycol having a molecular weight of about 300 to about 6000, about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose, stearyl acetate, and the like. Examples of suitable cellulose compounds include cellulose acetate, polysorbate 80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth gum, gum arabic, guar gum, and xanthan gum, sugars, cellulose compounds such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, and povidone.

[0372] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, 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. Surfactants are sometimes included to improve physical stability or for other purposes.

[0373] 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.

[0374] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E TPGS, and ammonium salts.

[0375] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic use. In some embodiments, the pharmaceutical compositions are administered once daily, twice daily, three or more times daily. The pharmaceutical compositions are administered daily, every day, 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.

[0376] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at intervals. 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 intervals (e.g., a first pharmaceutical composition is administered on day 1, followed by at least a second pharmaceutical composition at least 1, 2, 3, 4, 5, or more days later).

[0377] In some embodiments, two or more different pharmaceutical compositions are co-administered.In some cases, two or more different pharmaceutical compositions are co-administered simultaneously.In some cases, two or more different pharmaceutical compositions are co-administered consecutively without any time gap between administrations.In other cases, two or more different pharmaceutical compositions are co-administered consecutively with about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days or more gap between administrations.

[0378] If the patient's condition improves, the administration of the composition may be continued at the discretion of the physician. Alternatively, the administered dose of the composition may be temporarily reduced or temporarily discontinued for a certain period of time (i.e., a "drug holiday"). In some cases, the length of the drug holiday may vary from 2 days to 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 from 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%.

[0379] Once the patient's condition has improved, a maintenance dose is administered if necessary, after which the dosage or frequency of administration, or both, may be reduced, depending on the symptoms, to a level at which improvement in the disease, disorder, or condition is maintained.

[0380] In some embodiments, the amount of a given agent that corresponds to such an amount will vary depending on factors such as the particular compound, the severity of the disease, the identity (e.g., weight, sex) of the subject or host requiring treatment, and the like, but is nevertheless routinely determined in a manner known in the art depending on the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the disease being treated, and the subject or host being treated. In some instances, the desired dose is conveniently provided in a single dose or as divided doses administered simultaneously (i.e., over a short period of time) or administered at appropriate intervals, for example, as two, three, four or more sub-doses daily.

[0381] The foregoing ranges are only suggestive, since the amount of variability associated with individual treatment regimens is great 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 individual subject, the severity of the disease or condition being treated, and the judgment of the health-care practitioner.

[0382] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining 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 of LD50 to ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a dosage range for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form employed and the route of administration utilized.

[0383] Kit / manufactured product In certain aspects herein, kits and articles of manufacture are disclosed for use with one or more of the compositions and methods described herein. Such kits include a compartmentalized carrier, package, or container for receiving one or more containers, such as vials or tubes, each of which contains one of the individual components to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from various materials, such as glass or plastic.

[0384] The articles of manufacture provided herein include packaging materials, including, but not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging suitable for the selected formulation, intended dosage form, and treatment.

[0385] For example, the container contains a target nucleic acid molecule described herein. Such kits include identification, labels, or instructions relating to use in the methods described herein.

[0386] The kit typically includes a label and / or instructions listing the contents, as well as a package insert with the instructions. A set of instructions is also typically included.

[0387] In one embodiment, a label is on or associated with a container. In one embodiment, a label is on a container when letters, numbers, or other characters forming the label are applied, molded, or etched into the container itself. A label is associated with a container when it is present on 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. A label also provides instructions for using the contents, such as in the methods described herein.

[0388] In certain aspects, the pharmaceutical compositions are provided in a pack or dispenser containing one or more unit dosage forms containing a compound provided herein. For example, the pack, such as a blister pack, contains metal or plastic foil. In one embodiment, the pack or dispenser is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied by a notice attached to the container in a format 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 administration to humans or animals. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for prescriptions or an approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0389] 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 claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of all claimed subject matter. In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0390] As used herein, ranges and amounts can be expressed as a particular value or range with "about." "About" also includes the exact amount. Thus, "about 5 μL" means "about 5 μL" as well as "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

[0391] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0392] 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 non-human. None of these terms require or are limited to situations characterized by the supervision (e.g., regular or intermittent) of a healthcare professional (e.g., a physician, registered nurse, nurse practitioner, medical assistant, orderly, or hospice worker).

[0393] 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. Optionally, this amount is administered to a patient (such as a human) in single or multiple doses 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 employed to provide a therapeutically effective amount will vary depending on the type of injury, the subject's age, weight, sex, condition, severity of the disease, route of administration, and the particular inhibitor employed. In some examples, therapeutically effective amounts of the polynucleic acid molecule conjugates described herein are initially estimated from cell culture and animal models. For example, IC determined by cell culture methods. 50 The IC value can optionally serve as a starting point in an animal model, while the IC value determined for the animal model 50The values ​​are optionally used to find therapeutically effective amounts for humans.

[0394] Skeletal muscles, or voluntary muscles, are generally anchored to bones by tendons and are commonly used to accomplish skeletal movements such as locomotion or to maintain posture. While some skeletal muscle control is maintained as a totally involuntary reflex (e.g., postural muscles or the diaphragm), skeletal muscles respond to conscious control. Smooth muscles, or involuntary muscles, are found in the walls of organs and structures such as the esophagus, stomach, intestines, uterus, urethra, and blood vessels.

[0395] Skeletal muscle is further divided into two broad types: type I (i.e., "slow-twitch") and type II (i.e., "fast-twitch"). Type I muscle fibers are densely packed with capillaries and rich in mitochondria and myoglobin, giving type I muscle tissue its characteristic red color. In some cases, type I muscle fibers carry more oxygen and use fats or hydrocarbons for fuel to maintain aerobic activity. Type I muscle fibers contract with little force for extended periods of time. Type II muscle fibers are further subdivided into three main subtypes (IIa, IIx, and IIb) that differ in both the speed of contraction and the force generated. Type II muscle fibers contract immediately and forcefully but very rapidly, resulting in only a brief burst of anaerobic activity before the muscle contracts painfully.

[0396] Unlike skeletal muscle, smooth muscle is not under conscious control.

[0397] Cardiac muscle is also an involuntary muscle, but its structure is very similar to skeletal muscle and it is found only in the heart. Cardiac and skeletal muscle are striated in that they contain sarcomeres organized into highly regularly arranged bundles. In contrast, the myofibrils of smooth muscle cells are not arranged in sarcomeres and are therefore not striated.

[0398] Muscle cells include all cells that contribute to muscle tissue. Typical muscle cells include myoblasts, satellite cells, myotubes, and myofibrillar tissue.

[0399] As used herein, muscle strength is proportional to cross-sectional area (CSA), and muscle velocity is proportional to muscle fiber length. Therefore, comparing the cross-sectional area and muscle fiber length 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 in 2000. Another method for measuring muscle mass is to generate cross-sectional images of selected muscle tissue by computerized transverse tomography and sonographic evaluation.

[0400] The term antibody-oligonucleotide conjugate (AOC) refers to an antibody conjugated to a nucleotide.

[0401] The term "siRNA conjugate" or "siRNA-antibody conjugate" refers to an antibody conjugated to an siRNA.

[0402] "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.

[0403] The term "DUX4-AOC" refers to an antibody conjugated to an siRNA that hybridizes to a target sequence of human DUX4 mRNA.

[0404] Embodiment Embodiment 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, wherein the polynucleic acid molecule mediates RNA interference against DUX4.

[0405] Embodiment 2. The polynucleic acid molecule conjugate of embodiment 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.

[0406] Embodiment 3 The polynucleic acid molecule conjugate of embodiment 1 or 2, wherein the antibody or antigen-binding fragment thereof is an anti-transferrin receptor antibody or antigen-binding fragment thereof.

[0407] Embodiment 4. The polynucleic acid molecule conjugate of any one of embodiments 1-3, 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.

[0408] Embodiment 5. The polynucleic acid molecule conjugate of any one of embodiments 1 to 4, wherein the polynucleotide hybridizes to at least 8 contiguous bases of the target sequence of DUX4.

[0409] Embodiment 6. The polynucleic acid molecule conjugate of any one of embodiments 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.

[0410] Embodiment 7. The polynucleic acid molecule conjugate of any one of embodiments 1 to 6, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and wherein the sense strand 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: 1-70 or SEQ ID NOs: 141-210.

[0411] Embodiment 8. The polynucleic acid molecule conjugate of any one of embodiments 1 to 7, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and wherein the sense strand 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: 142, 146, 196, or 201-206.

[0412] Embodiment 9. The polynucleic acid molecule conjugate of any one of embodiments 1 to 8, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and wherein the antisense strand 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.

[0413] Embodiment 10. The polynucleic acid molecule conjugate of any one of embodiments 1 to 9, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and wherein the antisense strand 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: 412-420 and SEQ ID NOs: 430-438.

[0414] Embodiment 11. The polynucleic acid molecule conjugate of any one of embodiments 1 to 10, wherein the polynucleic acid molecule comprises at least one 2'-modified nucleotide, and further wherein the 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-methylacetamide (2'-O-NMA) modified nucleotide, or comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA), or a combination thereof.

[0415] Embodiment 12. The polynucleic acid molecule conjugate of any one of embodiments 1 to 11, wherein at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.

[0416] Embodiment 13. The polynucleic acid molecule conjugate of any one of embodiments 1 to 12, wherein the polynucleic acid molecule comprises three or more 2'-modified nucleotides selected from 2'-O-methyl and 2'-deoxy-2'-fluoro.

[0417] Embodiment 14. The polynucleic acid molecule conjugate of any one of embodiments 1 to 13, wherein the polynucleic acid molecule comprises a 5'-terminal vinylphosphonate modified nucleotide.

[0418] Embodiment 15. The 5'-terminal vinylphosphonate modified nucleotide is

[0419] [ka] is selected from wherein B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or alkoxy; 15. The polynucleic acid molecule conjugate of any one of embodiments 1 to 14, wherein J is an internucleotide linking group linking adjacent nucleotides of the polynucleotide.

[0420] Embodiment 16. The polynucleic acid molecule conjugate of any one of embodiments 1 to 15, 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.

[0421] Embodiment 17. The polynucleic acid molecule conjugate of embodiment 16, wherein the 2'-O-methyl is a purine nucleotide.

[0422] Embodiment 18. The polynucleic acid molecule conjugate of embodiment 16, wherein the 2'-O-methyl is a pyridine nucleotide.

[0423] Embodiment 19. The polynucleic acid molecule conjugate of any one of embodiments 16 to 18, wherein the sense strand and / or the antisense strand comprises at least two, at least three, or at least four consecutive 2'-O-methyl modified nucleotides at the 5' end.

[0424] Embodiment 20. The polynucleic acid molecule conjugate of any one of embodiments 1 to 19, comprising a linker connecting the antibody or antigen-binding fragment thereof to the polynucleic acid molecule.

[0425] Embodiment 21. The polynucleic acid molecule conjugate of embodiment 20, wherein the linker is a C1-C6 alkyl linker.

[0426] Embodiment 22. The polynucleic acid molecule conjugate of embodiment 20, 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.

[0427] Embodiment 23 The polynucleic acid molecule conjugate of embodiment 20, wherein the linker is a cleavable or non-cleavable linker.

[0428] Embodiment 24. The polynucleic acid molecule conjugate of any one of embodiments 1 to 23, wherein the ratio of polynucleic acid molecule to antibody or antigen-binding fragment thereof is about 1:1, 2:1, 3:1, or 4:1.

[0429] Embodiment 25 The polynucleic acid molecule conjugate of any one of embodiments 1 to 24, wherein the polynucleic acid molecule mediates RNA interference against human DUX4 and modulates muscle wasting in a subject.

[0430] Embodiment 26. The polynucleic acid molecule conjugate of embodiment 25, wherein the RNA interference comprises reducing 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.

[0431] Embodiment 27. The polynucleic acid molecule conjugate of any one of embodiments 25 to 26, wherein the RNA interference affects expression in the cell of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX.

[0432] Embodiment 28. The polynucleic acid molecule conjugate of any one of embodiments 25 to 26, wherein the RNA interference affects expression in the cell of a marker gene selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD.

[0433] Embodiment 29. The polynucleic acid molecule conjugate of embodiment 28, wherein affecting expression of the marker gene reduces expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more.

[0434] Embodiment 30. The polynucleic acid molecule conjugate of any one of embodiments 25 to 29, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0435] Embodiment 31. Formula (I): AXB (Formula I) wherein: A is an 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; 31. The polynucleic acid molecule conjugate of any one of embodiments 1 to 30, wherein X is conjugated to a cysteine ​​residue of A.

[0436] Embodiment 32. A pharmaceutical composition comprising the polynucleic acid molecule conjugate of any one of embodiments 1 to 31 and a pharmaceutically acceptable excipient.

[0437] Embodiment 33 The pharmaceutical composition of embodiment 32, formulated as a nanoparticle formulation.

[0438] Embodiment 34. The pharmaceutical composition of any one of embodiments 32-33, formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intrathecal administration.

[0439] Embodiment 35. A method for treating muscular dystrophy in a subject in need thereof, comprising providing a polynucleic acid conjugate of any one of embodiments 1 to 34, and administering the polynucleic acid conjugate to the subject to treat the muscular dystrophy, wherein the polynucleic acid conjugate reduces the amount of human DUX4 mRNA transcript.

[0440] Embodiment 36 The method of embodiment 35, wherein the polynucleic acid portion mediates RNA interference against human DUX4 and regulates muscle atrophy in the subject.

[0441] Embodiment 37. The method of embodiment 36, wherein the RNA interference affects expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD in cells affected by muscular dystrophy.

[0442] Embodiment 38. The method of any one of embodiments 35-37, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0443] Embodiment 39. Use of the polynucleic acid molecule conjugate of any one of embodiments 1 to 30, or the pharmaceutical composition of any one of embodiments 32 to 34, for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

[0444] Embodiment 40. Use of the polynucleic acid molecule conjugate of any one of embodiments 1 to 30, or the pharmaceutical composition of any one of embodiments 32 to 34, in the manufacture of a medicament for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

[0445] Embodiment 41. A kit comprising the polynucleic acid molecule conjugate of any one of embodiments 1 to 31, or the pharmaceutical composition of any one of embodiments 32 to 34. [Example]

[0446] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0447] Example 1. Design of a bioinformatic siRNA library against the human full-length DUX4 transcript Figure 2 shows a flowchart of the in silico selection process for DUX4 siRNAs. The sequences of all siRNAs capable of binding to DUX4 or a predetermined region of DUX4 are collected to generate a starting set of DUX4 siRNAs. From the starting set of DUX4 siRNAs, the first elimination step involves excluding one or more DUX4 siRNAs with single nucleotide polymorphisms (SNPs) and / or MEFs below -5. The second elimination step involves excluding DUX4 siRNAs with zero or one mismatch (MM) within the human transcriptome (so that the only hits allowed are DUX, DUX5, and DBET). The third elimination step involves excluding DUX4 siRNAs with zero mismatch (MM) within the human intragenic region (so that the only hits allowed are DUX1, DUX5, and DBET pseudogenes). The next elimination step involves excluding DUX4 siRNAs with a mismatch to the DUX4 human sequence used in the FLExDUX4 FSHD mouse model. The next step is to select only one or more DUX4 siRNAs with a predicted viability of 60 or greater. The elimination step then involves filtering out one or more polynucleic acid molecules with matches to the seed region 1-1000 of known miRNAs. The elimination step then continues by filtering out regions 1-1000 with a GC content of 75 or greater. The final selection process then excludes the region 295-1132, containing no more than eight predicted off-target hits with two MMs, with a maximum of 12 hits allowed. Using this series of selection steps, we were able to select 70 candidate DUX4 siRNAs from the starting set of 1694 DUX4 siRNAs. Figure 3 shows the location and number of these selected DUX4 siRNAs within the DUX4 mRNA transcript (NM_001306068).

[0448] The identified siRNA candidates share common features in their sequences, as shown in Table 10 below. The identified siRNAs mostly have 2'-O-Me, with only 2'-F modifications located on the sense strand at positions 7, 8, and 9 in all three DUX4 templates. 2'-O-Me modifications with 2'-F modifications are located on the antisense strand at positions 1, 2, 6, 14, and 16 in DUX4 template 1, and at positions 2, 6, 14, and 16 in DUX4 templates 1 and 2. The identified siRNAs also contain four phosphorothioate modifications on each strand, located at the final two linkages at the 5' and 3' ends, respectively. The identified siRNAs further contain "Uf" at position 1 of the 5' end of the antisense strand for DUX4 template 1 and "vpN" at position 1 of the 5' end of the antisense strand for DUX4 template 3, regardless of the actual target mRNA sequence (linked to "a" at the final position of the 3' end of the sense strand). The identified siRNAs further contain a "uu" overhang at the 3' end of only the antisense strand, but no overhang at the 3' end of the sense strand. Optimization of the identified siRNAs can include vinyl phosphonate nucleotides, inverted abasic moieties, or amine linkers to the passenger strand or guide strand.

[0449] [Table 10]

[0450] Tables 11, 12, 13, 14, and 15 illustrate siRNA candidates identified for the regulation of human DUX4.

[0451] [Table 11-1]

[0452] [Table 11-2]

[0453] [Table 12-1]

[0454] [Table 12-2]

[0455] [Table 12-3]

[0456] [Table 12-4]

[0457] [Table 13]

[0458] [Table 14]

[0459] [Table 15]

[0460] Example 2. siRNA Sequence and Synthesis All siRNA single strands were fully assembled on solid phase using standard phosphoramidite chemistry and purified by HPLC. The purified single strands were then duplexed to obtain duplex siRNAs. For the vinylphosphonate-modified guide strand, a guide strand with a vinylphosphonate-modified nucleotide structure at the 5' end (VpUq) was produced. All siRNA passenger strands contained different formats of conjugation handles, C6-NH2 and / or C6-SH, at each end of the strand, respectively. The conjugation handle(s) were connected to the siRNA passenger strand or siRNA guide strand via inverted abasic phosphodiester or phosphorothioate. Figures 5A-5F show representative structures of the formats used in in vivo experiments. Figure 5A illustrates a representative structure of an siRNA with a C6-NH2 conjugation handle at the 5' end of the passenger or guide strand and a C6-SH at the 3' end. Figure 5B illustrates a typical structure of an siRNA passenger or guide strand having a C6-NH2 conjugation handle at the 5'-end and a C6-S-PEG at the 3'-end. Figure 5C illustrates a typical structure of an siRNA passenger or guide strand having a C6-NH2 conjugation handle at the 5'-end and a C6-S-NEM at the 3'-end. Figure 5D illustrates a typical structure of an siRNA passenger or guide strand having a C6-N-SMCC conjugation handle at the 5'-end and a C6-S-NEM at the 3'-end. Figure 5E illustrates a typical structure of an siRNA passenger or guide strand having a PEG at the 5'-end and a C6-SH at the 3'-end. Figure 5F illustrates a typical structure of an siRNA passenger or guide strand having a C6-S-NEM at the 5'-end and a C6-NH2 conjugation handle at the 3'-end.

[0461] Example 3. Synthesis of the conjugate Figures 6A-6F illustrate exemplary structures of the A-X1-B-X2-Y (Formula I) architecture described herein. Figure 6A illustrates an antibody-Cys-SMCC-5'-passenger chain (Architecture 1). This conjugate was generated by antibody interchain cysteine ​​conjugation to a maleimide (SMCC) at the 5' end of the passenger chain. Figure 6B illustrates an antibody-Cys-SMCC-3'-passenger chain (Architecture 2). This conjugate was generated by antibody interchain cysteine ​​conjugation to a maleimide (SMCC) at the 3' end of the passenger chain. Figure 6C illustrates an antibody-Cys-bisMal-3'-passenger chain (ASC Architecture 3). This conjugate was generated by antibody interchain cysteine ​​conjugation to a bismaleimide (bisMal) linker at the 3' end of the passenger chain. Figure 6D illustrates a model structure of a Fab-Cys-bisMal-3'-passenger strand (ASC architecture 4). This conjugate was generated by Fab interchain cysteine ​​conjugation to a bismaleimide (bisMal) linker at the 3' end of the passenger strand. Figure 6E illustrates a model structure of an antibody-siRNA conjugate (ASC architecture 5) in which two different siRNAs are attached to one antibody molecule. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNA to reduced mAb interchain cysteines to a bismaleimide (bisMal) linker at the 3' end of the passenger strand of each siRNA. Figure 6F illustrates a model structure of an antibody-siRNA conjugate (ASC architecture 6) in which two different siRNAs are attached. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNA to a reduced mAb interchain cysteine ​​to maleimide (SMCC) linker at the 3' end of the passenger strand of each siRNA.

[0462] Example 3.1 Synthesis of Antibody-siRNA Conjugates Using SMCC Linkers FIG. 7A illustrates an exemplary synthetic scheme (Synthetic Scheme 1) of antibody-Cys-SMCC-siRNA-PEG conjugates via antibody-cysteine ​​conjugation.

[0463] Step 1: Antibody interchain disulfide reduction with TCEP

[0464] The antibody was buffer exchanged into borax buffer (pH 8) to a maximum concentration of 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 containing 5 mM EDTA, pH 7.4, and then 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 containing 5 mM EDTA, pH 7.4, at room temperature and rotated overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed the presence of antibody-siRNA conjugates along with unreacted antibody and siRNA.

[0465] Step 2: Purification

[0466] The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1 described in Example 3.4. Fractions containing antibody-siRNA-PEG conjugates with DAR1 and DAR>2 were isolated, concentrated, and buffer exchanged with PBS, pH 7.4.

[0467] Step 3: Analysis of purified conjugates

[0468] The isolated conjugates were characterized by SEC, SAX chromatography, and SDS-PAGE. The purity of the conjugates was examined 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 by analytical SAX and were greater than 90% pure. A typical DAR>2 cysteine ​​conjugate contains more than 85% DAR2 and less than 15% DAR3.

[0469] Example 3.2. Synthesis of antibody-siRNA conjugates using bismaleimide (BisMal) linkers FIG. 7B illustrates an exemplary synthetic scheme for antibody-Cys-BisMal-siRNA-PEG conjugates (Synthetic Scheme 2).

[0470] Step 1: Antibody reduction with TCEP

[0471] The antibody was buffer exchanged with 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 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 4°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.

[0472] Step 2: Purification

[0473] The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method 1. The fractions containing DAR1 and DAR2 antibody-siRNA conjugates were isolated, concentrated, and buffer exchanged with PBS, pH 7.4.

[0474] Step 3: Analysis of purified conjugates

[0475] The isolated conjugates were characterized by either mass spectrometry or SDS-PAGE. The purity of the conjugates was examined by analytical HPLC using either anion exchange chromatography method-2 or 3 plus size exclusion chromatography method-1.

[0476] Example 3.3. Fab' Generation from mAb and Conjugation to siRNA FIG. 7C illustrates a typical synthetic scheme (Synthetic Scheme 3) for producing Fab-siRNA conjugates.

[0477] Step 1: Antibody digestion with pepsin

[0478] Th...

Claims

1. A polynucleic acid molecule conjugate comprising: comprising an antibody or antigen-binding fragment thereof conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DUX4; the polynucleic acid molecule comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 72, 76, 126, or 131-136, and comprises 2'-F modified nucleotides at positions 2, 6, 14, and 16; the polynucleic acid molecule mediates RNA interference against the DUX4; Polynucleic acid molecule conjugates.

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

4. The polynucleic acid molecule conjugate of any one of claims 1 to 3, wherein the polynucleic acid molecule is about 16 to about 30 nucleotides in length.

5. 5. The polynucleic acid molecule conjugate of claim 1, wherein the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand comprises at least one nucleic acid sequence of UfsNfsnnnNfnnnnnnnnNfnNfnnnsusu, usNfsnnnNfnnnnnnnnNfnNfnnnsusu, or vpNsNfsnnnNfnnnnnnnnNfnNfnnnsus, where vpN = vinylphosphonate VpUq, lowercase (n) = 2'-O-Me modification, Nf = 2'-F modification, and s = phosphorothioate backbone modification.

6. 5. The polynucleic acid molecule conjugate of any one of claims 1 to 4, wherein the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438.

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 an antisense strand, and the sense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 2, 6, 56, or 61-66, and comprises at least two or at least three consecutive 2'-F modified nucleotides.

8. 7. The polynucleic acid molecule conjugate of any one of claims 1 to 6, wherein the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 2, 6, 56, or 61-66.

9. The polynucleic acid molecule conjugate of any one of claims 1 to 8, wherein the polynucleic acid molecule comprises phosphorothioate or phosphorodithioate linkages.

10. The polynucleic acid molecule conjugate of any one of claims 1 to 9, wherein the polynucleic acid molecule comprises six or more 2'-modified nucleotides selected from 2'-O-methyl and 2'-deoxy-2'-fluoro.

11. The polynucleic acid molecule conjugate of any one of claims 1 to 10, wherein the polynucleic acid molecule comprises a 5'-terminal vinylphosphonate modified nucleotide.

12. The 5'-terminal vinylphosphonate modified nucleotide is 【Chemistry 1】 is selected from wherein B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or alkoxy; 12. The polynucleic acid molecule conjugate of claim 1, wherein J is an internucleotide linking group connecting adjacent nucleotides of said polynucleic acid molecule.

13. The polynucleic acid molecule conjugate of any one of claims 10 to 12, wherein the sense strand and / or the antisense strand comprises at least two, at least three, or at least four consecutive 2'-O-methyl modified nucleotides at the 5'-end or 3'-end.

14. 14. The polynucleic acid molecule conjugate of any one of claims 1 to 13, comprising a linker connecting the antibody or antigen-binding fragment thereof to the polynucleic acid molecule via a cysteine ​​or lysine residue on the antibody or antigen-binding fragment thereof.

15. The linker is C １ -C ６ The polynucleic acid molecule conjugate of claim 14, wherein the linker is an alkyl linker.

16. 15. The polynucleic acid molecule conjugate of claim 14, 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.

17. The polynucleic acid molecule conjugate of claim 14, wherein the linker is a cleavable or non-cleavable linker.

18. 18. The polynucleic acid molecule conjugate of any one of claims 1 to 17, wherein the ratio of said polynucleic acid molecule to said antibody or antigen-binding fragment thereof is about 1:1, 2:1, 3:1, or 4:

1.

19. The polynucleic acid molecule conjugate of any one of claims 1 to 18, wherein the polynucleic acid molecule mediates RNA interference against human DUX4 and regulates muscle wasting in a subject.

20. The polynucleic acid molecule conjugate of claim 19, 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.

21. 21. The polynucleic acid molecule conjugate of claim 19 or 20, 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.

22. 21. The polynucleic acid molecule conjugate of claim 19 or 20, wherein the RNA interference comprises affecting the expression of a marker gene selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD in a cell.

23. The polynucleic acid molecule conjugate of claim 22, 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.

24. The polynucleic acid molecule conjugate according to any one of claims 19 to 23, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

25. A polynucleic acid molecule conjugate according to any one of claims 1 to 24; a pharmaceutically acceptable excipient; 10. A pharmaceutical composition comprising:

26. 26. The pharmaceutical composition of claim 25, formulated as a nanoparticle formulation.

27. 27. The pharmaceutical composition of claim 24 or 26, formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intrathecal administration.

28. 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 24; and treating the muscular dystrophy by administering the polynucleic acid conjugate to the subject, wherein the polynucleic acid conjugate reduces the amount of human DUX4 mRNA transcript.

29. 29. The method of claim 28, wherein the polynucleic acid conjugate mediates RNA interference against the human DUX4 and regulates muscular dystrophy in the subject.

30. 30. The method of claim 29, wherein the RNA interference affects the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD in cells affected by muscular dystrophy.

31. 31. The method of any one of claims 28 to 30, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

32. Use of the polynucleic acid molecule conjugate of any one of claims 1 to 24, or the pharmaceutical composition of any one of claims 25 to 27, for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

33. Use of the polynucleic acid molecule conjugate of any one of claims 1 to 24, or the pharmaceutical composition of any one of claims 25 to 27, in the manufacture of a medicament for treating a subject diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD).

34. A kit comprising the polynucleic acid molecule conjugate of any one of claims 1 to 24 or the pharmaceutical composition of any one of claims 25 to 27.

35. A polynucleic acid molecule that mediates RNA interference against DUX4, wherein the polynucleic acid molecule comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438.

36. 1. A double-stranded polynucleic acid molecule that mediates RNA interference against DUX4, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 412-420 or 430-438, and the sense strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

37. 1. A double-stranded polynucleic acid molecule that mediates RNA interference against DUX4, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence comprising at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from a sequence selected from SEQ ID NOs: 412-420 or 430-438, and the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

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