Compositions and methods for treating facioscapulohumeral 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 on September 16, 2021, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Muscular atrophy is the loss of muscle mass or the progressive weakening and degeneration of muscles such as skeletal muscles, voluntary muscles, cardiac muscles, and smooth muscles that control movement. Various pathophysiological conditions including disuse, starvation, cancer, diabetes, and renal failure, or treatment with glucocorticoids, result in muscular atrophy and loss of strength. The phenotypic effects of muscular atrophy are induced by various molecular events including inhibition of muscle protein synthesis, improvement of muscle protein turnover, abnormal regulation of satellite cell differentiation, and abnormal conversion of muscle fiber types.

[0003] FSHD is a rare, progressive, physically disabling disorder for which there is no approved treatment. FSHD is one of the most common forms of muscular dystrophy, equally affecting both genders and typically presenting in young people and young adults. FSHD is characterized by progressive skeletal muscle loss that initially causes weakness in the muscles of the face, shoulders, arms, and torso and progresses to weakness in the muscles of the lower limbs and pelvic girdle. The weakness of the skeletal muscles results in significant physical limitations, including progressive loss of facial muscles that can prevent smiling and communication, make daily activities using the arms difficult, and make it difficult to get up, and many patients ultimately rely on wheelchair use in their daily activities. The onset of chronic pain, anxiety, and depression has also been reported in a number of FSHD patients.

[0004] FSHD is caused by the abnormal expression of the DUX4 gene in skeletal muscle, leading to the inappropriate presence of the DUX4 protein. RNA-induced gene repression results in several control levels, including transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some cases, RNA interference (RNAi) produces effects that persist for extended periods across multiple cell divisions. Therefore, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease treatment.

[0005] Reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually referred to. [Overview of the project]

[0006] In some aspects of this specification, a polynucleic acid molecule conjugate is described, comprising an antibody or its antigen-binding fragment 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 the polynucleic acid molecule mediates RNA interference with DUX4. In some embodiments, the antibody or its antigen-binding fragment includes 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-strand variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is an anti-transferrin receptor antibody or its antigen-binding fragment. In some embodiments, the polynucleic acid molecule is about 16 to about 30 nucleotides long. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand comprising at least one nucleic acid sequence from UfsNfsnnnNfnnnnnnnNfnNfnnnsusu, usNfsnnnNfnnnnnnnnNfnNfnnnsusu, or vpNsNfsnnnNfnnnnnnnNfnNfnnnsus, where vpN = vinyl phosphonate VpUq, lowercase (n) = 2'-O-Me modification, Nf = 2'-F modification, and s = phosphorothioate skeleton modification. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand 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.In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand 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. 2, 6, 56, or 61-66, and comprising 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, the sense strand 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. 2, 6, 56, or 61-66. In some embodiments, the polynucleic acid molecule comprises a phosphorothioate linkage or a phosphorodithioate linkage. 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] The formula is selected from, where B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links to adjacent nucleotides of the polynucleic acid molecule. In some embodiments, the sense strand and / or antisense strand include 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 includes a linker that connects the antibody or its antigen-binding fragment to the polynucleic acid molecule via a cysteine ​​residue or a lysine residue on the antibody or its 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 includes a maleimide group, a dipeptide moiety, a benzoic acid group, or a derivative 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 approximately 1:1, 2:1, 3:1, or 4:1 between the polynucleic acid molecule and the antibody or its antigen-binding fragment. In some embodiments, the polynucleic acid molecule mediates RNA interference against human DUX4 to modulate the muscular atrophy of the subject. In some embodiments, the RNA interference includes reducing the expression of DUX4 gene mRNA transcription by at least 50%, at least 60%, or at least 70% or more compared to the amount of DUX4 gene mRNA transcription in untreated cells. In some embodiments, the RNA interference includes affecting the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX in cells. In some embodiments, the RNA interference includes affecting the expression of a marker gene selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD in cells. In some embodiments, affecting the expression of a marker gene means reducing the expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or more. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0008] In some embodiments of this specification, pharmaceutical compositions comprising a polynucleic acid molecule conjugate described herein and a pharmaceutically acceptable excipient are described. 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 intracoagulant administration.

[0009] In some embodiments of this specification, a method for treating a muscular dystrophy in a subject requiring treatment is described, comprising the steps of: providing a polynucleic acid conjugate described herein; and treating the muscular dystrophy by administering the polynucleic acid conjugate to a subject, wherein the polynucleic acid conjugate reduces the amount of mRNA transcription of human DUX4. In some embodiments, the polynucleic acid conjugate mediates RNA interference with human DUX4 to modulate the muscular dystrophy in the subject. In some embodiments, the RNA interference includes 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] In some embodiments of this specification, the use of the polynucleic acid molecule conjugate or the pharmaceutical composition described herein for treating subjects diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD) is described. In some embodiments, the use of the polynucleic acid molecule conjugate or the pharmaceutical composition described herein in the manufacture of a drug for treating subjects diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD) is described. In some embodiments of this specification, a kit comprising the polynucleic acid molecule conjugate or the pharmaceutical composition described herein is described.

[0011] In some aspects of this specification, polynucleic acid molecules mediating RNA interference with DUX4 are described, which include 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 aspects of this specification, a double-stranded polynucleic acid molecule mediating RNA interference to DUX4 is described, 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] In some aspects of this specification, a double-stranded polynucleic acid molecule for mediating RNA interference against DUX4 is described, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence having at least 15 consecutive nucleotides that differ by only one, two, or three or fewer nucleotides from a sequence selected from SEQ ID NOs. 412-420 or 430-438, and the sense strand comprises at least 15 consecutive nucleotides that differ by only one, two, or three or fewer nucleotides from a sequence selected from SEQ ID NOs. 142, 146, 196, or 201-206.

[0014] In certain aspects of this specification, polynucleic acid molecules and pharmaceutical compositions for modulating genes associated with muscular atrophy, particularly facioscapulohumeral muscular dystrophy (FSHD), are disclosed. In some aspects of this specification, methods for treating muscular atrophy, particularly FSHD, with polynucleic acid molecules or polynucleic acid molecule conjugates disclosed herein are also described.

[0015] In certain embodiments of this specification, polynucleic acid molecule conjugates are disclosed that mediate RNA interference with DUX4, comprising an antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DUX4. In certain embodiments, the antibody or its antigen-binding fragment includes 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-strand 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 polynucleotide molecule comprises a sense strand and / or an antisense strand, each independently comprising at least one 2' modified nucleotide, at least one modified nucleotide ligation, or at least one inverted abasic moiety. In certain embodiments, the polynucleotide hybridizes to at least eight adjacent bases of the target sequence of DUX4. In certain embodiments, the polynucleotide is approximately 8 to approximately 50 nucleotides long, or approximately 10 to approximately 30 nucleotides long. In certain embodiments, the polynucleotide molecule comprises a sense strand and / or an antisense strand, and 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%, and at least 99% identical to a sequence selected from SEQ ID NOs: 1-70 or SEQ ID NOs: 141-210. Alternatively and / or additionally, the polynucleic acid molecule includes a sense strand and / or an antisense strand, the antisense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to a sequence selected from SEQ ID NOs. Alternatively and / or additionally, the polynucleic acid molecule includes a sense strand and / or an antisense strand, the antisense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to a sequence selected from SEQ ID NOs. 142, 146, 196, 201-206, 412-420, or 430-438. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, the antisense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and 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, the sense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% 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 a 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide, or comprising locked nucleic acid (LNA) or ethylene nucleic acid (ENA), or a combination thereof. In certain embodiments, the linkage between at least one modified nucleotide comprises a phosphorothioate bond or a 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 5'-terminal vinyl phosphonate-modified nucleotides.

[0018] Another embodiment is a polynucleic acid molecule conjugate in which at least one 5'-vinylphosphonate-modified non-natural nucleotide is

[0019] [ka] selected from, wherein B is a heterocyclic base moiety, a polynucleic acid molecule is provided.

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

[0021]

Chemical formula

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

[0023]

Chemical formula

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

[0025]

Chemical formula

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

[0027] Another embodiment is a polynucleic acid molecule conjugate in which at least one 5'-vinylphosphonate-modified non-natural nucleotide is

[0028] [ka] Selected from, The present invention provides a polynucleotide molecule in which B is a heterocyclic base portion and J is an internucleotide linking group that links to adjacent nucleotides of the polynucleotide.

[0029] Another embodiment is a polynucleic acid molecule conjugate in which at least one 5'-vinylphosphonate-modified non-natural nucleotide is

[0030] [ka] The present invention provides a polynucleotide molecule selected from the following, in which B is a heterocyclic base moiety and J is an internucleotide linking group that links to adjacent nucleotides of the polynucleotide.

[0031] Another embodiment is a polynucleic acid molecule conjugate in which at least one 5'-vinylphosphonate-modified non-natural nucleotide is

[0032] [ka] Selected from, The present invention provides a polynucleotide molecule in which B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links to adjacent nucleotides of the polynucleotide.

[0033] Another embodiment is a polynucleic acid molecule conjugate in which at least one 5'-vinylphosphonate-modified non-natural nucleotide is

[0034] [ka] Provides polynucleotide molecules selected from the following.

[0035] In certain embodiments, the 2'-modified nucleotide is a 2'-O-methyl-modified nucleotide, and the 2'-O-methyl-modified nucleotide is located at the 5' end of the sense strand and / or 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 antisense strand contains at least two, three, or four consecutive 2'-O-methyl-modified nucleotides at the 5' end.

[0036] In certain embodiments, the polynucleic acid molecule concert includes a linker connecting the target cell-binding portion to the polynucleic acid portion. In such embodiments, the linker is a C1-C6 alkyl linker, or a homobifunctional or heterobifunctional linker, comprising a maleimide group, a dipeptide portion, a benzoic acid group, or a derivative thereof. Alternatively and / or additionally, the linker is cleavable or non-cleavable. In certain embodiments, the ratio of the polynucleic acid portion to the target cell-binding portion is about 1:1, 2:1, 3:1, or 4:1.

[0037] In certain embodiments, the polynucleic acid moiety mediates RNA interference with human DUX4 to modulate the symptoms of the muscular dystrophy or muscular atrophy in question. In some embodiments, the RNA interference includes reducing the expression of DUX4 gene mRNA transcription by at least 50%, at least 60%, or at least 70% compared to the amount of DUX4 gene mRNA transcription in untreated cells. Alternatively and / or additionally, the RNA interference includes affecting the expression of marker genes in cells, including or selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX. In some embodiments, affecting marker gene expression means reducing the expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). Alternatively and / or additionally, RNA interference includes affecting the expression of marker genes in cells, including or selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD. In some embodiments, affecting marker gene expression means reducing the expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, or 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 its antigen-binding fragment, B is a polynucleic acid molecule that hybridizes to a target sequence of DUX4, and X is a conjugate or nonpolymeric linker that is conjugated to a cysteine ​​residue of A.

[0039] In certain embodiments of this specification, pharmaceutical compositions comprising a polynucleic acid molecule conjugate described herein and a pharmaceutically acceptable excipient are disclosed. 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 intracoagulant administration.

[0040] Symptoms of FSHD include effects on skeletal muscles. Skeletal muscles affected by FSHD include the muscles around the eyes and mouth, shoulder muscles, upper arm muscles, lower leg muscles, abdominal muscles, and gluteal muscles. In some cases, symptoms of FSHD also affect vision and hearing. In some cases, symptoms of FSHD also affect heart or lung function. In some specific cases, symptoms of FSHD include muscle weakness, muscular atrophy, muscular dystrophy, pain and inflammation, spasticity, scoliosis, lordosis, hypoventilation, retinal abnormalities, exposure to keratitis, mild hearing loss, and EMG abnormalities. As used herein, the term muscular atrophy refers to the broad range of muscle-related effects of FSHD.

[0041] In certain embodiments of this specification, methods are disclosed for treating muscular dystrophy in subjects requiring treatment of muscular dystrophy by providing the 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 with human DUX4 to modulate muscular dystrophy in the subject. In certain embodiments, the RNA interference includes affecting the expression of a marker gene for DUX4, including or selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX, in cells affected by muscular dystrophy. In certain embodiments, the RNA interference includes affecting the expression of a marker gene for DUX4, including or selected from the group consisting of WFDC3, ILVBL, SLC15A2, and SORD, in cells affected by muscular dystrophy.

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

[0043] In certain aspects of this specification, the use of polynucleic acid molecule conjugates or pharmaceutical compositions described herein for treating subjects diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD) is disclosed. Also in certain aspects of this specification, the use of polynucleic acid molecule conjugates or pharmaceutical compositions described herein in the manufacture of agents for treating subjects diagnosed with or suspected of having facioscapulohumeral muscular dystrophy (FSHD) is described.

[0044] In certain embodiments of this specification, kits comprising polynucleic acid molecular conjugates or pharmaceutical compositions described herein are disclosed. [Brief explanation of the drawing]

[0045] Various aspects of this disclosure are described, in particular, by the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description illustrating exemplary embodiments in which the principles of this disclosure are used, and by referring to the appended drawings.

[0046] [Figure 1] A diagram illustrating the symptoms of FSHD is provided. [Figure 2] A flowchart illustrating the 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] This figure shows a graph of in vivo downregulation of DUX4 target genes in the skeletal muscle of a mouse model of FSHD. [Figure 4B] This figure shows a graph of the in vivo concentration of DUX-4 siRNA in muscle tissue. [Figure 5A]A typical structure of siRNA having a C6-NH2 conjugation handle at the 5' end of the passenger or guide strand and a C6-SH at the 3' end is shown. [Figure 5B] 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 is shown. [Figure 5C] 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, is shown. [Figure 5D] A typical structure of an siRNA passenger chain, having a C6-N-SMCC conjugation handle at the 5' end and a C6-S-NEM at the 3' end, is shown. [Figure 5E] A typical structure of an siRNA passenger or guide strand having PEG at the 5' end and C6-SH at the 3' end is shown. [Figure 5F] 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 is shown. [Figure 6A] The antibody-Cys-SMCC-5'-passenger chain (architecture 1) is illustrated. This conjugate was generated by inter-antibody chain cysteine ​​conjugation to maleimide (SMCC) at the 5' end of the passenger chain. [Figure 6B] The antibody-Cys-SMCC-3'-passenger chain (architecture 2) is illustrated. This conjugate was generated by inter-antibody chain cysteine ​​conjugation to maleimide (SMCC) at the 3' end of the passenger chain. [Figure 6C] The antibody-Cys-bisMal-3'-passenger chain (ASC architecture 3) is illustrated. This conjugate was generated by inter-antibody chain cysteine ​​conjugation to the bismaleimide (bisMal) linker at the 3' end of the passenger chain. [Figure 6D] The model structure of the Fab-Cys-bisMal-3'-passenger chain (ASC architecture 4) is illustrated. This conjugate was generated by Fab-chain inter-cysteine ​​conjugation to the bismaleimide (bisMal) linker at the 3' end of the passenger chain. [Figure 6E] A model structure of an antibody-siRNA conjugate (ASC architecture 5), in which two different siRNAs are attached to a single antibody molecule, is illustrated. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNA to a reduced mAb interchain cysteine ​​relative to a bismaleimide (bisMal) linker at the 3' end of the passenger chain of each siRNA. [Figure 6F] A model structure (ASC architecture 6) of an antibody-siRNA conjugate with two different siRNAs attached is illustrated. This conjugate was generated by conjugating a mixture of SSB and HPRT siRNA to a reduced mAb interchain cysteine ​​relative to a maleimide (SMCC) linker at the 3' end of the passenger chain of each siRNA. [Figure 7A] A typical synthesis scheme (synthesis scheme 1) of the antibody-Cys-SMCC-siRNA-PEG conjugate via antibody-cysteine ​​conjugation is illustrated. [Figure 7B] A typical synthesis scheme (synthesis scheme 2) for the antibody-Cys-BisMal-siRNA-PEG conjugate is illustrated. [Figure 7C] A typical synthesis scheme (synthesis scheme 3) for generating Fab-siRNA conjugates is illustrated. [Modes for carrying out the invention]

[0047] FSHD is caused by the abnormal expression of the DUX4 gene in skeletal muscle, leading to the inappropriate presence of the DUX4 protein. DUX4 itself is a transcription factor that induces the expression of other genes, and these inappropriately expressed downstream genes contribute to the muscular pathology. Normally, DUX4-driven gene expression is limited to germline and early stem cell development. In FSHD patients, the DUX4 protein in skeletal muscle regulates other gene products, some of which are toxic to muscle. Evidence of abnormal DUX4-driven gene expression is the main molecular signature that distinguishes FSHD-affected muscle tissue from healthy muscle. Abnormal DUX4 expression in FSHD results in muscle death and replacement of muscle with fat, leading to skeletal muscle weakness and progressive physical disability. Data suggest that reducing DUX4 gene expression and its downstream transcriptional programs may provide disease-modifying therapeutic approaches for treating FSHD with DUX4 as the underlying cause.

[0048] Two methods exist to release the silencing or repression of the DUX4 gene. In FSHD1, which accounts for approximately 95% of FSHD patients, there is a mutation that shortens the DNA array in a region near the end of the long arm of chromosome 4, known as D4Z4, which has repeats in the subtelomeric region of the chromosome. The D4Z4 region is abnormally shortened, containing repeats between 1 and 10 instead of the normal 11 to 100 repeats. This contraction leads to hypomethylation of the D4Z4 region and depression of DUX4. FSHD2 patients do not have significant D4Z4 repeat contraction, but usually have a mutation in a regulatory gene known as the SMCHD1 gene, which contributes to the repression of the DUX4 gene via DNA methylation. If this repression is lost due to a mutation in the SMCHD1 gene that causes hypomethylation of the D4Z4 region, DUX4 is improperly expressed, inducing the disease state. Figure 1 shows an illustrative diagram of the FSHD pathology.

[0049] Nucleic acid (e.g., RNAi) therapy is a highly selective and specific targeted therapy. However, in some cases, nucleic acid therapy is also hindered by insufficient intracellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications of nucleic acid compositions are being explored, such as novel linkers for good stabilization and / or low toxicity, optimization of binding sites for increased target specificity and / or target delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0050] In some embodiments, the arrangement or order of the various components constituting the nucleic acid composition further affects intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, if the nucleic acid components include binding sites, polymers, and polynucleotide molecules, the order or arrangement of the binding sites, polymers, and / or polynucleotide molecules (e.g., binding site-polynucleotide molecule-polymer, binding site-polymer-polynucleotide molecule, or polymer-binding site-polynucleotide molecule) further affects intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0051] In some aspects of this specification, polynucleic acid molecules and polynucleic acid molecule conjugates are described herein, among some aspects, for the treatment of facioscapulohumeral muscular dystrophy (FSHD), in particular muscular dystrophy and / or muscular atrophy associated therewith. In some examples, the polynucleic acid molecule conjugates described herein enhance intracellular uptake, stability, and / or efficacy. Optionally, the polynucleic acid molecule conjugate comprises an antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule. Optionally, a polynucleic acid molecule hybridizing to a target sequence of DUX4, preferably human DUX4. Optionally, a nucleic acid molecule hybridizing to a target sequence of human DUX4 having accession number NM_001306068. Optionally, a nucleic acid molecule hybridizing to a target sequence of human DUX4 having sequence number 439.

[0052] Further embodiments described herein include methods for treating FSHD, comprising the step of administering a polynucleic acid molecule or polynucleic acid molecule conjugate as described herein.

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

[0054] In some embodiments, the polynucleic acid molecule contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 1 to 70. In some embodiments, the polynucleic acid molecule contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 141 to 210. In some embodiments, the polynucleic acid molecule contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. In some embodiments, the polynucleic acid molecule contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs.

[0055] In some embodiments, the polynucleic acid molecule contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. 142, 146, 196, 201-206, 412-420, or 430-438.

[0056] In some embodiments, the polynucleotide 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 with a sequence selected from SEQ ID NOs. 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 with a sequence selected from SEQ ID NOs. Optionally, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. In some cases, the second polynucleotide contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs.

[0057] In some cases, the first polynucleotide contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. In some cases, the second polynucleotide contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs.

[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) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. In some examples, the antisense strand (e.g., the guide strand) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. 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) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. 211–280, the antisense strand (e.g., the guide strand) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. 211–280. In some examples, the sense strand (e.g., passenger strand) contains 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 sequence numbers 142, 146, 196, or 201–206. In some examples, the antisense strand (e.g., guide strand) contains 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 sequence numbers 412–420 or 430–438.

[0059] In some examples, the sense strand contains a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from sequence numbers 1, 2, 3, 6, 14, 36, 52, 56, 61, 62, 63, 65, or 66. In some examples, the antisense strand contains 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 contains the sense and antisense strands presented in Table 11.

[0060] In some examples, the sense strand contains a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from sequence numbers 141, 142, 143, 146, 176, 192, 196, 201, 202, 203, 205, or 206. In some examples, the antisense strand contains 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 contains the sense and antisense strands presented in Table 12.

[0061] In some examples, the sense strand contains a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from sequence numbers 142, 146, 196, or 201–206 in Tables 14 and 15.

[0062] In some examples, the antisense strand contains a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from sequence numbers 412-420 or 430-438 in Tables 14 and 15.

[0063] In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, the antisense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to a sequence selected from SEQ ID NOs: 412-420 or SEQ ID NOs: 430-438. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, the antisense strand being 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, the sense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and 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, the sense strand being identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201–206.

[0064] In some embodiments, the sequenced polynucleic acid molecule has at least 14, 15, 16, 17, 18, or 19 adjacent nucleotides that differ by 3 or fewer nucleotides, 2 or fewer nucleotides, or 1 or fewer nucleotides from any one of sequence numbers 142, 146, 196, or 201-206, or sequence numbers 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 include RNA or DNA. In some cases, the polynucleic acid molecules include RNA. In some examples, RNA includes small interfering RNA (siRNA), short hairpin RNA (shRNA), μRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heteronuclear RNA (hnRNA). In some examples, RNA includes shRNA. In some examples, RNA includes miRNA. In some examples, RNA includes dsRNA. In some examples, RNA includes tRNA. In some examples, RNA includes rRNA. In some examples, RNA includes hnRNA. In some examples, oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), which are short single-stranded oligonucleotide analogs constructed with a morpholine ring backbone linked by phosphorodithioate ligation. In some examples, RNA includes siRNA. In some examples, the polynucleic acid molecule includes siRNA.

[0066] In some embodiments, the polynucleotide molecule is about 8 to about 50 nucleotides long. In some embodiments, the polynucleotide molecule is about 10 to about 50 nucleotides long. In some examples, the polynucleotide 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 long.

[0067] In some embodiments, the polynucleotide molecule is approximately 50 nucleotides long. In some examples, the polynucleotide molecule is approximately 45 nucleotides long. In some examples, the polynucleotide molecule is approximately 40 nucleotides long. In some examples, the polynucleotide molecule is approximately 35 nucleotides long. In some examples, the polynucleotide molecule is approximately 30 nucleotides long. In some examples, the polynucleotide molecule is approximately 25 nucleotides long. In some examples, the polynucleotide molecule is approximately 20 nucleotides long. In some examples, the polynucleotide molecule is approximately 19 nucleotides long. In some examples, the polynucleotide molecule is approximately 18 nucleotides long. In some examples, the polynucleotide molecule is approximately 17 nucleotides long. In some examples, the polynucleotide molecule is approximately 16 nucleotides long. In some examples, the polynucleotide molecule is approximately 15 nucleotides long. In some examples, the polynucleotide molecule is approximately 14 nucleotides long. In some examples, the polynucleotide molecule is approximately 13 nucleotides long. In some examples, the polynucleotide molecule is approximately 12 nucleotides long. In some examples, the polynucleotide molecule is approximately 11 nucleotides long. In some cases, polynucleotide molecules are approximately 10 nucleotides long. In some cases, polynucleotide molecules are approximately 8 nucleotides long. In some cases, polynucleotide molecules are between approximately 8 and 50 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 50 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 45 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 40 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 35 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 30 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 25 nucleotides long. In some cases, polynucleotide molecules are between approximately 10 and 20 nucleotides long. In some cases, polynucleotide molecules are between approximately 15 and 25 nucleotides long. In some cases, polynucleotide molecules are between approximately 15 and 30 nucleotides long. In some cases, polynucleotide molecules have a nucleotide length between approximately 12 and 30.

[0068] In some embodiments, the polynucleotide molecule includes a first polynucleotide. In some examples, the polynucleotide molecule includes a second polynucleotide. In some examples, the polynucleotide molecule includes both a first and a second polynucleotide. In some examples, the first polynucleotide is a sense strand or passenger strand. In some examples, the second polynucleotide is an antisense strand or 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 long. In some embodiments, the first polynucleotide is about 10 to about 50 nucleotides long. 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 long.

[0070] In some cases, the first polynucleotide is approximately 50 nucleotides long. In some cases, the first polynucleotide is approximately 45 nucleotides long. In some cases, the first polynucleotide is approximately 40 nucleotides long. In some cases, the first polynucleotide is approximately 35 nucleotides long. In some cases, the first polynucleotide is approximately 30 nucleotides long. In some cases, the first polynucleotide is approximately 25 nucleotides long. In some cases, the first polynucleotide is approximately 20 nucleotides long. In some cases, the first polynucleotide is approximately 19 nucleotides long. In some cases, the first polynucleotide is approximately 18 nucleotides long. In some cases, the first polynucleotide is approximately 17 nucleotides long. In some cases, the first polynucleotide is approximately 16 nucleotides long. In some cases, the first polynucleotide is approximately 15 nucleotides long. In some cases, the first polynucleotide is approximately 14 nucleotides long. In some cases, the first polynucleotide is approximately 13 nucleotides long. In some cases, the first polynucleotide is approximately 12 nucleotides long. In some cases, the first polynucleotide is approximately 11 nucleotides long. In some cases, the first polynucleotide is approximately 10 nucleotides long. In some cases, the first polynucleotide is approximately 8 nucleotides long. In some cases, the first polynucleotide is between approximately 8 and 50 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 50 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 45 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 40 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 35 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 30 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 25 nucleotides long. In some cases, the first polynucleotide is between approximately 10 and 20 nucleotides long. In some examples, the first polynucleotide has a nucleotide length between approximately 15 and 25.In some examples, the first polynucleotide has a nucleotide length between approximately 15 and 30. In other examples, the first polynucleotide has a nucleotide length between approximately 12 and 30.

[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 long. In some embodiments, the second polynucleotide is about 10 to about 50 nucleotides long. 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 long.

[0072] In some cases, the second polynucleotide is approximately 50 nucleotides long. In some cases, the second polynucleotide is approximately 45 nucleotides long. In some cases, the second polynucleotide is approximately 40 nucleotides long. In some cases, the second polynucleotide is approximately 35 nucleotides long. In some cases, the second polynucleotide is approximately 30 nucleotides long. In some cases, the second polynucleotide is approximately 25 nucleotides long. In some cases, the second polynucleotide is approximately 20 nucleotides long. In some cases, the second polynucleotide is approximately 19 nucleotides long. In some cases, the second polynucleotide is approximately 18 nucleotides long. In some cases, the second polynucleotide is approximately 17 nucleotides long. In some cases, the second polynucleotide is approximately 16 nucleotides long. In some cases, the second polynucleotide is approximately 15 nucleotides long. In some cases, the second polynucleotide is approximately 14 nucleotides long. In some cases, the second polynucleotide is approximately 13 nucleotides long. In some cases, the second polynucleotide is approximately 12 nucleotides long. In some cases, the second polynucleotide is approximately 11 nucleotides long. In some cases, the second polynucleotide is approximately 10 nucleotides long. In some cases, the second polynucleotide is approximately 8 nucleotides long. In some cases, the second polynucleotide is between approximately 8 and 50 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 50 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 45 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 40 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 35 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 30 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 25 nucleotides long. In some cases, the second polynucleotide is between approximately 10 and 20 nucleotides long. In some cases, the second polynucleotide has a nucleotide length between approximately 15 and 25.In some examples, the second polynucleotide has a nucleotide length between approximately 15 and 30. In other examples, the second polynucleotide has a nucleotide length between approximately 12 and 30.

[0073] In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the polynucleotide 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. In some cases, the overhang is a 5' overhang, a 3' overhang, or both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the overhang comprises 1 non-base-paired nucleotide. In some cases, the overhang comprises 2 non-base-paired nucleotides. In some cases, the overhang comprises 3 non-base-paired nucleotides. In some cases, the overhang contains four non-base-paired nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand containing two non-base-paired nucleotides as an overhang at its 3' end, while the sense strand has no overhang. Optionally, in such embodiments, the non-base-paired nucleotides have a TT, dTdT, or UU sequence. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand having one or more nucleotides complementary to the antisense sequence at its 5' end.

[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 the target sequence of DUX4. In some embodiments, the target sequence of DUX4 is a nucleic acid sequence in DUX4 that is approximately 10–50 base pairs long, approximately 15–50 base pairs long, 15–40 base pairs long, 15–30 base pairs long, or 15–25 base pairs long, where the first nucleotide of the target sequence begins at any nucleotide in the DUX4 mRNA transcript, either 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 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 region (5' or 3' untranslated region) of the DUX mRNA.In some embodiments, the first nucleotide of the target sequence is nal10~nal15, nal10~nal20, nal50~nal60, nal55~nal65, nal75~nal85, nal95~nal105, nal135~nal145, nal155~nal165, nal225~nal235, nal265~nal275, nal275~nal285, nal285~nal295, nal325~nal335, nal335~n 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 position 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 polynucleotide molecule is at least 50% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 60% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 70% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 80% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 90% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 99% complementary to the target sequence described herein. In some examples, the sequence of the polynucleotide molecule is 100% complementary to the target sequence described herein.

[0076] In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with respect to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with respect to the target sequence described herein. In some examples, the sequence of the polynucleotide molecule has three or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has two or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has one or fewer mismatches with respect to the target sequence described herein.

[0077] In some embodiments, the bases of all polynucleotide molecules in all polynucleotide molecules potentially bind to target sequences of DUX4 selected to generate a polynucleotide molecule library. In certain embodiments, such selection processes are carried out in silico by one or more steps to exclude less desirable polynucleotide molecules from the candidates. For example, in some embodiments, the selection process includes an exclusion step for one or more polynucleotide molecules having single nucleotide polymorphisms (SNPs) and / or MEFs less than -5. Alternatively and / or additionally, in some embodiments, the selection process includes an exclusion step for one or more polynucleotide molecules having 0 and 1 mismatches (MMs) in the human transcriptome (such as only DUX, DUX5, and DBET hits). Alternatively and / or additionally, in some embodiments, the selection process includes an exclusion step for one or more polynucleotide molecules having 0 MMs in the human intragene region (such as only DUX1, DUX5, and DBET pseudogenes). Alternatively and / or additionally, in some embodiments, the selection process includes a step of excluding one or more polynucleotide molecules having MMs against the DUX4 human sequence used in the FLExDUX4 FSHD mouse model. Alternatively and / or additionally, in some embodiments, the selection process includes a step of excluding one or more polynucleotide molecules with a predicted viability of less than 60. Alternatively and / or additionally, such a selection process includes carrying over one or more polynucleotide molecules with a predicted viability of 60 or more. Alternatively and / or additionally, in some embodiments, the selection process includes a step of excluding one or more polynucleotide molecules having matches against known miRNA species regions 1 to 1000. Alternatively and / or additionally, in some embodiments, the selection process includes a step of excluding one or more polynucleotide molecules with a GC content % of 75 or more. Alternatively and / or additionally, in some embodiments, the selection process includes a step of excluding eight or fewer predicted off-target hits having two MMs. In some embodiments, for regions 295-1132 (nal295-1132), 12 or fewer predicted off-target hits with two memory mills are permitted.

[0078] In some embodiments, the selection process is carried out in silico by one or more sequential steps that exclude less desirable polynucleotide molecules from the candidates. For example, in some embodiments, the selection process begins with the collection of candidate polynucleotide molecules to generate a library. From this library, a first exclusion step includes excluding one or more polynucleotide molecules having single nucleotide polymorphisms (SNPs) and / or MEFs less than -5. A second exclusion step then includes excluding one or more polynucleotide molecules having 0 and 1 MM in the human transcriptome (such that only the acceptable hits are DUX, DUX5, and DBET). A third exclusion step then includes excluding one or more polynucleotide molecules having 0 MM in the human intragene region (such that only the acceptable hits are DUX1, DUX5, and DBET pseudogenes). A subsequent exclusion step includes excluding one or more polynucleotide molecules having MM for the DUX4 human sequence to be used in the FLExDUX4 FSHD mouse model. Next, the following step is to carry over only one or more polynucleotide molecules with a predicted viability of 60 or higher. The next exclusion step includes excluding one or more polynucleotide molecules that have matches to known miRNA species regions 1-1000. The exclusion step then continues by excluding one or more polynucleotide molecules with a GC content % of 75 or higher. The final selection process then excludes regions 295-1132 and includes eight or fewer predicted off-target hits with two MMs, where a maximum of 12 hits are allowed.

[0079] In some embodiments, the specificity of the polynucleic acid molecule hybridizing to the 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 performed under highly strict hybridization conditions.

[0080] In some embodiments, polynucleic acid molecules have few off-target effects. In some cases, “off-target” or “off-target effect” refers to all instances in which a polynucleic acid polymer directed to a given target produces unintended effects through direct or indirect interactions with another mRNA sequence, DNA sequence, or cellular protein or other part. In some cases, “off-target effect” occurs when another transcript is simultaneously degraded due to partial homology or complementarity between the other transcript and the sense and / or antisense strand of the polynucleic acid molecule.

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

[0082] In some embodiments, nucleotide analogs or artificial nucleotide bases comprise nucleic acids with modifications to the 2' hydroxyl group of the ribose moiety. In some examples, the modification comprises H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Typical alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. In some examples, the alkyl moiety comprises further modifications. In some examples, the modifications comprise azo groups, keto groups, aldehyde groups, carboxyl groups, nitro groups, nitroso groups, nitrile groups, heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) groups, isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxides, sulfones, sulfides, and disulfides). In some examples, the alkyl moiety comprises further heterosubstitutions. In some cases, the carbon atoms of the heterocyclic group are substituted with nitrogen, oxygen, or sulfur. Examples of heterocyclic substitutions include, but are not limited to, morpholinos, imidazoles, and pyrrolidinos.

[0083] In some cases, the modification of the 2'-hydroxyl group is either 2'-O-methyl modification or 2'-O-methoxyethyl (2'-O-MOE) modification. Depending on the case, 2'-O-methyl modification adds a methyl group to the 2'-hydroxyl group of the ribose moiety, while 2'-O-methoxyethyl modification adds a methoxyethyl group to the 2'-hydroxyl group of the ribose moiety. Typical chemical structures of adenosine molecules with 2'-O-methyl modification and uridine with 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 derived from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group for each sugar, thereby improving its cellular uptake properties due to its zwitterionic nature. Typical chemical structures of 2'-O-aminopropyl nucleoside phosphoramidites are illustrated below.

[0086] [ka]

[0087] In some cases, modifications at the 2' hydroxyl group are locked or cross-linked ribose modifications (e.g., locked nucleic acids, i.e., LNA) in which the oxygen molecule bonded at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxymethylene-linked bicyclic ribonucleotide monomer. Typical representations of the chemical structure of LNA are shown below. The representation on the left emphasizes the chemical linkage of the LNA monomer. The representation on the right emphasizes the locked 3'-endo(3E) conformation of the furanose ring of the LNA monomer.

[0088] [ka]

[0089] In some examples, modifications at the 2'-hydroxyl group include ethylene nucleic acids (ENAs), such as 2'-4'-ethylene-bridged nucleic acids, which lock the sugar conformation into the C3'-endo sugar puckering conformation. ENAs are further part of the bridged nucleic acid class of modified nucleic acids, including LNAs. Typical chemical structures of ENAs and bridged nucleic acids are illustrated below.

[0090] [ka]

[0091] In some embodiments, the additional modification of the 2'-hydroxyl group includes 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 are, 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 having a modification at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halolysine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, 7-deaza-adenosine, deazanucleotides, 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, quosin, archeosin, naphthyl, and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, phenyl and modified phenyl groups such as 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, aminophenol, or 2,4,6-trimethoxybenzene, modified cytosine acting as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include nucleotides modified in relation to the sugar moiety, as well as nucleotides having a non-ribosyl sugar or its analogue. For example, the sugar portion may be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocyclic or carbocyclic, or based thereon. The term nucleotide also includes those known in the art as universal bases.Examples of universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

[0093] In some embodiments, nucleotide analogs further include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof. Morphorino or phosphoramidite morpholino oligos (PMOs) include synthetic molecules that mimic native nucleic acid structures by deviating from normal sugar and phosphate structures. In some examples, a five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is bonded by a phosphoramidate group instead of a phosphate group. In such cases, the modification of the skeleton removes all positive and negative charges that would make a morpholino neutral molecule capable of crossing the cell membrane without the need for the aid of cell delivery agents, such as those used by charged oligonucleotides.

[0094] [ka]

[0095] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar backbone rings or phosphate links, and the bases are linked by oligoglycine-like molecules with appropriate spacing, thereby eliminating the backbone charge.

[0096] [ka]

[0097] In some embodiments, one or more modifications occur optionally in internucleotide linkages. In some examples, the modified internucleotide linkages include phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, borontrifluorides, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thioalkylphosphotriesters, hydrogen phosphonate linkages, alkylphosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, aminoalkylphosphoramidates, thionophosphoramidates, phosphoropiperadates, phosphoranilothioates, and phosphoranilidetes. Examples include, but are not limited to, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazos, methylenedimethylhydrazos, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamides, linkages with riboacetyl groups, aminoethylglycines, silyl or siloxane linkages, alkyl or cycloalkyl linkages containing or not containing 1 to 10 heteroatoms, such as saturated or unsaturated, substituted, and / or heteroatoms, linkages having a morpholino structure, amides, polyamides in which a base is directly or indirectly bonded to the aza nitrogen of the skeleton, and combinations thereof. Phosphothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate linkages. Typical PS ASOs are exemplified below.

[0098] [ka]

[0099] In some cases, the modifications are methyl or thiol modifications, such as methylphosphonate or thiolphosphonate modifications. Typical thiolphosphonate nucleotides (left) and methylphosphonate nucleotides (right) are illustrated below.

[0100] [ka]

[0101] In some cases, the modified nucleotides are

[0102] [ka] Examples include, but are not limited to, 2'-fluoroN3-P5'-phosphoramidites.

[0103] In some cases, as modified nucleotides,

[0104] [ka] Examples include, but are not limited to, 5'-vinylphosphonate modified non-natural nucleotides selected from the above, where B is the heterocyclic base moiety.

[0105] In some cases, as modified nucleotides,

[0106] [ka] Examples include, but are not limited to, a 5'-vinylphosphonate modified non-natural nucleotide selected from the following, where 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 to adjacent nucleotides of the polynucleotide.

[0107] In some cases, as modified nucleotides,

[0108] [ka] Examples include, but are not limited to, a single 5'-vinylphosphonate modified non-natural nucleotide selected from the following, where 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 to adjacent nucleotides of the polynucleotide.

[0109] In some cases, as modified nucleotides,

[0110] [ka] Examples include, but are not limited to, a 5'-vinylphosphonate modified non-natural nucleotide selected from the following, where B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links to an adjacent nucleotide of the polynucleotide.

[0111] In some examples, the modified nucleotide may be a non-natural nucleotide with a single 5'-vinylphosphonate modification, selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA).

[0112] In some cases, as modified nucleotides,

[0113] [ka] A single 5'-vinylphosphonate modified non-natural nucleotide selected from the following, but not limited to these, is a heterocyclic base moiety, and J is an internucleotide linking group that links to an adjacent nucleotide of the polynucleotide.

[0114] In some cases, as modified nucleotides,

[0115] [ka] A single 5'-vinylphosphonate modified non-natural nucleotide selected from the following, but not limited to these, is a heterocyclic base moiety, and J is an internucleotide linking group that links to an adjacent nucleotide of the polynucleotide.

[0116] In some cases, as modified nucleotides,

[0117] [ka] Examples include, but are not limited to, a 5'-vinylphosphonate modified non-natural nucleotide selected from the following, where B is a heterocyclic base moiety, R6 is selected from hydrogen, halogen, alkyl, or alkoxy, and J is an internucleotide linking group that links to an adjacent nucleotide of the polynucleotide.

[0118] In some cases, as modified nucleotides,

[0119] [ka] Examples include, but are not limited to, a single 5'-vinylphosphonate modified non-natural nucleotide.

[0120] In some cases, as modified nucleotides,

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

[0122] In some embodiments, one or more modifications may further optionally include modifications of a ribose moiety, phosphate skeleton, and nucleoside, or modifications of a nucleotide analog at the 3' or 5' end. For example, the 3' end may optionally include a 3' cationic group, or the nucleoside may be reversed at the 3' end by a 3'-3' linkage. In another alternative, the 3' end may optionally be conjugated with an aminoalkyl group, e.g., 3'C5-aminoalkyldT. In yet another alternative, the 3' end may optionally be conjugated with a debasic site, e.g., a depurine or depyrimidine site. In some examples, the 5' end may be conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent. In some cases, the 5' end may be conjugated with a debasic site, e.g., a depurine or depyrimidine site.

[0123] In some embodiments, the polynucleic acid molecule comprises one or more of the artificial nucleotide analogs described herein. In some examples, the polynucleic acid molecule comprises one or more of the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some cases, polynucleic acid molecules 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), and It contains 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'-fluoroN3-P5'-phosphoramidites, or combinations thereof. In some examples, polynucleic acid molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2'-O-methyl modified nucleotides.In some cases, polynucleic acid molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 25 or more of 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some cases, polynucleic acid molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 25 or more of thiol phosphonate nucleotides.

[0124] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 5% to approximately 100%, approximately 10% to approximately 100%, approximately 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, and approximately 90% to approximately 100%.

[0125] Depending on the case, polynucleic acid molecules may contain at least one of the following modification levels: approximately 10% to approximately 90%, approximately 20% to approximately 90%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, and approximately 80% to approximately 100%.

[0126] Depending on the case, polynucleic acid molecules may contain at least one of the following modifications: approximately 10% to approximately 80%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 80%, approximately 50% to approximately 80%, approximately 60% to approximately 80%, and approximately 70% to approximately 80%.

[0127] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to 70%, approximately 20% to 70%, approximately 30% to 70%, approximately 40% to 70%, approximately 50% to 70%, and approximately 60% to 70%.

[0128] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to 60%, approximately 20% to 60%, approximately 30% to 60%, approximately 40% to 60%, and approximately 50% to 60%.

[0129] Depending on the case, polynucleic acid molecules may contain at least one of the following modifications: approximately 10% to approximately 50%, approximately 20% to approximately 50%, approximately 30% to approximately 50%, and approximately 40% to approximately 50%.

[0130] Depending on the case, polynucleic acid molecules may contain at least one of the following modifications: approximately 10% to 40%, approximately 20% to 40%, and approximately 30% to 40%.

[0131] In some cases, polynucleic acid molecules contain at least one of approximately 10% to 30% modifications and approximately 20% to 30% modifications.

[0132] Depending on the case, polynucleotide molecules contain approximately 10% to 20% modifications.

[0133] Depending on the case, polynucleic acid molecules may contain approximately 15% to 90%, 20% to 80%, 30% to 70%, or 40% to 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% modification.

[0135] In some embodiments, the polynucleic acid molecule contains 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 cases, polynucleic acid molecules contain 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 molecule contains the 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 molecule contains the artificial nucleotide analog described herein. In some examples, about 5% of the polynucleic acid molecule contains the artificial nucleotide analog described herein. In some examples, about 10% of the polynucleic acid molecule contains the artificial nucleotide analog described herein. In some examples, about 15% of the polynucleic acid molecule contains the artificial nucleotide analog described herein. In some examples, about 20% of the polynucleic acid molecule contains the artificial nucleotide analog described herein. In some examples, about 25% of the polynucleic acid molecule contains the artificial nucleotide analog described herein. In some examples, about 30% of the polynucleic acid molecule contains the artificial nucleotide analog described herein. In some examples, approximately 35% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 40% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 45% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 50% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 55% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 60% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 65% ​​of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 70% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 75% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 80% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 85% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 90% of the polynucleotide molecules contain the artificial nucleotide analogs described herein.In some examples, approximately 95% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 96% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 97% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 98% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 99% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

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

[0139] In some embodiments, a polynucleic acid molecule is assembled from two distinct polynucleotides, one containing a sense strand and the other containing the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is linked to the antisense strand via a linker molecule, which in some examples 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, 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, 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 embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the pyrimidine nucleotide being a 2'-deoxy-2'-fluoropyrimidine nucleotide when present in the antisense strand, and the purine nucleotide being a 2'-O-methylpurine nucleotide when present in the antisense strand.

[0142] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the pyrimidine nucleotide being a 2'-deoxy-2'-fluoropyrimidine nucleotide when present in the antisense strand, and the purine nucleotide being a 2'-deoxy-purine nucleotide when present in the antisense strand.

[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 multiple (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, etc.) 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, at least two of the multiple 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 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 antisense strand. In some embodiments, the sense strand of the polynucleic acid molecule contains 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 includes at least one, at least two, at least three, or at least four 2'-deoxy-2'-fluoro-modified nucleotides at the 3' end of at least four, at least five, or at least six consecutive 2'-O-methyl-modified nucleotides at the 5' end of the polynucleotide, or at the 5' end of at least four, at least five, or at least six consecutive 2'-O-methyl-modified nucleotides at the 3' end of the polynucleotide. Further optionally, such 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 / or antisense strand has a 2'-O-methyl modified nucleotide located at the 5' end of the sense strand and / or antisense strand. In some embodiments, at least one of the sense strand and / or antisense strand has a 2'-O-methyl modified nucleotide located at the 3' end of the sense strand and / or antisense strand. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5' end of the sense strand and / or 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 antisense strand is a pyridine nucleotide.

[0145] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having two or more consecutive 2'-deoxy-2'-fluoromodified nucleotides at its 5' end. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having two or more consecutive 2'-O-methylmodified nucleotides at its 3' end. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having at least 2, 3, 4, 5, 6, or 7 consecutive 2'-O-methylmodified nucleotides.

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

[0147] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand includes terminal capping portions at the 5' end, 3' end, or both the 5' and 3' ends. In other embodiments, the terminal capping portions are inverted deoxydecate portions.

[0148] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having a glyceryl modification at its 3' end.

[0149] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising 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 nucleotide 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) universally modified nucleotides, and optionally the 3' end, 5' end, or both of the 3' and 5' ends of the sense strand. The antisense chain contains a terminal cap molecule, and the antisense chain comprises approximately 1 to approximately 10 or more, specifically approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleotide linkages, and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or approximately one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally contains a terminal cap molecule at the 3' end, 5' end, or both the 3' and 5' ends of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 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, by interphosphothioate nucleotide linkages and / or terminal cap molecules located at the 3' end, 5' end, or both the 3' and 5' ends.

[0150] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising 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 nucleotide 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) universally modified nucleotides, as well as optionally the 3' end, 5' end, or both of the 3' and 5' ends of the sense strand. The antisense chain contains a terminal cap molecule, and the antisense chain comprises approximately 1 to approximately 25 or more phosphorothioate nucleotide linkages, e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 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, by interphosphothioate nucleotide linkages and / or terminal cap molecules located at the 3' end, 5' end, or both the 3' and 5' ends.

[0151] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand comprising 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 nucleotide linkages and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand, and optionally comprising a terminal cap molecule at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand. In some embodiments, the antisense chain comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate nucleotide 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) universally modified nucleotides, as well as optionally a terminal cap molecule at the 3' end, 5' end, or both the 3' and 5' ends of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, 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, 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, by interphosphothioate nucleotide linkages and / or terminal cap molecules located at the 3' end, 5' end, or both the 3' and 5' ends.

[0152] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand comprising 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 nucleotide 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 the 3' end, 5' end, or 3' and 5' ends of the sense strand Both contain terminal cap molecules, and the antisense strand comprises approximately 1 to approximately 25 or more phosphorothioate nucleotide linkages, e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. And / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or approximately one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally contains terminal cap molecules at the 3' end, 5' end, or both the 3' and 5' ends of the antisense strand. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 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, by about 1 to about 5, e.g., about 1, 2, 3, 4, 5 or more, phosphorothioate nucleotide interlinks and / or terminal cap molecules located at the 3' end, 5' end, or both the 3' and 5' ends.

[0153] In some embodiments, the polynucleic acid molecules described herein are chemically modified small interfering nucleic acid molecules having about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleotide linkages in each chain of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having 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, the sense strand having a phosphate backbone modification at the 5' end of the antisense strand. In some examples, the phosphate backbone modification is a phosphorothioate. In some embodiments, the sense strand or antisense strand has three consecutive nucleosides linked via two phosphorothioate backbones.

[0154] In another embodiment, the polynucleic acid molecules described herein include 2'-5' internucleotide links. In some examples, the 2'-5' internucleotide links are located at the 3' end, 5' end, or both the 3' and 5' ends of one or both sequence strands. In further examples, the 2'-5' internucleotide links are present at various other positions within one or both sequence strands, for example, one or both strands of the polynucleic acid molecule containing all the internucleotide links of pyrimidine nucleotides, with about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more including 2'-5' internucleotide links, or one or both strands of the polynucleic acid molecule containing all the internucleotide links of purine nucleotides, with about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more including 2'-5' internucleotide links.

[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 complementary to a target nucleic acid sequence, one or more pyrimidine nucleotides present in the polynucleic acid being 2'-deoxy-2'-fluoropyrimidine nucleotides (e.g., all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and all purine nucleotides present in the polynucleic acid are 2'-deoxypurine nucleotides (e.g., all purine nucleotides The polynucleic acid molecule further comprises a rheotide (which is a 2'-deoxypurine nucleotide, or alternatively, multiple purine nucleotides which are 2'-deoxypurine nucleotides), and a terminal cap modification optionally present at the 3' end, 5' end, or both the 3' and 5' ends of the antisense strand, and the polynucleic acid molecule further comprises optionally about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxyribonucleotides at the 3' end of the polynucleic acid molecule, and the terminal nucleotide further comprises one or more (e.g., 1, 2, 3, or 4) phosphorothioate nucleoside linkages, and the polynucleic acid molecule further comprises optionally terminal phosphate groups such as a 5' terminal phosphate group.

[0156] Depending on the circumstances, one or more of the artificial nucleotide analogs described herein may be resistant to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonucleases or 3'-5' exonucleases, compared to natural polynucleic acid molecules. In some cases, artificial nucleotide analogs, including LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA), are used, such as RNase. These molecules exhibit resistance to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some cases, 2'-O-methyl-modified polynucleic acid molecules show nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleic acid molecules show nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, LNA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, morpholino exhibits nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiolphosphonate-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, the 5' conjugate described herein inhibits 5'-3' exonuclease cleavage. In some cases, the 3' conjugate described herein inhibits 3'-5' exonuclease cleavage.

[0157] In some embodiments, one or more of the artificial nucleotide analogs described herein exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. One or more artificial nucleotide analogs, including LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoroN3-P5'-phosphoramidites, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA), exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-methyl-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-deoxy-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-deoxy-2'-fluoro-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE)-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules.In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, LNA modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, ENA modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, PNA modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, HNA modified polynucleic acid molecules show increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, morpholino-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, methylphosphonate-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, thiolphosphonate-modified polynucleic acid molecules exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite exhibit increased binding affinity to mRNA compared to equivalent natural polynucleic acid molecules. In some cases, the increase in 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 chiral-pure (or stereopure) polynucleic acid molecules, or polynucleic acid molecules containing a single enantiomer. In some examples, the polynucleic acid molecule contains an L-nucleotide. In some examples, the polynucleic acid molecule contains a D-nucleotide. In some examples, the polynucleic acid molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of its 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 the polynucleic acid molecule described in U.S. Patent Application Publication No. 2014 / 194610 and No. 2015 / 211006 and International Publication WO2015107425.

[0159] In some embodiments, the polynucleic acid molecules described herein are further modified to include a moiety that conjugates an aptamer. In some examples, the aptamer-conjugate moiety is a moiety that conjugates a DNA aptamer. In some examples, the aptamer-conjugate moiety is an Alphamer (Centauri Therapeutics) and includes an aptamer moiety that recognizes a specific cell surface target and a moiety that presents a specific epitope for attachment to a circulating antibody. In some examples, the polynucleic acid molecules described herein are further modified to include a moiety that conjugates an aptamer, as described in U.S. Patents No. 8,604,184, 8,591,910, and 7,850,975.

[0160] In further embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some examples, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2' hydroxyl position by modifications such as 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), or by locked or cross-linked ribose conformations (e.g., LNA or ENA). In some cases, polynucleotide molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, polynucleotide molecules also contain morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphoramidite to increase their stability. In some examples, polynucleotide molecules are chiral-pure (or stereopure) polynucleotide molecules. In some examples, chiral-pure (or stereopure) polynucleotide molecules are modified to increase their stability. Suitable modifications to RNA for increasing stability for delivery will be obvious to those skilled in the art.

[0161] In some examples, a polynucleic acid molecule is a double-stranded polynucleotide molecule containing a self-complementary sense region and an antisense region, where the antisense region contains a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some examples, a polynucleic acid molecule is assembled from two distinct polynucleotides, where one strand is the sense strand and the other is the antisense strand, and the antisense and sense strands are self-complementary (for example, each strand contains a nucleotide sequence complementary to the nucleotide sequence in the other strand, for example, the antisense and sense strands form a double-stranded or double-stranded structure, for example, the double-stranded region has about 19, 20, 21, 22, 23 or more base pairs), where the antisense strand contains a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense strand contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, polynucleotide molecules can be assembled from a single oligonucleotide, in which the self-complementary sense and antisense regions of the polynucleotide molecule are linked by nucleic acid-based or non-nucleic acid-based linkers.

[0162] In some cases, the polynucleotide molecule is a polynucleotide comprising a double, asymmetrical double, hairpin-shaped, or asymmetrical hairpin-shaped secondary structure having a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the polynucleotide molecule is a cyclic single-stranded polynucleotide having two or more loop structures and a stem comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the cyclic polynucleotide is processed in vivo or in vitro to produce an active polynucleotide molecule capable of mediating RNAi. In further cases, the polynucleic acid molecule also comprises a single-stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof (for example, such a polynucleic acid molecule does not need to be present in a polynucleic acid molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-stranded polynucleotide further comprises terminal phosphate groups such as 5'-phosphate or 5',3'-diphosphate.

[0163] In some cases, an asymmetric hairpin is a linear polynucleic acid molecule comprising an antisense region, a loop region containing nucleotides or non-nucleotides, and a sense region containing fewer nucleotides than the antisense region, having nucleotides sufficiently complementary to the base pair with the antisense region and forming a double helix 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 containing about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, an asymmetric hairpin polynucleic acid molecule also contains a chemically modified 5'-terminal phosphate group. In further cases, the loop region of an asymmetric hairpin polynucleic acid molecule contains nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0164] In some embodiments, an asymmetric double helix is ​​a polynucleic acid molecule having two distinct strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region, to the extent that it has nucleotides sufficiently complementary to the base pair with the antisense region and forms a double helix with the loop. For example, an asymmetric double polynucleic acid molecule comprises an antisense region having a length sufficient to mediate RNAi in a cellular system 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 bases refer to nucleotide base analogs that form base pairs with either natural DNA or RNA bases, where the distinction between the two is minimal. Non-exclusive examples of universal bases include C-phenyl, C-naphthyl, and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives known in the art, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0166] Synthesis of polynucleotide molecules In some embodiments, the polynucleic acid molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, polynucleic acid molecules are chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecule or the physical stability of the double helix formed between the polynucleic acid molecule and the target nucleic acid. Typical methods are described in U.S. Patents Nos. 5,142,047, 5,185,444, 5,889,136, 6,008,400, and 6,111,086, PCT Publication WO2009099942, or European Patent Application Publication No. 1579015.Further typical methods include "2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides" by Griffey et al., J.Med.Chem. 39(26): pp. 5100-5109 (1997); "Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar puckering." by Obika et al., Tetrahedron Letters 38(50): pp. 8735 (1997); "ENA oligonucleotides as therapeutics." by Koizumi, M., Current opinion in molecular therapeutics 8(2): pp. 144-149 (2006); and "Novel oligonucleotide analogues based on morpholino nucleoside subunits-antisense technologies: new chemical" by Abramova et al. Possibilities can be found in "Possibilities," Indian Journal of Chemistry 48B: pp. 1721-1726 (2009). Alternatively, polynucleotide molecules can be biologically generated using expression vectors in which the polynucleotide molecule is subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted polynucleotide molecule is in an antisense orientation toward the target polynucleotide molecule of interest).

[0167] In some embodiments, the polynucleic acid molecule is synthesized by a tandem synthesis method, in which both strands are separated by a cleavable linker that substantially cleaves the double helix to give separate fragments or strands that allow for their purification, and is synthesized as a single contiguous oligonucleotide fragment or strand.

[0168] In some cases, polynucleic acid molecules are also assembled from two distinct nucleic acid chains or fragments, one fragment containing the sense region and the other fragment containing the antisense region of the molecule.

[0169] For example, as further modification methods for incorporating sugar, base, and phosphate modifications, see the international publication WO92 / 07065 by Eckstein et al., Nature, 1990, pp. 344, 565-568 by Perrault et al., Science, 1991, pp. 253, 314-317 by Pieken et al., and Trends in Usman and Cedergren. Biochem.Sci.,1992,17,334~339pp, International Publication WO93 / 15187 by Usman et al., U.S. Patent No. 5,334,711 by Sproat, 1995, J.Biol.Chem.,270,25702pp by Beigelman et al., International Publication WO97 / 26270 by Beigelman et al., U.S. Patent No. 5,716,824 by Beigelman et al., U.S. Patent No. 5,627,053 by Usman et al., International Publication WO98 / 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.,39,1131pp by Karpeisky et al., 1998, Earnshaw and Gait References include Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. These publications describe general methods and strategies for determining the sites of introducing sugar, base, and / or phosphate modifications into nucleic acid molecules without catalytic modification.

[0170] In some cases, chemical modification of the nucleotide linkages of polynucleic acid molecules with phosphorothioates, phosphorodithioates, and / or 5'-methylphosphonates improves stability; however, excessive modification can lead to toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these nucleotide linkages is sometimes minimized. In such cases, reducing the concentration of these linkages reduces toxicity, increases the efficacy of these molecules, and improves specificity.

[0171] Polynucleic acid molecule conjugate In some embodiments, a polynucleic acid molecule (B) is further conjugated to a 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 B. In some examples, at least one polypeptide A is conjugated to at least two, three, four, five, six, seven, eight or more B.

[0172] In some cases, a polynucleic acid molecule is conjugated to a polypeptide (A) and optionally to 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 its terminal (e.g., 5' or 3' terminal) or via an internal site. In some cases, at least one C is conjugated to at least one polypeptide A directly or indirectly via at least one B. If done indirectly via at least one B, at least one C is conjugated at the same terminal as at least one polypeptide A on B, at the opposite terminal 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 further examples, at least one D is conjugated directly or indirectly to at least one polypeptide A, at least one B, or at least one C, optionally. If conjugation occurs directly to 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. If conjugated indirectly to 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, the binding portion A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is an antigen-binding fragment. In some examples, the antibody or its antigen-binding fragment includes a humanized antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a monoclonal antibody or its antigen-binding fragment, 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, monovalent Fab', bivalent Fab2, F(ab)'3 fragment, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide-stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or its antigen-binding fragment, bispecific antibody or its binding fragment, or chemically modified derivatives thereof.

[0175] In some embodiments, binding site A is a bispecific antibody or its antigen-binding fragment. In some examples, the bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, the bispecific antibody is a trifunctional antibody. In some examples, the trifunctional antibody is a full-length monoclonal antibody containing binding sites for two different antigens.

[0176] In some cases, a bispecific antibody is a bispecific mini-antibody. In some examples, a bispecific mini-antibody includes a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE). In some embodiments, a bispecific T cell engager is a fusion protein containing two single-strand variable fragments (scFv) in which the two scFv target two different antigen epitopes.

[0177] In some embodiments, the binding site A is a bispecific mini-antibody. In some examples, A is a bispecific Fab2. In some examples, A is a bispecific F(ab)'3 fragment. In some cases, A is a bispecific bis-scFv. In some cases, 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, the binding site A is a trispecific antibody. In some examples, the trispecific antibody contains an F(ab)'3 fragment or a triabody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, A is a triabody. In some embodiments, A is a trispecific antibody as described by Dimas et al., "Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells," Mol. Pharmaceutics, 12(9): pp. 3490-3501 (2015).

[0179] In some embodiments, binding portion A is an antibody or its antigen-binding fragment that recognizes a cell surface protein. In some examples, binding portion A is an antibody or its antigen-binding fragment that recognizes a cell surface protein on muscle cells. In some cases, binding portion A is an antibody or its antigen-binding fragment that recognizes a cell surface protein on skeletal muscle cells.

[0180] In some embodiments, typical 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 the transferrin receptor (TfR), preferably specifically to transferrin receptor 1 (TfR1), or more preferably specifically to human transferrin receptor 1 (TfR1) (or human CD71).

[0182] In some examples, the anti-transferrin receptor antibody includes a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and the HCDR3 sequence containing SEQ ID NO: 283.

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

[0184] [Table 1]

[0185] In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 281, an HCDR2 sequence containing sequence number 282, 284, or 285, and an HCDR3 sequence containing sequence number 283. In some examples, the VH region includes an HCDR1 sequence containing sequence number 281, an HCDR2 sequence containing sequence number 282, and an HCDR3 sequence containing sequence number 283. In some examples, the VH region includes an HCDR1 sequence containing sequence number 281, an HCDR2 sequence containing sequence number 284, and an HCDR3 sequence containing sequence number 283. In some examples, the VH region includes an HCDR1 sequence containing sequence number 281, an HCDR2 sequence containing sequence number 285, and an HCDR3 sequence containing sequence number 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, where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, and if present, is F.

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

[0188] [Table 2]

[0189] In some examples, the VL region includes the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence containing sequence numbers 287, 289, or 292, and the LCDR3 sequence containing sequence number 288 or 290, where X3 is selected from N or S.

[0190] In some examples, the VL region includes the LCDR1 sequence containing sequence number 286 or 291, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence containing sequence number 288 or 290, where X4 is selected from A or G and X5 is selected from D or E.

[0191] In some examples, the VL region includes the LCDR1 sequence containing sequence number 286 or 291, the LCDR2 sequence containing sequence number 287, 289, or 292, and the LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, it is F.

[0192] In some examples, the VL region includes the LCDR1 sequence containing sequence number 286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 may or may not be present, if present it is F.

[0193] In some examples, the VL region includes the LCDR1 sequence containing sequence number 286, the LCDR2 sequence containing sequence number 287, and the LCDR3 sequence containing sequence number 288.

[0194] In some examples, the VL region includes the LCDR1 sequence containing sequence number 286, the LCDR2 sequence containing sequence number 289, and the LCDR3 sequence containing sequence number 290.

[0195] In some examples, the VL region includes the LCDR1 sequence containing sequence number 291, the LCDR2 sequence containing sequence number 292, and the LCDR3 sequence containing sequence number 290.

[0196] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence comprising SEQ ID NO: 281, an HCDR2 sequence comprising 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, the VL region comprising an LCDR1 sequence comprising RTSENIYX3NLA, an LCDR2 sequence comprising AX4TNLAX5, and an LCDR3 sequence comprising 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, if present being F.

[0197] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 283; the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence including SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence including 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, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 283; the VL region comprising an LCDR1 sequence including SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence including 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, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 283; the VL region comprising an LCDR1 sequence including SEQ ID NO: 286 or 291, an LCDR2 sequence including SEQ ID NO: 287, 289, or 292, and an 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, the VH region comprising the HCDR1 sequence including SEQ ID NO: 281, the HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and the HCDR3 sequence including SEQ ID NO: 283; the VL region comprising the LCDR1 sequence including SEQ ID NO: 286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 may or may not be present, if present being F.

[0201] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 286, an LCDR2 sequence including SEQ ID NO: 287, and an LCDR3 sequence including SEQ ID NO: 288.

[0202] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 286, an LCDR2 sequence including SEQ ID NO: 289, and an LCDR3 sequence including SEQ ID NO: 290.

[0203] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 291, an LCDR2 sequence including SEQ ID NO: 292, and an LCDR3 sequence including SEQ ID NO: 290.

[0204] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 282, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence containing SEQ ID NO: 288 or 290, where X3 is selected from N or S.

[0205] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 282, and an HCDR3 sequence containing SEQ ID NO: 283; the VL region including an LCDR1 sequence containing SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 288 or 290, where X4 is selected from A or G and X5 is selected from D or E.

[0206] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 2, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region including an LCDR1 sequence containing SEQ ID NO: 286 or 291, an LCDR2 sequence containing SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, it is F.

[0207] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region includes the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 282, and the HCDR3 sequence containing SEQ ID NO: 283, the VL region includes the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, X5 is selected from D or E, and X6 is present or absent, if present it is F.

[0208] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 282, and the HCDR3 sequence containing SEQ ID NO: 283, and the VL region including the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence containing SEQ ID NO: 287, and the LCDR3 sequence containing SEQ ID NO: 288.

[0209] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 282, and the HCDR3 sequence containing SEQ ID NO: 283, and the VL region including the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence containing SEQ ID NO: 9, and the LCDR3 sequence containing SEQ ID NO: 290.

[0210] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 282, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 291, an LCDR2 sequence containing SEQ ID NO: 292, and an LCDR3 sequence containing SEQ ID NO: 290.

[0211] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 284, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence containing SEQ ID NO: 288 or 290, where X3 is selected from N or S.

[0212] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 284, and an HCDR3 sequence containing SEQ ID NO: 283; the VL region comprising an LCDR1 sequence containing SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 288 or 290, where X4 is selected from A or G and X5 is selected from D or E.

[0213] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 284, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region including an LCDR1 sequence containing SEQ ID NO: 286 or 291, an LCDR2 sequence containing SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, it is F.

[0214] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region includes the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 284, and the HCDR3 sequence containing SEQ ID NO: 283, the VL region includes the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, X5 is selected from D or E, and X6 is present or absent, if present it is F.

[0215] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 284, and the HCDR3 sequence containing SEQ ID NO: 283, and the VL region including the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence containing SEQ ID NO: 287, and the LCDR3 sequence containing SEQ ID NO: 288.

[0216] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 284, and the HCDR3 sequence containing SEQ ID NO: 283, and the VL region including the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence containing SEQ ID NO: 289, and the LCDR3 sequence containing SEQ ID NO: 290.

[0217] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 284, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 291, an LCDR2 sequence containing SEQ ID NO: 292, and an LCDR3 sequence containing SEQ ID NO: 290.

[0218] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 285, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 287, 289, or 29, and an LCDR3 sequence containing SEQ ID NO: 288 or 290, where X3 is selected from N or S.

[0219] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 285, and an HCDR3 sequence containing SEQ ID NO: 283; the VL region comprising an LCDR1 sequence containing SEQ ID NO: 286 or 291, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 288 or 290, where X4 is selected from A or G and X5 is selected from D or E.

[0220] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 285, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region including an LCDR1 sequence containing SEQ ID NO: 286 or 291, an LCDR2 sequence containing SEQ ID NO: 287, 289, or 292, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, it is F.

[0221] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region includes the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 285, and the HCDR3 sequence containing SEQ ID NO: 283, the VL region includes the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, X5 is selected from D or E, and X6 is present or absent, if present it is F.

[0222] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 285, and the HCDR3 sequence containing SEQ ID NO: 283, and the VL region including the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence containing SEQ ID NO: 287, and the LCDR3 sequence containing SEQ ID NO: 288.

[0223] In some examples, the anti-transferrin receptor antibody includes a VH region and a VL region, the VH region including the HCDR1 sequence containing SEQ ID NO: 281, the HCDR2 sequence containing SEQ ID NO: 285, and the HCDR3 sequence containing SEQ ID NO: 283, and the VL region including the LCDR1 sequence containing SEQ ID NO: 286, the LCDR2 sequence containing SEQ ID NO: 289, and the LCDR3 sequence containing SEQ ID NO: 290.

[0224] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 281, an HCDR2 sequence containing SEQ ID NO: 285, and an HCDR3 sequence containing SEQ ID NO: 283, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 291, an LCDR2 sequence containing SEQ ID NO: 292, and an LCDR3 sequence containing 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 contains approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 293-296, and the sequence of the VL region contains approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 298-301.

[0226] In some embodiments, the VH region includes sequences selected from sequence numbers 293-296 (Table 3), and the VL region includes sequences selected from sequence numbers 298-301 (Table 4). In Tables 3 and 4, underlined regions represent the corresponding CDR1, CDR2, or CDR3 sequences.

[0227] [Table 3]

[0228] [Table 4]

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

[0230] [Table 5]

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

[0232] In some cases, anti-transferrin receptor antibodies contain one or more mutations in the framework region, e.g., the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some cases, one or more mutations stabilize the antibody and / or increase its half-life. In some cases, one or more mutations modulate Fc receptor interactions to reduce or eliminate Fc effector functions such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further cases, one or more mutations modulate glycosylation.

[0233] In some embodiments, one or more mutations are located in the Fc region. In some examples, the Fc region contains mutations at residue positions L234, L235, or a combination thereof. In some examples, the mutations include L234 and L235. In some examples, the mutations include L234A and L235A. In some cases, the residue positions are related to IgG1.

[0234] In some cases, the Fc region contains mutations at residue positions L234, L235, D265, N297, K322, L328, or P329, or combinations thereof. In some cases, the mutations include L234 and L235 in combination with mutations at residue positions K322, L328, or P329. In some cases, the Fc region contains mutations at L234, L235, and K322. In some cases, the Fc region contains mutations at L234, L235, and L328. In some cases, the Fc region contains mutations at L234, L235, and P329. In some cases, the Fc region contains mutations at D265 and N297. In some cases, the residue positions are related to IgG1.

[0235] In some examples, the Fc region comprises L234A, L235A, D265A, N297G, K322G, L328R, or P329G, or combinations 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 related to IgG1.

[0236] In some examples, the Fc region comprises a mutation at residue positions L235, L236, D265, N297, K322, L328, or P329, or combinations of such 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 a mutation at residue position 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 related to IgG2.

[0237] In some embodiments, the Fc region comprises L235A, L236A, D265A, N297G, K322G, L328R, or P329G, or combinations 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 related to IgG2.

[0238] In some embodiments, the Fc region comprises mutations 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 mutations at L233 and L234. In some examples, the Fc region comprises mutations at L233 and L234 in combination with mutations at residue positions K321, L327, or P328. Optionally, the Fc region comprises mutations at L233, L234, and K321. Optionally, the Fc region comprises mutations at L233, L234, and L327. 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 the IgG1, IgG2, IgG3, or IgG fourth framework are contemplated. Optionally, mutations to residues corresponding to residues L233, L234, D264, N296, K321, L327, or P328 of SEQ ID NO: 23 in the IgG1, IgG2, or IgG fourth framework are also contemplated.

[0239] In some embodiments, the Fc region includes residue positions L233A, L234A, D264A, N296G, K321G, L327R, or P328G, 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 includes L233A and L234A. In some examples, the Fc region includes L233A and L234A in combination with K321G, L327R, or P328G. In some cases, the Fc region includes L233A, L234A, and K321G. In some cases, the Fc region includes L233A, L234A, and L327R. In some cases, the Fc region contains L233A, L234A, and K321G. In some cases, the Fc region contains L233A, L234A, and P328G. In some examples, the Fc region contains mutations in D264A and N296G.

[0240] In some embodiments, the constant region of human IgG is, for example, Natsume et al., 2008 Cancer Res, 68(10): pp. 3863-72; Idusogie et al., 2001 J Immunol, 166(4): pp. 2571-75; Moore et al., 2010 mAbs, 2(2): pp. 181-189; Lazar et al., 2006 PNAS, 103(11): pp. 4005-4010; Shields et al., 2001 JBC, 276(9): pp. 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): pp. 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: pp. 152-164; Alegre et al., 1992 J The amino acid modifications described in Immunol, 148: pp. 3461-3468, and Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): pp. 1-11, modify the antibody-dependent cytotoxin (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). The heavy chain (HC) may contain a sequence selected from Table 6. The light chain (LC) may contain a sequence selected from Table 7. 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 an improved serum half-life 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, the binding moiety A is non-specifically conjugated to the polynucleic acid molecule (B). In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) via a lysine residue or a cysteine residue in a non-site-specific manner. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) via a lysine residue (e.g., a lysine residue present in the binding moiety A) in a non-site-specific manner. Optionally, the binding moiety A is conjugated to the polynucleic acid molecule (B) via a cysteine residue (e.g., a cysteine residue present in the binding moiety A) in a non-site-specific manner.

[0251] In some embodiments, binding site A is conjugated to polynucleic acid molecule (B) in a site-specific manner. In some examples, binding site A is conjugated to polynucleic acid molecule (B) by a site-specific method via a lysine residue, a cysteine ​​residue, at the 5' end, at the 3' end, via a non-native amino acid, or via an enzymatically modified or enzymatically catalyzed residue. In some examples, binding site A is conjugated to polynucleic acid molecule (B) by a site-specific method via a lysine residue (e.g., a lysine residue present in binding site A). In some examples, binding site A is conjugated to polynucleic acid molecule (B) by a site-specific method via a cysteine ​​residue (e.g., a cysteine ​​residue present in binding site A). In some examples, binding site A is conjugated to polynucleic acid molecule (B) at the 5' end by a site-specific method. In some examples, binding site A is conjugated to polynucleic acid molecule (B) at the 3' end by a site-specific method. In some cases, binding site A is conjugated to the polynucleic acid molecule (B) via a non-native amino acid in a site-specific manner. In other cases, binding site A is conjugated to the polynucleic acid molecule (B) via an enzymatic modification or an enzymatic catalyst residue in a site-specific manner.

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

[0253] In some embodiments, the number of polynucleotide molecules (B) conjugated to binding site 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 polynucleotide molecule (B). In some examples, the DAR ratio between polynucleotide molecule (B) and binding site A is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more. In some examples, the DAR ratio between polynucleotide molecule (B) and binding site A is approximately 1 or more. In some examples, the DAR ratio between polynucleotide molecule (B) and binding site A is approximately 2 or more. In some examples, the DAR ratio between polynucleotide molecule (B) and binding site A is approximately 3 or more. In some examples, the DAR ratio between polynucleotide molecule (B) and binding site A is approximately 4 or more. In some examples, the DAR ratio between polynucleotide molecule (B) and binding site A is approximately 5 or more. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 6 or higher. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 7 or higher. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 8 or higher. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 9 or higher. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 10 or higher. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 11 or higher. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 12 or higher.

[0254] In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 1. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 2. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 3. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 4. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 5. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 6. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 7. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 8. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 9. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 10. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 11. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 12. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 13. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 14. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 15. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is approximately 16.

[0255] In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 1. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 2. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 4. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 6. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 8. In some cases, the DAR ratio between the polynucleotide molecule (B) and binding site A is 12.

[0256] In some cases, a conjugate containing a polynucleotide molecule (B) and binding site A exhibits improved activity compared to a conjugate containing the polynucleotide molecule (B) but without binding site A. In some cases, the improved activity results in enhanced biologically relevant functions in treating or preventing disease conditions, such as improved stability, affinity, binding, functional activity, and efficacy. In some cases, the disease condition is the result of one or more mutated exons in a gene. In some cases, a conjugate containing a polynucleotide molecule (B) and binding site A results in increased exon skipping of one or more mutated exons compared to a conjugate containing the polynucleotide molecule (B) but without binding site A. In some cases, 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 containing the polynucleotide molecule (B) but without binding site 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, either 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 the Fc receptor. In some examples, one or more modifications are described, for example, in International Publication WO97 / 34631, which discloses that amino acid residues are involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing modifications to the nucleic acid sequence underlying the amino acid sequence of the antibody or antigen-binding fragment are well known to those skilled in the art.

[0258] In some cases, the antigen-binding fragment further includes its derivatives and comprises a polypeptide sequence containing at least one CDR.

[0259] In some examples, the term “single-stranded” as used herein means that the first and second domains of a bispecific single-stranded construct are covalently linked in the form of a colinear amino acid sequence that can be encoded by a single nucleic acid molecule.

[0260] In some examples, a bispecific single-chain antibody construct relates to a construct containing binding domains derived from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some examples, the scFv contains VH and VL domains linked by a linker peptide. In some examples, the linker is of sufficient length and sequence to ensure that each of the first and second domains can independently maintain their specific binding specificity.

[0261] In some embodiments, binding or interaction as used herein defines binding / interaction between at least two antigen interaction sites. In some examples, an antigen interaction site defines a polypeptide motif that exhibits the ability to specifically interact with a specific antigen or group of specific antigens. In some cases, 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 a target molecule. For example, specific recognition relates to the specificity of an antibody molecule or its ability to discriminate a specific region of a target molecule. In further embodiments, the specific interaction between an antigen interaction site and its specific antigen results in the initiation of a signal, for example, due to the induction of a conformational change of the antigen, oligomerization of the antigen, etc. In further embodiments, binding is illustrated by the specificity of a "key-lock principle." For this reason, in some examples, antigen interaction sites and specific motifs in the amino acid sequence of an antigen bind to each other as a result of their primary, secondary, or tertiary structures, as well as secondary modifications of those structures. In such cases, the specific interaction between the antigen interaction site and its specific antigen similarly results in the site's simple binding to the antigen.

[0262] In some cases, specific interactions further refer to the reduction of cross-reactivity of an antibody or antigen-binding fragment, or the reduction of off-target effects. For example, an antibody or antigen-binding fragment that binds to a polypeptide / protein of interest but does not bind to any other polypeptides, or does not bind to any other polypeptides, is considered specific to the polypeptide / protein of interest. Examples of specific interactions between an antigen interaction site and a specific antigen include the specificity of a ligand to its receptor, e.g., the interaction between an antigenic determinant (epitope) and the antigenic binding site of an antibody.

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

[0264] In some cases, the binding site is a steroid. Typical steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, and carbohydrates, which are saturated, unsaturated, substituted, or a combination thereof. In some cases, the steroid is cholesterol. In some cases, the binding site is cholesterol. In some cases, cholesterol is conjugated to a polynucleic acid molecule by one or more of the conjugation chemistry described herein. In some cases, cholesterol is conjugated to a polynucleic acid molecule by native ligation chemistry. In some cases, cholesterol is conjugated to a polynucleic acid molecule by lysine conjugation.

[0265] In some examples, the binding site is a polymer, including but not limited to polynucleotide aptamers that bind to specific surface markers on cells. In this example, the binding site is a polynucleotide that does not hybridize to the target gene or mRNA, but instead is selectively able to bind to the cell surface marker, similar to antibody binding to the cell surface marker's specific epitope.

[0266] In some cases, the binding site is a peptide. In some cases, the peptide contains between approximately 1 and 3 kDa. In some cases, the peptide contains between approximately 1.2 and 2.8 kDa, between approximately 1.5 and 2.5 kDa, or between approximately 1.5 and 2 kDa. In some examples, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some examples, the binding site is a bicyclic peptide (e.g., bicycles in Bicycle Therapeutics).

[0267] In further cases, the binding site is a small molecule. In some examples, the small molecule is an antibody-recruiting small molecule. In some cases, 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 cell surface receptors. For example, in some cases, the target-binding end, which includes a glutamate urea compound, enables interaction with PSMA, thereby improving antibody interaction with cells expressing PSMA. In some cases, the binding site is a small molecule described in "A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules" by Zhang et al., J Am Chem Soc. 132(36): pp. 12711-12716 (2010), or "Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease" by McEnaney et al., ACS Chem Biol. 7(7): pp. 1139-1151 (2012).

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

[0269] In some cases, antibodies or their antigen-binding fragments are expressed by recombination, and the nucleic acids encoding the antibodies or their antigen-binding fragments are assembled from chemically synthesized oligonucleotides (e.g., those described by Kutmeier et al. in "BioTechniques 17:242" in 1994), requiring the synthesis of duplicate oligonucleotides containing multiple portions of the antibody-coding sequence, annealing and ligation of these oligonucleotides, and subsequent PCR amplification of the ligated oligonucleotides.

[0270] Alternatively, the nucleic acid molecules encoding antibodies can be optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing immunoglobulin) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence.

[0271] In some cases, the antibody or its antigen-binding fragment is optionally generated by immunizing an animal such as a rabbit to produce a polyclonal antibody, or more preferably by generating a monoclonal antibody as described, for example, by Kohler and Milstein (1975, Nature 256: pp. 495-497), by Kozbor et al. (1983, Immunology Today 4: p. 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 an antibody can be optionally obtained by screening a Fab expression library for clones of Fab fragments that bind to a specific antigen (e.g., as described by Huse et al. in Science 246:1275-1281, 1989) or by screening an antibody library (e.g., see Clackson et al. in Nature 352:624, 1991, and Hane et al. in Proc.Natl.Acad.Sci.USA 94:4937, 1997).

[0272] In some embodiments, techniques developed to produce "chimeric antibodies" by splicing genes from appropriately bioactive human antibody molecules with genes from appropriately antigen-specific mouse antibody molecules are used (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81: pp. 851-855; Neuberger et al., 1984, Nature 312: pp. 604-608; Takeda et al., 1985, Nature 314: pp. 452-454). Chimeric antibodies are molecules in which various parts originate from different animal species, such as humanized antibodies, which have a variable region derived from a mouse monoclonal antibody and a constant region of human immunoglobulin.

[0273] In some embodiments, techniques described for the production of single-chain antibodies (U.S. Patent No. 4,694,778, Science 242:423-42 by Bird, Proc.Natl.Acad.Sci.USA 85:5879-5883 by Huston et al., and Nature 334:544-54 by Ward et al., 1989) are suitable for producing single-chain antibodies. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv site via amino acid crosslinking, resulting in a single-chain polypeptide. Techniques for assembling functional Fv fragments in E. coli are also used optionally (Science 242:1038-1041 by Skerra et al., 1988).

[0274] In some embodiments, an expression vector containing the antibody nucleotide sequence, or the antibody nucleotide sequence itself, 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 for antibody production. In certain embodiments, antibody expression is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0275] In some embodiments, various host expression vector systems are used to express the antibodies or antigen-binding fragments described herein. Such host expression systems represent not only vehicles that produce and then purify the antibody coding sequence, but also cells that express the antibody or antigen-binding fragment in situ when transformed or transfected with a suitable nucleotide coding sequence. 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 coding sequence of the antibody or antigen-binding fragment; and yeasts (e.g., Saccharomyces picia) transformed with recombinant yeast expression vectors containing the coding sequence of the antibody or antigen-binding fragment. Examples include, but are not limited to, Pichia); insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence of an antibody or its antigen-binding fragment; plant cell lines 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 of an antibody or its antigen-binding fragment; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) containing recombinant expression constructs derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a mammalian virus (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 manipulated by arbitrary selection. Host cells are transformed with DNA controlled by appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers, rather than using an expression vector containing the viral replication origin. After introduction of the exogenous DNA, the manipulated cells are grown in concentrated medium for 1-2 days, then switched to selective medium. The selectable markers in the recombinant plasmid confer resistance to selection, allowing the cells to grow to form a foci that stably integrates the plasmid into its chromosomes and expands into a cell line for cloning. This method can be advantageously used to manipulate cell lines expressing antibodies or their antigen-binding fragments.

[0277] Many 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 adopted into tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance is also associated with the following genes: DHFR, which imparts resistance to methotrexate (Proc.Natl.Acad.Sci.USA 77:357 by Wigler et al., 1980; Proc.Natl.Acad.Sci.USA 78:1527 by O'Hare et al., 1981); GPT, which imparts resistance to mycophenolate (Proc.Natl.Acad.Sci.USA 78:2072 by Mulligan and Berg, 1981); and neo, which imparts resistance to aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Biotherapy by Wu and Wu, 1991). It is used as a selection criterion for hygromycin-resistant hygro (Santerre et al., Gene 30:147, 1984).Generally known methods in the field of recombinant DNA techniques that are available for use are described in Ausubel et al. (eds.), *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 1993; Kriegler, *Gene Transfer and Expression*, A Laboratory Manual, Stockton Press, NY, 1990; Chapters 12 and 13 of *Current Protocols in Human Genetics*, John Wiley & Sons, NY, 1994, by Dracopoli et al. (eds.); and Colberre-Garapin et al., *J.Mol.Biol.150:p.1*, 1981.

[0278] In some cases, antibody expression levels increase with vector amplification (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) for commentary). If the marker in the antibody-expressing vector system is amplified, the number of copies of the marker gene increases due to an increase in the level of the inhibitor present in the host cell culture. Since the amplified site is associated with the antibody's nucleotide sequence, antibody production also increases (see Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0279] In some cases, any method known in the field for the purification or analysis of antibodies or antibody conjugates is used, for example, by chromatography (e.g., by ion exchange, affinity, specifically affinity of protein A to specific antigens, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. Typical chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and restricted protein liquid chromatography.

[0280] Conjugation Chemistry In some embodiments, the polynucleic acid molecule (B) is conjugated to a binding site. In some embodiments, the polynucleic acid molecule B is conjugated to a binding site in the formula AXB (where X is a linker that conjugates A and B). In some examples, the binding site includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, hydrocarbons, polymers such as polyethylene glycol and polypropylene glycol, as well as all analogs or derivatives of these classes of substances. Further examples of binding sites also include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and steroids such as polysaccharides. In some examples, the binding site is an antibody or its antigen-binding fragment. In some examples, the polynucleic acid molecule is further conjugated to a polymer and, optionally, to an endosomal soluble site.

[0281] In some embodiments, polynucleic acid molecules are conjugated to the binding site by a chemical ligation process. In some examples, polynucleic acid molecules are conjugated to the binding site by native ligation. In some cases, the conjugation is as described in "Synthesis of proteins by native chemical ligation" by Dawson et al., Science 1994, pp. 266, 776-779; "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives" by Dawson et al., J.Am.Chem.Soc. 1997, pp. 119, 4325-4329; "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology" by Hackeng et al., Proc.Natl.Acad.Sci.USA 1999, pp. 96, 10068-10073; or "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol" by Wu et al., Angew.Chem.Int.Ed. 2006, pp. 45, 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, polynucleic acid molecules are site-specifically or non-specifically conjugated to the binding site via native ligation chemistry.

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

[0283] In some cases, polynucleic acid molecules are conjugated to a binding site by a site-specific method that utilizes unnatural amino acids incorporated into the binding site. In some cases, the unnatural amino acid includes p-acetylphenylalanine (pAcPhe). In some cases, the keto group of pAcPhe is selectively joined to the alkoxyamine-derived conjugation site 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 cases, polynucleic acid molecules are conjugated to the binding site by a site-specific method utilizing an enzyme-catalyzed process. In some cases, the site-specific method utilizes SMARTag® technology (Catalent, Inc.). In some cases, SMARTag® technology involves an oxidation process in the presence of an aldehyde tag, followed by the conjugation of formylglycine (FGly) to alkylhydrazine-functionalized polynucleic acids via hydrazino-Pictet-Spengler (HIPS) ligation, which includes the generation of FGly residues from cysteine ​​by formylglycinase (FGE) (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), and Agarwal et al., "A Pictet-Spengler ligation for protein chemical modification," PNAS 110(1):46-51 (2013)).

[0285] In some cases, the enzyme-catalyzed process involves microbial transglutaminase (mTG). In some instances, polynucleic acid molecules are conjugated to the binding site using the microbial transglutaminase-catalyzed process. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and the primary amine of the functionalized polynucleic acid molecule. In some cases, mTG is produced from Streptomyces mobarensis (see Strop et al., "Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates," Chemistry and Biology 20(2) pp. 161-167 (2013)).

[0286] In some cases, polynucleic acid molecules are conjugated to the binding site by a method described in International Publication WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0287] In some cases, polynucleic acid molecules are conjugated to the binding portion by the method described in U.S. Patent Applications No. 2015 / 0105539 and No. 2015 / 0105540.

[0288] Polymer conjugation portion In some embodiments, polymer moiety C is further conjugated to a polynucleic acid molecule, a binding moiety, or a combination thereof as described herein. In some examples, polymer moiety C is conjugated to a polynucleic acid molecule in the formula A-X1-B-X2-C (where X1 and X2 are two linkers conjugating A and B, and B and C, respectively). In some cases, polymer moiety C is conjugated to a binding moiety. In other cases, polymer moiety C is conjugated to a polynucleic acid molecule-binding moiety molecule. In further cases, 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 a cross-bonded network of two-dimensional or three-dimensional monomers. In some examples, polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C includes, but is not limited to, alpha- or omega-dihydroxypolyethylene glycol, biodegradable lactone 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, or mixtures thereof. As used herein, a mixture refers to the use of different polymers in relation to a block copolymer, as well as within the same compound. 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 moiety C contains polyalkylene oxide. In some examples, polymer moiety C contains PEG. In some examples, polymer moiety C contains polyethyleneimide (PEI) or hydroxyethyl starch (HES).

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

[0291] In some embodiments, polyalkylene oxides (e.g., PEG) are polydisperse or monodisperse compounds. In some examples, polydisperse materials include a dispersion distribution of materials of different molecular weights, characterized by average weight (weight-average) size and degree of dispersion. In some examples, monodisperse PEG contains molecules of size 1. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weight represents the average molecular weight of the polyalkylene oxide, e.g., PEG.

[0292] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is approximately 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 100,000Da.

[0293] In some embodiments, C is a polyalkylene oxide (e.g., PEG), and the values ​​are approximately 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, 2 It has 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 approximately 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, It has molecular weights 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 approximately 200 Da. In some examples, the molecular weight of C is approximately 300 Da. In some examples, the molecular weight of C is approximately 400 Da. In some examples, the molecular weight of C is approximately 500 Da. In some examples, the molecular weight of C is approximately 600 Da. In some examples, the molecular weight of C is approximately 700 Da. In some cases, the molecular weight of C is approximately 800 Da. In some cases, the molecular weight of C is approximately 900 Da. In some cases, the molecular weight of C is approximately 1000 Da. In some cases, the molecular weight of C is approximately 1100 Da. In some cases, the molecular weight of C is approximately 1200 Da. In some cases, the molecular weight of C is approximately 1300 Da. In some cases, the molecular weight of C is approximately 1400 Da. In some cases, the molecular weight of C is approximately 1450 Da.In some cases, the molecular weight of C is approximately 1500 Da. In some cases, the molecular weight of C is approximately 1600 Da. In some cases, the molecular weight of C is approximately 1700 Da. In some cases, the molecular weight of C is approximately 1800 Da. In some cases, the molecular weight of C is approximately 1900 Da. In some cases, the molecular weight of C is approximately 2000 Da. In some cases, the molecular weight of C is approximately 2100 Da. In some cases, the molecular weight of C is approximately 2200 Da. In some cases, the molecular weight of C is approximately 2300 Da. In some cases, the molecular weight of C is approximately 2400 Da. In some cases, the molecular weight of C is approximately 2500 Da. In some cases, the molecular weight of C is approximately 2600 Da. In some cases, the molecular weight of C is approximately 2700 Da. In some cases, the molecular weight of C is approximately 2800 Da. In some cases, the molecular weight of C is approximately 2900 Da. In some cases, the molecular weight of C is approximately 3000 Da. In some cases, the molecular weight of C is approximately 3250 Da. In some cases, the molecular weight of C is approximately 3350 Da. In some cases, the molecular weight of C is approximately 3500 Da. In some cases, the molecular weight of C is approximately 3750 Da. In some cases, the molecular weight of C is approximately 4000 Da. In some cases, the molecular weight of C is approximately 4250 Da. In some cases, the molecular weight of C is approximately 4500 Da. In some cases, the molecular weight of C is approximately 4600 Da. In some cases, the molecular weight of C is approximately 4750 Da. In some cases, the molecular weight of C is approximately 5000 Da. In some cases, the molecular weight of C is approximately 5500 Da. In some cases, the molecular weight of C is approximately 6000 Da. In some cases, the molecular weight of C is approximately 6500 Da. In some cases, the molecular weight of C is approximately 7000 Da. In some cases, the molecular weight of C is approximately 7500 Da. In some cases, the molecular weight of C is approximately 8000 Da. In some cases, the molecular weight of C is approximately 10,000 Da. In some cases, the molecular weight of C is approximately 12,000 Da. In some cases, the molecular weight of C is approximately 20,000 Da. In some cases, the molecular weight of C is approximately 35,000 Da. In some cases, the molecular weight of C is approximately 40,000 Da. In some cases, the molecular weight of C is approximately 50,000 Da. In some cases, the molecular weight of C is approximately 60,000 Da.In some cases, the molecular weight of C is approximately 100,000 Da.

[0294] In some embodiments, polyalkylene oxides (e.g., PEG) contain discrete ethylene oxide units (e.g., 4 to about 48 ethylene oxide units). In some examples, polyalkylene oxides containing discrete ethylene oxide units are linear. In other cases, polyalkylene oxides containing discrete ethylene oxide units are branched.

[0295] In some examples, polymer portion C is a polyalkylene oxide (e.g., PEG) containing discrete ethylene oxide units. In some cases, polymer portion C contains between about 4 and about 48 ethylene oxide units. In some cases, polymer portion C contains 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 cases, polymer portion C is a discrete PEG containing, for example, about 4 to about 48 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units. In some cases, polymer portion C is a 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. In some cases, polymer portion C is a discrete PEG containing, for example, about 13 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 14 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 15 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 16 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 17 ethylene oxide units. In some cases, 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. In some cases, polymer portion C is a discrete PEG containing, for example, about 37 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 38 ethylene oxide units.In some cases, polymer portion C is a discrete PEG containing, for example, about 39 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 40 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 41 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 42 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 43 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 44 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 45 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 46 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 47 ethylene oxide units. In some cases, the polymer portion C is, for example, a discrete PEG containing about 48 ethylene oxide units.

[0297] In some cases, polymer portion C is dPEG® (Quanta Biodesign Ltd).

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

[0299] [ka] It is expressed as follows.

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

[0301] In some cases, cMAP is conjugated to the PEG portion to produce cMAP-PEG copolymers, mPEG-cMAP-PEGm triblock polymers, or cMAP-PEG-cMAP triblock polymers. In some cases, the PEG portion is in the range of approximately 500 Da to approximately 50,000 Da. In some cases, the PEG portion is in the range of approximately 500 Da to approximately 1000 Da, over 1000 Da to approximately 5000 Da, over 5000 Da to approximately 10,000 Da, over 10,000 Da to approximately 25,000 Da, over 25,000 Da to approximately 50,000 Da, or any combination of two or more of these ranges.

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

[0303] In some embodiments, polymer portion C is conjugated with a polynucleic acid molecule, a binding portion, and optionally an endosomal soluble portion as described above.

[0304] Endosomal lytic portion or cell membrane permeable portion 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 endosomal soluble moiety and / or a cell membrane permeable moiety. The endosomal soluble moiety is, in some cases, a cellular compartmental release component, such as a compound that can be released from any of the cellular compartments known in the art, including endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other cellular compartments. The endosomal soluble moiety may comprise an endosomal soluble polypeptide, an endosomal soluble polymer, an endosomal soluble lipid, or an endosomal soluble small molecule. The endosomal soluble moiety may comprise an endosomal soluble polypeptide. In other cases, the endosomal soluble moiety comprises an endosomal soluble polymer. The cell membrane permeable moiety may comprise a cell permeable peptide (CPP). In other cases, the cell membrane permeable moiety comprises a cell permeable lipid. In other cases, the cell membrane penetration portion contains cell-penetrating small molecules.

[0305] Endosomal-soluble polypeptides and cell membrane-penetrating polypeptides In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further conjugated with an endosomal soluble polypeptide. In some cases, the endosomal soluble polypeptide is a pH-dependent membrane-active peptide. In some cases, the endosomal soluble polypeptide is an amphiphilic polypeptide. Further cases, the endosomal soluble polypeptide is a peptide mimetic. In some examples, the endosomal soluble polypeptide comprises INF, melittin, meucin, or their corresponding derivatives. In some examples, the endosomal soluble polypeptide comprises INF or its derivatives. In other cases, the endosomal soluble polypeptide comprises melittin or its derivatives. Further cases, the endosomal soluble polypeptide comprises meucin or its derivatives.

[0306] In some cases, INF7 is a 24-residue polypeptide whose sequence includes CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 331) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 332). In some cases, INF7 or its derivatives include the following sequences: 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 cases, melittin is a 26-residue polypeptide whose sequence includes CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 336) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 337). In some examples, melittin includes the polypeptide sequence described in U.S. Patent No. 8,501,930.

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

[0309] In some examples, the endosomal soluble polypeptide comprises a polypeptide having a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity with INF7 or its derivatives, melittin or its derivatives, or meusin or its derivatives. In some examples, the endosomal soluble portion comprises INF7 or its derivatives, melittin or its derivatives, or meusin or its derivatives.

[0310] In some cases, the endosomal soluble portion is INF or a derivative thereof. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 331-335. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 331. In some cases, the endosomal soluble portion contains polypeptides having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 331 may be included. 332-335 may be included. 331 may be included. 431 may be included. 531 may be included. 631 may be included. 731 may be included.

[0311] In some cases, the endosomal soluble portion is melittin or a derivative thereof. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 336 or 337. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 337. In some cases, the endosomal soluble portion contains SEQ ID NO: 286. In some cases, the endosomal soluble portion contains SEQ ID NO: 337. In some cases, the endosomal soluble portion consists of SEQ ID NO: 336. In some cases, the endosomal soluble portion consists of SEQ ID NO: 337.

[0312] In some cases, the endosomal soluble portion is meusin or a derivative thereof. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 338 or 339. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 339. In some cases, the endosomal soluble portion contains SEQ ID NO: 338. In some cases, the endosomal soluble portion contains SEQ ID NO: 339. In some cases, the endosomal soluble portion consists of SEQ ID NO: 338. In some cases, the endosomal soluble portion consists of SEQ ID NO: 339. In some examples, the endosomal soluble portion contains sequences illustrated in Table 8.

[0313] [Table 8-1]

[0314] [Table 8-2]

[0315] In some cases, the endosomal lysate portion contains Bcl-2 and / or Bcl- XLIt contains a Bak BH3 polypeptide that induces apoptosis by antagonizing suppressor target genes such as [specific examples of suppressor targets]. In some cases, the endosomal lytic portion contains a Bak BH3 polypeptide described by Albarran et al., "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive & Functional Polymers 71:261-265 (2011).

[0316] In some cases, the endosomal lytic portion includes polypeptides described in PCT Publication WO2013 / 166155 or WO2015 / 069587 (e.g., cell-penetrating polypeptides).

[0317] Endosomal soluble lipids In some embodiments, the endosomal soluble portion is a lipid (e.g., a fusion lipid). In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further conjugated with an endosomal soluble lipid (e.g., a fusion lipid). Typical fusion lipids include 1,2-dioleoyl-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)methaneamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethaneamine (XTC).

[0318] In some cases, the endosomal soluble portion is a lipid (e.g., a fusion lipid) as described in International Publication WO09 / 126,933.

[0319] Endosomal soluble small molecules In some embodiments, the endosomal soluble portion is a small molecule. In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further conjugated with an endosomal soluble small molecule. Typical small molecules suitable as the endosomal soluble portion include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), amopyroquines, primaquine, mefloquine, nivaquines, halophanthrin, quinone imines, or combinations thereof. In some cases, the quinoline endosome-soluble moiety is 7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline (chloroquine), 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline (hydroxychloroquine), 7-fluoro-4-(4-diethylamino-1-methylbutylamino)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-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline, 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, hydroxychloroquine phosphate 7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline ((desmethylhydroxychloroquine) 7-Fluoro-4-(4-ethyl-(2-H (Droxyethyl)-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 (Xyethyl)-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] Examples include, but are not limited to, butyl-amino]-6-methoxyquinoline, 3,4-dihydro-1-(2H)-quinoline carboxyaldehyde, 1,1'-pentamethylenediquinoleinium diiodide, 8-quinolinol sulfate, and their amino, aldehyde, carboxyl, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives, or analogs thereof. In some examples, the endosomal soluble portion is a small molecule described by Naisbitt et al. (1997, J Pharmacol Exp Therapy 280: pp. 884-893) and U.S. Patent No. 5,736,557.

[0320] Cell-penetrating polypeptide (CPP) In some embodiments, the cell-penetrating polypeptide comprises a short, positively charged peptide having 5 to 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 or combination thereof listed in Table 9.

[0321] [Table 9]

[0322] Linker In some embodiments, the linkers described herein are either severable or inseverable linkers. In some examples, the linker is a severable linker. In other examples, the linker is an inseverable linker.

[0323] In some cases, the linker is a nonpolymer linker. A nonpolymer linker refers to a linker that does not contain repeating units of monomers produced by the polymerization process. Typical nonpolymer linkers include, but are not limited to, C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-sacrificing linkers, maleimide linkers, or combinations thereof. In some cases, a nonpolymer linker includes C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-sacrificing linkers, maleimide linkers, or combinations thereof. In further cases, a nonpolymer linker does not contain two or more linkers of the same type, e.g., two or more homobifunctional crosslinkers or two or more peptide linkers. In further cases, the non-polymeric linker optionally includes one or more reactive functional groups.

[0324] In some cases, the non-polymer linker does not include the polymers described above. In some cases, the non-polymer linker does not include the polymer encompassed by polymer portion C. In some cases, the non-polymer linker does not include polyalkylene oxides (e.g., PEG). In some cases, the non-polymer linker does not include PEG.

[0325] In some cases, the linker includes homobifunctional linkers. Typical homobifunctional linkers include Lomant reagent dithiobis(succinimidylpropionate) DSP, 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS), disuccinimidyltetralate (DST), disulfosuccinimidyltetralate (sulfoDST), and ethylene-g Lycobis(succinimidyl succinate (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamide) Examples include, but are not limited to, tan (DPDPB), bismaleimide hexane (BMH), aryl halide-containing compounds (DFDNB), e.g., 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 cases, the linker includes heterobifunctional linkers. Typical 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), and sulfosuccinimidyl-6-[α-methyl-α -(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimid ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimid 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)succinimidide ester (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimidide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexa Noates (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-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, for example,4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), amine-reactive and photoreactive crosslinkers, e.g., N-hydroxysuccinimidyl-4-azidosalicylic acid (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamide)hexanoate ( Sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamide)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-nitrobenzamide)-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 Synimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyrubate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers, e.g., 1-(ρ-azidosalicylamide)-4-(iodoacetamido)butane (AsIB),Examples include, but are not limited to, N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive crosslinkers, e.g., ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, e.g., 4-(ρ-azidosalicylamide)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, e.g., ρ-azidophenylglyoxal (APG).

[0327] In some cases, the linker contains a reactive functional group. In some instances, the reactive functional group contains a nucleophile that reacts to an electrophile present on the bond. Typical electrophiles 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 nucleophiles include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0328] In some cases, the linker contains a maleimide group. In some cases, the maleimide group is also called a maleimide spacer. In some cases, the maleimide group further contains caproic acid to form maleimidocaproyl (mc). In some cases, the linker contains maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other cases, the maleimide group contains a maleimidomethyl group such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) as 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 prevent the maleimide from undergoing elimination via a retro-Michael reaction by incorporating a basic amino group adjacent to the maleimide to provide an intramolecular catalyst for thiosuccinimide ring hydrolysis. In some examples, the self-stabilizing maleimide is the maleimide group described by Lyon et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10): pp. 1059-1062 (2014). In some examples, the linker contains a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0330] In some examples, the linker contains a peptide moiety. In some examples, the peptide moiety contains at least 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety contains up to 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety contains approximately 2, approximately 3, approximately 4, approximately 5, or approximately 6 amino acid residues. In some examples, the peptide moiety is cleavable (e.g., enzymatically or chemically). In some examples, the peptide moiety is incleavable. In some examples, the peptide portion includes 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 cases, the linker contains peptide moieties 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). In some cases, the linker contains Val-Cit. In some cases, the linker is Val-Cit.

[0331] In some embodiments, the linker includes a benzoic acid group or a derivative thereof. In some examples, the benzoic acid group or a derivative thereof includes para-aminobenzoic acid (PABA). In some examples, the benzoic acid group or a derivative thereof includes gamma-aminobutyric acid (GABA).

[0332] In some embodiments, the linker comprises one or more of the maleimide group, the peptide moiety, and / or benzoic acid groups in any combination. In some embodiments, the linker comprises a combination of the maleimide group, the peptide moiety, and / or benzoic acid groups. 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 an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0333] In some embodiments, the linker is a self-sacrificing linker or a self-excreting linker. In some cases, the linker is a self-sacrificing linker. In other cases, the linker is a self-excreting linker (e.g., a cyclizing self-excreting linker). In some examples, the linker includes the linkers 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 contains a branched, polyfunctional 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 contains a PAMAM dendrimer.

[0335] In some embodiments, the linker is a traceless linker or a linker that leaves no linker moiety (e.g., atomic group or linker group) behind the binding site A, polynucleotide B, polymer C, or endosomal soluble site D after cleavage. Typical traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. 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): pp. 2493-2497 (2013). In some cases, the linker is a traceless linker as described in "Traceless solid-phase organic synthesis" by Blaney et al., Chem. Rev. 102: pp. 2607-2024 (2002). In some cases, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0336] In some cases, the linker is linked to U.S. Patent Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688, U.S. Patent Publication Nos. 2014 / 0127239, 2013 / 028919, and 2014 / 28697 Linkers described in Specification No. 0, Specification No. 2013 / 0309256, Specification No. 2015 / 037360, or Specification No. 2014 / 0294851, or PCT Publications WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0337] In some embodiments, X1 and X2 are independently bonds or nonpolymer linkers. In some examples, X1 and X2 are independently bonds. In some cases, X1 and X2 are independently nonpolymer linkers.

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

[0339] In some examples, X2 is a bond or a nonpolymer linker. In some examples, X2 is a bond. In other cases, X2 is a linker. In further cases, X2 is a nonpolymer linker. In some embodiments, X1 is a C1-C6 alkyl group. In some examples, X2 comprises the homobifunctional linker or heterobifunctional linker described above. In some examples, X2 is the homobifunctional linker described above. In some examples, X2 is the heterobifunctional linker described above. In some examples, X2 comprises a maleimide group such as maleimidocaproyl (mc) or a 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 further examples, X2 comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In further examples, X2 comprises an mc group. In further examples, X2 comprises an mc-val-cit group. In further examples, X2 contains a val-cit-PABA group. In even further examples, X2 contains an 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 muscles. In some cases, the loss of muscle mass, and / or the progressive weakening and degeneration of muscles, results from a high rate of protein breakdown, a low rate of protein synthesis, or a combination of both. In some cases, the high rate of muscle protein breakdown is due to the catabolism of muscle proteins (i.e., the breakdown of muscle proteins to use amino acids as substrates for gluconeogenesis).

[0341] In one embodiment, muscular atrophy refers to a substantial loss of muscle strength. A substantial loss of muscle strength means a decrease in the strength of the affected, injured, or unused muscle tissue of a subject compared to the same muscle tissue of a control subject. In one embodiment, a substantial loss of muscle strength is a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the same muscle tissue of a control subject. In another embodiment, a substantial loss of muscle strength means a decrease in the strength of the unused muscle tissue compared to the muscle strength of the same muscle tissue of the same subject before the period of unavailability. In one embodiment, a substantial loss of muscle strength is a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the muscle strength of the same muscle tissue of the same subject before the period of unavailability.

[0342] In another embodiment, muscular atrophy refers to a substantial loss of muscle mass. A substantial loss of muscle mass means a decrease in the muscle mass of the affected, damaged, or unused muscle tissue of a subject compared to the same muscle tissue of a control subject. In one embodiment, substantial loss of muscle mass is a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the same muscle tissue of a control subject. In another embodiment, substantial loss of muscle mass means a decrease in the muscle mass of unused muscle tissue compared to the muscle mass of the same muscle tissue of the same subject before the period of unavailability. In one embodiment, substantial loss of muscle tissue is a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the muscle mass of the same muscle tissue of the same subject before the period of unavailability. Muscle mass is optionally measured by evaluating the cross-sectional area of ​​the muscle using methods such as magnetic resonance imaging (e.g., muscle mass / cross-sectional area (CSA) MRI).

[0343] In some embodiments, this specification describes a method for treating a target muscular atrophy, comprising the steps of obtaining a polynucleic acid molecule described herein and administering a therapeutically effective dose of the polynucleic acid molecule or a polynucleic acid molecule conjugate described herein to a target in order to reduce the amount of human DUX4 mRNA transcript. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, the antisense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to a sequence selected from SEQ ID NOs: 412-420 or SEQ ID NOs: 430-438. In some embodiments, the polynucleic acid molecule comprises a sense strand and / or an antisense strand, the antisense being 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, the sense strand being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to a sequence selected from SEQ ID NOs: 142, 146, 196, or 201-206.

[0344] In some cases, muscular atrophy is associated with facioscapulohumeral muscular dystrophy (FSHD). The polynucleic acid moiety mediates RNA interference with human DUX4 to modulate the muscular atrophy in question. In some embodiments, the expression of one or more marker genes affected by DUX4 expression is also modified or regulated (e.g., reduced) by the reduction in human DUX4 expression. Examples of 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, the 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, this specification describes a method for treating a target muscular atrophy, comprising the steps of: obtaining an siRNA-antibody conjugate described herein; administering the siRNA-antibody conjugate described herein to a target in a therapeutically effective dose; and reducing the level of the target human DUX4 mRNA transcript. In some examples, the muscular atrophy is associated with FSHD. The siRNA-antibody conjugate mediates RNA interference against human DUX4 mRNA to treat the target muscular atrophy, and the methods include administering the siRNA-antibody conjugate described herein to a target in a therapeutically effective dose and reducing the level of the target human DUX4 mRNA transcript.

[0346] In some embodiments, this specification describes a method for treating a target muscular atrophy, comprising the steps of: obtaining a DUX4 siRNA-antibody conjugate (DUX4 siRNA-conjugate or DUX4-AOC) as described herein; administering the DUX4 siRNA-antibody conjugate as described herein to a subject in a therapeutically effective dose; and reducing the level of the subject's human DUX4 mRNA transcript. In some examples, the muscular atrophy is associated with FSHD. The DUX4 siRNA-antibody conjugate mediates RNA interference against human DUX4 mRNA to treat the target muscular atrophy, and the process involves administering the DUX4 siRNA-antibody conjugate as described herein to a subject in a therapeutically effective dose and reducing the level of the subject's human DUX4 mRNA transcript.

[0347] In some embodiments, this specification describes a method for treating a target FSHD, comprising the steps of: obtaining a DUX4 siRNA-antibody conjugate described herein (DUX4 siRNA-conjugate or DUX4-AOC); administering the DUX4 siRNA-antibody conjugate described herein to a subject in a therapeutically effective dose; and reducing the level of the subject's human DUX4 mRNA transcript. 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 the target FSHD, and the process involves administering the DUX4 siRNA-antibody conjugate described herein to a subject in a therapeutically effective dose and reducing the level of the subject's human DUX4 mRNA transcript. 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 the reduction in human DUX4 expression levels. Examples of DUX4 biomarker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD.

[0348] In some embodiments, this specification describes a method for alleviating symptoms of a subject with FSHD, comprising the steps of: obtaining a DUX4 siRNA-antibody conjugate (DUX4-siRNA-conjugate or DUX4-AOC) as described herein; and administering the siRNA conjugate as described herein to the subject in a therapeutically effective dose by reducing the level of the mRNA transcript of the subject's human DUX4. In some examples, the FSHD is FSHD type 1 (FSHD1). In some examples, the FSHD is FSHD type 2 (FSHD2). In other embodiments, this specification describes a method for alleviating symptoms of a patient with FSHD, comprising the steps of: obtaining an siRNA conjugate as described herein; and administering the siRNA conjugate as described herein to the patient with FSHD in a therapeutically effective dose by reducing the level of the mRNA transcript of human DUX4 or the level of the DUX4 protein.

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

[0350] In some embodiments, this specification describes a method for improving skeletal muscle function in patients with FSHD, comprising the step of administering a therapeutically effective dose of the siRNA conjugate described herein to a patient with FSHD by reducing the level of human DUX4 mRNA transcript or the level of DUX4 protein. In some examples, FSHD is FSHD type 1 (FSHD1). In some examples, FSHD is type 2. In some embodiments, this specification describes a method for improving skeletal muscle function, vision, tonic strength, cardiac function, or pulmonary function in a patient with FSHD, comprising the step of administering a therapeutically effective dose of the siRNA conjugate described herein to a patient with FSHD by reducing the level of human DUX4 mRNA transcript or the level of DUX4 protein.

[0351] In some embodiments, this specification describes a method for treating a target FSHD, comprising the steps of: obtaining an antisense oligonucleotide (ASO) antibody conjugate described herein; administering the ASO antibody conjugate described herein to a subject in a therapeutically effective dose; and reducing the level of the subject's human DUX4 mRNA transcript. 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 the target FSHD, and the process includes administering the ASO antibody conjugate described herein to a subject in a therapeutically effective dose and reducing the level of the subject's human DUX4 mRNA transcript. In some embodiments, the expression levels of one or more marker genes affected by DUX4 expression are also modified or regulated by the reduction in human DUX4 expression levels. Examples of DUX4 biomarker genes include, but are not limited to, MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, LEUTX, WFDC3, ILVBL, SLC15A2, and SORD.

[0352] In some embodiments, methods for treating a subject's FSHD are described herein. In some examples, the FSHD subject suffers from FSHD1. In other examples, the FSHD subject suffers from FSHD2. In another embodiment, the FSHD subject has muscle cells that abnormally express the DUX4 protein, resulting from 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 in the FSHD subject's chromosome 4, which normally contains 1 to 10 repeats instead of 11 to 100 repeats. The epigenetic molecular change in the muscle cells is a change that results in hypomethylation of the D4Z4 region site in the FSHD subject's chromosome 4. 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 target by a number of routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, rectal, or transdermal. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal or intra-abdominal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet another example, 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, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multiply particle formulations (e.g., nanoparticle formulations), and mixed formulations of immediate-release and controlled-release formulations.

[0355] In some cases, the pharmaceutical formulation includes multiply nanoparticle formulations. In some cases, the pharmaceutical formulation includes nanoparticle formulations. In some cases, the nanoparticles include cMAP, cyclodextrin, or lipids. Depending on the case, the nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Further typical nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently attached metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, nanoparticles are metallic 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, nanoparticles contain 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., with one or more of the polynucleic acid molecules or binding sites described herein). In some examples, the coating includes chondroitin sulfate, dextran sulfate, carboxymethyl dextran, 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 include graphene-coated nanoparticles.

[0358] In some cases, the nanoparticles have at least one dimension of approximately 500 nm, 400 nm, 300 nm, 200 nm, or less than 100 nm.

[0359] In some embodiments, the nanoparticle formulations include paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently attached metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, or quantum dots. In some examples, the polynucleic acid molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more of the polynucleic acid molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles.

[0360] In some embodiments, the pharmaceutical formulation includes a delivery vector, such as a recombinant vector, i.e., the delivery of a polynucleic acid molecule to a cell. 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 viruses, retroviruses, adenoviruses, or alphaviruses. In some examples, recombinant vectors capable of expressing a polynucleic acid molecule result in stable expression in target cells. In further examples, viral vectors that result in transient expression of a polynucleic acid molecule are used.

[0361] In some embodiments, the pharmaceutical formulation includes a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Typical carrier materials include, for example, binders, suspending agents, activators, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. Examples of pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, bacugadextrin, 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, and pregelatinized starch. For example, see 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, Pennsylvania, 1975; Liberman, H.A. and Lachman, L. (eds.), Pharmaceutical Dosage Forms, Marcel Decker, New York, New York, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition (Lippincott Williams & Wilkins, 1999).

[0362] In some cases, pharmaceutical formulations further contain pH adjusters or buffers, 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 amounts necessary to maintain the pH of the composition within an acceptable range.

[0363] In some cases, a pharmaceutical formulation contains one or more salts in amounts necessary to bring the osmolality of the composition within an acceptable range. Such salts include those having a sodium, potassium, or ammonium cation and a chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anion, and preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium sulfite, and ammonium sulfate.

[0364] In some cases, pharmaceutical formulations further include diluents used to stabilize the compound, as they provide a more stable environment. Salts dissolved in buffers (which also control or maintain pH) are used as diluents in the art, including but not limited to phosphate-buffered saline. In certain cases, the diluent increases the volume of the composition to facilitate compression or to create a sufficient volume for homogeneous mixing for capsule filling. Examples of such compounds include microcrystalline cellulose such as lactose, starch, mannitol, sorbitol, dextrose, and Avicel®; dicalcium phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch such as Di-Pac® (Amstar), compressible sugars; mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrose; 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 a substance. The term “disintegrates” includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches like corn starch and potato starch; pregelatinized starches such as National 1551 and Amijel®; or sodium starch glycolates such as Promogel® and Explotab®; cellulose from wood products, such as methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Examples include Tia® and Solka-Floc®, methylcellulose, croscarmellose, or cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked cellulose such as cross-linked carboxymethylcellulose or cross-linked croscarmellose, cross-linked starch such as sodium starch glycolate, cross-linked polymer such as crospovidone, cross-linked polyvinylpyrrolidone, alginates such as alginic acid or alginates such as sodium alginate, clay such as Veegum® HV (aluminum magnesium silicate), agar, guar gum, locust bean, karaya, pectin, or tragacanth gum, sodium starch glycolate, bentonite, natural sponge, surfactants, resins such as cation exchange resins, citrus fruit pulp, sodium lauryl sulfate, and sodium lauryl sulfate in starch formulations.

[0366] In some cases, pharmaceutical formulations contain fillers such as lactose, calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, and polyethylene glycol.

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

[0368] Plasticizers include compounds used to soften microencapsulated materials or film coatings to prevent them from becoming too brittle. Suitable plasticizers include, for example, polyethylene glycol such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers also function as dispersants or wetting agents.

[0369] The solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doxate, 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, glycoflor, transktol, propylene glycol, and dimethyl isosorbide.

[0370] Stabilizers include compounds such as antioxidants, buffers, acids, and preservatives.

[0371] The suspending agent is 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, hydroxypropyl methylcellulose, hydroxymethylcellulose, acetate stearate. The compounds include polysorbate 80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth gum, gum arabic, guar gum, xanthan gum, sugars, cellulose compounds such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

[0372] Surfactants include compounds such as sodium lauryl sulfate, sodium doxate, 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. Occasionally, surfactants are included for improved physical stability or other purposes.

[0373] Examples of viscosity enhancers include methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

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

[0375] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic purposes. 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 one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years or more.

[0376] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, consecutively, or at intervals. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered consecutively. In further cases, one or more pharmaceutical compositions are administered at intervals (for example, the first pharmaceutical composition is administered on day 1, followed by at least one, two, three, four, or five days, after which at least the second pharmaceutical composition is administered).

[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 gaps of approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or more between administrations.

[0378] If the patient's condition improves, the administration of the composition will be continued at the physician's discretion. Alternatively, the dose of the administered composition may be temporarily reduced or discontinued for a specific period (i.e., a “drug-free day”). In some cases, the length of the drug-free day may vary between 2 days and 1 year, including, but are not limited to, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. Dose reductions during drug-free days range from 10% to 100%, including, but are just a few examples, 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 improves, a maintenance dose is administered as needed. Subsequently, the dose, frequency, or both may be reduced, depending on the symptoms, to a level that maintains improvement in the disease, impairment, or illness.

[0380] In some embodiments, the amount of a given drug corresponding to such a quantity varies depending on factors such as the specific compound, the severity of the disease, and the identity of the subject or host requiring treatment (e.g., body weight, sex). Nevertheless, it is conventionally determined in a manner known in the art, depending on the specific circumstances surrounding the case, including the specific drug administered, the route of administration, the disease being treated, and the subject or host being treated. In some examples, the desired dose is conveniently provided as a single dose, or as a divided dose administered concurrently (i.e., over a short period) or as a subdose administered at appropriate intervals, for example, two, three, or four or more times a day.

[0381] Given the large number of variations in individual treatment regimens and the frequent deviations from these recommendations, the aforementioned ranges are merely suggestive. Such dosages are modified based on many variables, including but not limited to the activity of the compound used, the disease or illness being treated, the form of administration, the requirements of the individual patient, the severity of the disease or illness being treated, and the judgment of the healthcare professional.

[0382] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell culture or experimental animals, including but not limited to determining the LD50 (the dose at which 50% of the population dies) and ED50 (the dose at which 50% of the population is therapeutically effective). The dose-to-toxicity ratio is the therapeutic index, which is expressed as the ratio of LD50 to ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate dosage ranges for human use. Doses of such compounds are preferably within the range of circulating concentrations containing the ED50 with minimal toxicity. Doses vary within this range depending on the dosage form employed and the route of administration used.

[0383] Kit / Manufactured product In certain embodiments of this specification, kits and products are disclosed for use with one or more of the compositions and methods described herein. Such kits comprise a carrier, package, or container partitioned to receive one or more containers, such as vials or tubes, each container containing one of the distinct elements 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 manufacture of the products provided herein includes packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, and any packaging materials suitable for the selected formulation, intended dosage form, and treatment.

[0385] For example, a container contains the target nucleic acid molecule described herein. Such a kit includes identification markings, labels, or instructions relating to its use in the method described herein.

[0386] The kit typically includes labels and / or instructions for use listing the contents, as well as accompanying documentation with instructions for use. A set of instruction sheets is also typically included.

[0387] In one embodiment, the label is on or attached to the container. In one embodiment, the label is on the container if the letters, numbers, or other characters forming the label are attached to, molded into, or etched onto the container itself. The label is attached to the container if, for example, it is present on the receptacle or carrier that holds the container as an accompanying document. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic purpose. The label may also indicate instructions regarding the use of the contents, such as in the methods described herein.

[0388] In certain embodiments, the pharmaceutical composition is provided in a pack or dispenser containing one or more unit dosage forms containing the compounds provided herein. The pack, for example, 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, the notice reflecting the agency's approval of the form of the drug 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 the compounds provided herein are also prepared, formulated in a compatible pharmaceutical carrier, placed in a suitable container, and labeled with indications for treatment.

[0389] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the claimed subject matter pertains. It should be understood that the above general description and the following detailed description are illustrative and descriptive only and do not limit all claimed subject matter. In this application, the use of singular forms includes plural forms unless otherwise specified. Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural forms unless explicitly specified in the context. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of other forms of the term "including," such as "include," "includes," and "included," is not limited to this application.

[0390] As used herein, ranges and quantities can be expressed as specific values ​​or ranges preceded by "about." "About" also includes the exact quantity. Therefore, "about 5 μL" means "about 5 μL" plus "5 μL." Generally, the term "about" includes quantities that are expected to be within experimental error.

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

[0392] As used herein, the terms “individual,” “subject,” and “patient” mean any mammal. In some embodiments, mammals are humans. In some embodiments, mammals are non-human. None of these terms require, or are limited to, a situation characterized by supervision (e.g., periodic or intermittent) of a healthcare worker (e.g., physician, registered nurse, nurse practitioner, medical assistant, orderly or hospice worker).

[0393] The term "therapeutic dose" refers to the amount of polynucleotide conjugate sufficient to provide a desired therapeutic effect in a mammalian subject. In some cases, this amount is a single or multiple dose to a patient (such as a human) to treat, prevent, prevent the onset of, cure, delay, reduce the severity of, alleviate at least one symptom of, or extend the patient's survival beyond what would be expected without such treatment or recurrent disorder. Naturally, the dose level of a particular polynucleotide conjugate employed to provide a therapeutic dose will vary depending on the type of injury, the subject's age, weight, sex, condition, severity of the disease, route of administration, and the specific inhibitor employed. In some cases, the therapeutic dose of the polynucleotide conjugate described herein is initially estimated from cell cultures and animal models. For example, ICs determined by cell culture methods. 50 The value can optionally serve as a starting point in the animal model, while the IC (Information Control) determined for the animal model is also used. 50The values ​​are used at will to find the therapeutically effective dose for humans.

[0394] Skeletal muscles, or voluntary muscles, are generally fixed to bone by tendons and are typically used to achieve skeletal movements such as locomotion or to maintain posture. Some skeletal muscle control is maintained as an unconscious reflex (e.g., postural muscles or the diaphragm), but 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 contraction") and Type II (i.e., "fast contraction"). Type I muscle fibers are densely packed with capillaries and rich in mitochondria and myoglobin, giving them a characteristic red color. In some cases, Type I muscle fibers carry more oxygen and use fats or hydrocarbons as fuel to maintain aerobic activity. Type I muscle fibers contract over long periods with little effort. Type II muscle fibers are further subdivided into three main subtypes (IIa, IIx, and IIb) that differ in both contraction speed and the force they generate. Type II muscle fibers contract instantly and forcefully but tire very quickly, resulting in only short bursts of anaerobic activity before muscle contraction becomes painful.

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

[0397] Cardiac muscle is also involuntary muscle, but its structure is very similar to skeletal muscle and it is found only in the heart. Both cardiac and skeletal muscle are striated in that they contain sarcomeres, which are bundled together in a highly regular arrangement. In contrast, myofibrils of smooth muscle cells are not arranged in sarcomeres and are therefore not striated.

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

[0399] In this specification, muscle strength is proportional to the cross-sectional area (CSA), and muscle velocity is proportional to the muscle fiber length. Therefore, by comparing the cross-sectional area and muscle fibers across various types of muscle, it is possible to determine the indications for muscular 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. Further methods for measuring muscle mass involve creating tomographic images from selected muscle tissue by computer-aided tomography and ultrasound evaluation.

[0400] The term antibody-nucleotide conjugate (AOC) refers to an antibody that has been conjugated with a nucleotide.

[0401] The terms "siRNA conjugate" or "siRNA-antibody conjugate" refer to antibodies that have been conjugated with siRNA.

[0402] "DUX4 siRNA-conjugate" or "DUX4 siRNA-antibody conjugate" refers to an antibody conjugated to siRNA that hybridizes to the target sequence of human DUX4 mRNA.

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

[0404] Embodiment Embodiment 1. 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, wherein the polynucleic acid molecule mediates RNA interference against DUX4.

[0405] Embodiment 2. A polynucleic acid molecule conjugate of Embodiment 1, wherein 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-strand variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or its antigen-binding fragment.

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

[0407] Embodiment 4. A polynucleic acid molecule conjugate of any one of Embodiments 1 to 3, wherein the polynucleic acid molecule comprises a sense strand and / or an antisense strand, and the sense strand and / or antisense strand each independently comprises at least one 2' modified nucleotide, at least one modified nucleotide linkage, or at least one inverted debase moiety.

[0408] Embodiment 5. A polynucleotide molecular conjugate of any one of Embodiments 1 to 4, wherein the polynucleotide hybridizes to at least eight adjacent bases of the target sequence of DUX4.

[0409] Embodiment 6. A polynucleotide molecular conjugate of any one of Embodiments 1 to 5, wherein the polynucleotide is approximately 8 to approximately 50 nucleotides long, or approximately 10 to approximately 30 nucleotides long.

[0410] Embodiment 7. A 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 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%, and at least 99% identical to a sequence selected from SEQ ID NOs: 1 to 70 or SEQ ID NOs: 141 to 210.

[0411] Embodiment 8. A 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 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%, and at least 99% identical to a sequence selected from SEQ ID NOs. 142, 146, 196, or 201-206.

[0412] Embodiment 9. A 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 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%, and at least 99% identical to a sequence selected from SEQ ID NOs. 71 to 140 or SEQ ID NOs. 211 to 280.

[0413] Embodiment 10. A 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 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%, and at least 99% identical to a sequence selected from SEQ ID NOs. 412-420 and SEQ ID NOs. 430-438.

[0414] Embodiment 11. A 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 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 a 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide, or a locked nucleic acid (LNA) or ethylene nucleic acid (ENA), or a combination thereof.

[0415] Embodiment 12. A polynucleic acid molecule conjugate of any one of Embodiments 1 to 11, wherein at least one modified nucleotide linkage includes a phosphorothioate linkage or a phosphorodithioate linkage.

[0416] Embodiment 13. A 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. A polynucleic acid molecule conjugate of any one of Embodiments 1 to 13, wherein the polynucleic acid molecule contains a 5'-terminal vinyl phosphonate-modified nucleotide.

[0418] Embodiment 15. A 5'-terminal vinyl phosphonate-modified nucleotide is

[0419] [ka] Selected from, In the formula, B is the heterocyclic base portion. R6 is selected from hydrogen, halogen, alkyl, or alkoxy. A polynucleotide molecular conjugate according to any one of Embodiments 1 to 14, wherein J is an internucleotide linking group that links to an adjacent nucleotide of the polynucleotide.

[0420] Embodiment 16. A polynucleic acid molecular 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 located at the 5'-terminus of the sense strand and / or antisense strand.

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

[0422] Embodiment 18. A polynucleic acid molecular conjugate of Embodiment 16, wherein 2'-O-methyl is a pyridine nucleotide.

[0423] Embodiment 19. A polynucleic acid molecular conjugate of any one of Embodiments 16-18, wherein the sense strand and / or antisense strand each contain at least two, at least three, and at least four consecutive 2'-O-methyl-modified nucleotides at their 5' ends.

[0424] Embodiment 20. A polynucleic acid molecule conjugate from any one of Embodiments 1 to 19, comprising a linker that connects an antibody or its antigen-binding fragment to a 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. A polynucleic acid molecule conjugate of Embodiment 20, wherein the linker is a cleavable or non-cleavable linker.

[0428] Embodiment 24. A polynucleic acid molecule conjugate of any one of Embodiments 1 to 23, wherein the ratio of the polynucleic acid molecule to the antibody or its antigen-binding fragment is approximately 1:1, 2:1, 3:1, or 4:1.

[0429] Embodiment 25. A polynucleic acid molecule conjugate of any one of Embodiments 1 to 24, wherein the polynucleic acid molecule mediates RNA interference with human DUX4 and modulates the target muscular atrophy.

[0430] Embodiment 26. A polynucleic acid molecule conjugate of Embodiment 25, wherein RNA interference reduces the expression of the mRNA transcript of the DUX4 gene by at least 50%, at least 60%, or at least 70% compared to the amount of mRNA transcript of the DUX4 gene in untreated cells.

[0431] Embodiment 27. A polynucleic acid molecule conjugate of any one of Embodiments 25-26, comprising RNA interference affecting the expression of a marker gene selected from the group consisting of MBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX in a cell.

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

[0433] Embodiment 29. The polynucleic acid molecule conjugate of Embodiment 28, wherein the effect on the expression of the marker gene is to reduce the expression of the marker gene by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or more.

[0434] Embodiment 30. A 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) The formula includes the molecule, A is an antibody or its antigen-binding fragment, B is a polynucleic acid molecule that hybridizes to the target sequence of DUX4. X is a bond or a nonpolymer linker, A polynucleic acid molecule conjugate according to any one of embodiments 1 to 30, wherein X is conjugated to a cysteine ​​residue of A.

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

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

[0438] Embodiment 34. Any one of the pharmaceutical compositions from Embodiments 32 to 33, formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intracoagulant administration.

[0439] Embodiment 35. A method for treating muscular dystrophy in a subject requiring treatment for muscular dystrophy, comprising the steps of: providing a polynucleic acid conjugate of any one of Embodiments 1 to 34; and treating muscular dystrophy by administering the polynucleic acid conjugate to a subject, 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 with human DUX4 and modulates the target muscular atrophy.

[0441] Embodiment 37. The method of Embodiment 36, comprising the action of RNA interference 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.

[0442] Embodiment 38. Any one of Embodiments 35 to 37, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

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

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

[0445] Embodiment 41. A kit comprising a polynucleic acid molecule conjugate from any one of Embodiments 1 to 31, or a pharmaceutical composition from any one of Embodiments 32 to 34. [Examples]

[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 bioinformatics siRNA library for full-length human DUX4 transcripts. Figure 2 shows a flowchart of the in silico selection process for DUX4 siRNA. Sequences of DUX4, or all siRNAs capable of binding to a given region of DUX4, are collected to generate a starting set of DUX4 siRNAs. From the starting set of DUX4 siRNAs, the first exclusion step includes excluding one or more DUX4 siRNAs having single nucleotide polymorphisms (SNPs) and / or MEFs less than -5. The second exclusion step then includes excluding DUX4 siRNAs having 0 and 1 MM in the human transcriptome (so that only acceptable hits are DUX, DUX5, and DBET). The third exclusion step then includes excluding DUX4 siRNAs having 0 mismatches (MM) in the human intragene region (so that only acceptable hits are DUX1, DUX5, and DBET pseudogenes). The next exclusion step includes excluding DUX4 siRNAs having MMs against the DUX4 human sequence to be used in the FLExDUX4 FSHD mouse model. Next, the following step involves carrying over only one or more DUX4 siRNAs with a predicted viability of 60 or higher. The exclusion step then involves excluding one or more polynucleotide molecules that have matches to known miRNA species regions 1–1000. The exclusion step then continues by excluding 1–1000 with a GC content % of 75 or higher. Finally, the selection process excludes regions 295–1132 and includes eight or fewer predicted off-target hits with two miRNAs, where a maximum of 12 hits are allowed. Using this series of selection steps, we were able to select 70 candidate DUX siRNAs from a starting set of 1694 DUX siRNAs. Figure 3 shows the location and number of such selected DUX4 siRNAs within the DUX4 mRNA transcript (NM_001306068).

[0448] The identified siRNA candidates share common features in the sequences shown in Table 10 below. The identified siRNAs mostly contain 2'-O-Me, and in all three DUX4 templates, only 2'-F modifications are located on the sense strand at positions 7, 8, and 9. The 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. In addition, the identified siRNAs contain four phosphorothioate modifications on each strand, located at the 5' and 3' ends, respectively, forming two final links. The identified siRNAs further contain "Uf" at position 1 of the 5' end of the antisense strand in DUX4 template 1, and "vpN" at position 1 of the 5' end of the antisense strand in DUX4 template 3, regardless of the actual target mRNA sequence (which ligates to the "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 the antisense strand only, but no overhang at the 3' end of the sense strand. Optimization of the identified siRNAs may include vinyl phosphonate nucleotides, inverted debase moieties, or amine linkers to the passenger or guide strands.

[0449] [Table 10]

[0450] Tables 11, 12, 13, 14, and 15 illustrate candidate siRNAs 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 single-stranded siRNAs were fully assembled on a solid phase using standard phosphoramidite chemistry and purified by HPLC. The purified single-stranded siRNAs were doubled to obtain double-stranded siRNAs. For vinyl phosphonate-modified guide strands, guide strands with a vinyl phosphonate-modified nucleotide structure at the 5' end (VpUq) were produced. All siRNA passenger strands contained C6-NH2 and / or C6-SH conjugation handles of different formats, each at one end of the strand. The conjugation handle(s) were attached to the siRNA passenger strand or siRNA guide strand via inverse debasal phosphate diester or phosphorothioate. Figures 5A to 5F show typical structures of the formats used in in vivo experiments. Figure 5A illustrates a typical siRNA structure with a C6-NH2 conjugation handle at the 5' end and a C6-SH at the 3' end of the passenger strand or guide strand. 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 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 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 conjugates Figures 6A to 6F illustrate typical structures of the A-X1-B-X2-Y (Formula I) architecture described herein. Figure 6A illustrates the antibody-Cys-SMCC-5'-passenger chain (architecture 1). This conjugate was generated by inter-antibody chain cysteine ​​conjugation to maleimide (SMCC) at the 5' end of the passenger chain. Figure 6B illustrates the antibody-Cys-SMCC-3'-passenger chain (architecture 2). This conjugate was generated by inter-antibody chain cysteine ​​conjugation to maleimide (SMCC) at the 3' end of the passenger chain. Figure 6C illustrates the antibody-Cys-bisMal-3'-passenger chain (ASC architecture 3). This conjugate was generated by inter-antibody chain cysteine ​​conjugation to the bismaleimide (bisMal) linker at the 3' end of the passenger chain. Figure 6D illustrates the model structure of the Fab-Cys-bisMal-3'-passenger chain (ASC architecture 4). This conjugate was generated by Fab-chain inter-cysteine ​​conjugation to a bismaleimide (bisMal) linker at the 3' end of the passenger chain. Figure 6E illustrates the 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-chain inter-cysteine ​​to a bismaleimide (bisMal) linker at the 3' end of the passenger chain of each siRNA. Figure 6F illustrates the 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 ​​relative to a 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 linker Figure 7A illustrates a typical synthesis scheme (synthesis scheme 1) of the antibody-Cys-SMCC-siRNA-PEG conjugate via antibody-cysteine ​​conjugation.

[0463] Step 1: Inter-antibody chain disulfide reduction by TCEP

[0464] The antibody was buffer-exchanged with borax buffer (pH 8) to a maximum concentration of 10 mg / ml. Two equivalents of TCEP in water were added to this solution, and the mixture was stirred at room temperature for 2 hours. The resulting reaction mixture was buffer-exchanged with PBS containing 5 mM EDTA at pH 7.4, and then added to a solution of SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-XkDa (2 equivalents) (X=0.5kDa~10kDa) in PBS containing 5 mM EDTA at pH 7.4 at room temperature, and stirred overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed unreacted antibody and siRNA along with antibody-siRNA conjugates.

[0465] Step 2: Purification

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

[0467] Step 3: Analysis of the purified conjugate

[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-2 or anion exchange chromatography-3. Both methods are described in Example 3.4. The isolated DAR1 conjugates typically eluted in 9.0 ± 0.3 minutes by analytical SAX chromatography, with a purity exceeding 90%. Typical cysteine ​​conjugates with DAR>2 contain more than 85% DAR2 and less than 15% DAR3.

[0469] Example 3.2. Synthesis of antibody-siRNA conjugates using a bismaleimide (BisMal) linker Figure 7B illustrates a typical synthesis scheme (synthesis scheme 2) for the antibody-Cys-BisMal-siRNA-PEG conjugate.

[0470] Step 1: Antibody reduction by TCEP

[0471] The antibody was buffered 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 the mixture was rotated at room temperature for 2 hours. The resulting mixture was exchanged for PBS containing 5 mM EDTA at pH 7.4, and added to a solution of BisMal-C6-siRNA-C6-S-NEM (2 equivalents) in PBS containing 5 mM EDTA at room temperature. The mixture was maintained at 4°C overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed unreacted antibody and siRNA along with the antibody-siRNA conjugate.

[0472] Step 2: Purification

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

[0474] Step 3: Analysis of the purified conjugate

[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-2 or 3, in addition to size exclusion chromatography-1.

[0476] Example 3.3. Fab generation from mAb and conjugation to siRNA Figure 7C illustrates a typical synthesis scheme (synthesis scheme 3) for generating Fab-siRNA conjugates.

[0477] Step 1: Antibody digestion by pepsin

[0478] The antibody was buffered with 20 mM sodium acetate / acetic acid buffer at pH 4.0 to a maximum concentration of 5 mg / ml. Immobilized pepsin (Thermo Scientific, Prod#20343) was added, and the mixture was incubated at 37°C for 3 hours. The reaction mixture was filtered using a 30 kDa MWCO Amicon spin filter and pH 7.4 PBS. The retainate was collected and purified by size exclusion chromatography to isolate F(ab')2. The collected F(ab')2 was then reduced with 10 equivalents of TCEP and conjugated with SMCC-C6-siRNA-PEG5 in pH 7.4 PBS at room temperature. Analysis of the reaction mixture by SAX chromatography revealed the Fab-siRNA conjugate along with unreacted Fab and siRNA-PEG.

[0479] Step 2: Purification

[0480] The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography-1. The fractions containing the DAR1 and DAR2 Fab-siRNA conjugates were separated, concentrated, and buffered with PBS at pH 7.4.

[0481] Step 3: Analysis of the purified conjugate

[0482] The isolated conjugates were characterized and their purity examined by analytical HPLC using either anion exchange chromatography-2 or 3, in addition to SEC-1.

[0483] Example 3.4. Purification and Analysis Methods Anion exchange chromatography (SAX)-1. 1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5mm ID × 15cm, 13um 2. Solvent A: 20mM TRIS buffer, pH 8.0; Solvent B: 20mM TRIS, 1.5M NaCl, pH 8.0; Flow rate: 6.0ml / min. 3. Gradient: a. %A %B Column volume b. 100 0 1.00 c. 60 40 18.00 d. 40 60 2.00 e. 40 60 5.00 f. 0 100 2.00 g. 100 0 2.00

[0484] Anion exchange chromatography (SAX) method - 2 1. Column: Thermo Scientific, ProPac® SAX-10, Bio LC®, 4 × 250 mm 2. Solvent A: 80% 10mM TRIS at pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS at pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml / min. 3. Gradient: a. Time %A %B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 13.00 40 60 f. 15.00 90 10 g. 20.00 90 10

[0485] Anion exchange chromatography (SAX) method - 3 1. Column: Thermo Scientific, ProPac® SAX-10, Bio LC®, 4 × 250 mm 2. Solvent A: 80% 10mM TRIS at pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS at pH 8, 20% ethanol, 1.5M NaCl 3.Flow rate: 0.75ml / min. 4. Gradient: a. Time %A %B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 23.00 40 60 f. 25.00 90 10 g. 30.00 90 10

[0486] Size Exclusion Chromatography (SEC) Method - 1 1. Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8 × 300 mm, 5 μM 2. Mobile phase: 150 mM phosphate buffer 3. Flow rate: 1.0 ml / min over 15 minutes

[0487] Example 3.5. Synthesis of antibody-siRNA conjugates using a bismaleimide (BisMal) linker Antibody reduction by TCEP The antibody was buffered with 25 mM borate buffer (pH 8) containing 1 mM DTPA to a maximum concentration of 10 mg / ml. To this solu...

Claims

1. A conjugate comprising (i) an anti-transferrin receptor antibody or its antigen-binding fragment, (ii) siRNA including a guide strand and a passenger strand, and (iii) a linker. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region including HCDR1 containing the sequence of SEQ ID NO: 281, HCDR2 containing the sequence of SEQ ID NO: 284, and HCDR3 containing the sequence of SEQ ID NO:

283. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable light chain (VL) region including LCDR1 containing the sequence of SEQ ID NO: 286, LCDR2 containing the sequence of SEQ ID NO: 287, and LCDR3 containing the sequence of SEQ ID NO:

288. The guide strand of the siRNA consists of the sequence of SEQ ID NO: 413, the passenger strand of the siRNA consists of the sequence of SEQ ID NO: 146, and the anti-transferrin receptor antibody or its antigen-binding fragment binds to the end of the guide strand or the passenger strand via the linker. Conjugate.

2. The conjugate according to claim 1, wherein the linker is a severable linker or a non-severable linker.

3. The conjugate according to claim 1, wherein the linker is a homobifunctional linker or a heterobifunctional linker.

4. The conjugate according to claim 1, wherein the linker binds the anti-transferrin receptor antibody or its antigen-binding fragment to the 5' end of the passenger chain.

5. The conjugate according to claim 1, wherein the passenger chain is bound to a cysteine ​​residue of the anti-transferrin receptor antibody or its antigen-binding fragment.

6. The conjugate according to claim 1, wherein the ratio of the siRNA to the anti-transferrin receptor antibody or its antigen-binding fragment is approximately 1:1, 2:1, 3:1, or 4:

1.

7. The conjugate according to claim 1, wherein the anti-transferrin receptor antibody or its antigen-binding fragment is a full-length anti-transferrin receptor antibody.

8. The conjugate according to claim 7, wherein the full-length anti-transferrin receptor antibody is a humanized anti-transferrin receptor antibody.

9. The conjugate according to claim 7, wherein the full-length anti-transferrin receptor antibody further comprises a mutation selected from the group consisting of L233A, L234A, and L327R in the heavy chain constant region, the location of the mutation corresponds to an equivalent position in the amino acid sequence of SEQ ID NO:

303.

10. The conjugate according to claim 7, wherein the full-length anti-transferrin receptor antibody further comprises mutations L233A, L234A and L327R in the heavy chain constant region, the locations of the mutations corresponding to equivalent positions in the amino acid sequence of SEQ ID NO:

303.

11. The conjugate according to claim 1, wherein the anti-transferrin receptor antibody or its antigen-binding fragment is selected from the group consisting of monovalent Fab', bivalent Fab2, and single-stranded variable fragments (scFv).

12. The conjugate according to claim 1, wherein the siRNA comprises a 5'-terminal vinyl phosphonate-modified nucleotide.

13. A conjugate comprising (i) an anti-transferrin receptor antibody or its antigen-binding fragment, (ii) siRNA including a guide strand and a passenger strand, and (iii) a linker. The anti-transferrin receptor antibody or its antigen-binding fragment comprises the variable heavy chain (VH) sequence of SEQ ID NO: 294 and the variable light chain (VL) sequence of SEQ ID NO:

298. The guide strand of the siRNA consists of the sequence of SEQ ID NO: 413, the passenger strand of the siRNA consists of the sequence of SEQ ID NO: 146, and the anti-transferrin receptor antibody or its antigen-binding fragment binds to the end of the guide strand or the passenger strand via the linker. Conjugate.

14. The conjugate according to claim 13, wherein the linker is a severable linker or a non-severable linker.

15. The conjugate according to claim 13, wherein the linker is a homobifunctional linker or a heterobifunctional linker.

16. The conjugate according to claim 13, wherein the linker binds the anti-transferrin receptor antibody or its antigen-binding fragment to the 5' end of the passenger chain.

17. The conjugate according to claim 13, wherein the anti-transferrin receptor antibody or its antigen-binding fragment is a full-length anti-transferrin receptor antibody.

18. The conjugate according to claim 17, wherein the full-length anti-transferrin receptor antibody is a humanized anti-transferrin receptor antibody.

19. The conjugate according to claim 17, wherein the full-length anti-transferrin receptor antibody further comprises a mutation selected from the group consisting of L233A, L234A, and L327R in the heavy chain constant region, the location of the mutation corresponds to an equivalent position in the amino acid sequence of SEQ ID NO:

303.

20. The conjugate according to claim 17, wherein the full-length anti-transferrin receptor antibody further comprises mutations L233A, L234A and L327R in the heavy chain constant region, the locations of the mutations corresponding to equivalent positions in the amino acid sequence of SEQ ID NO:

303.

21. The conjugate according to claim 13, wherein the anti-transferrin receptor antibody or its antigen-binding fragment is selected from the group consisting of monovalent Fab', bivalent Fab2, and single-stranded variable fragment (scFv).

22. The conjugate according to claim 13, wherein the siRNA comprises a 5'-terminal vinyl phosphonate-modified nucleotide.

23. A conjugate comprising (i) an anti-transferrin receptor antibody, (ii) siRNA including a guide strand and a passenger strand, and (iii) a linker. The anti-transferrin receptor antibody comprises the heavy chain sequence of SEQ ID NO: 312 and the light chain sequence of SEQ ID NO:

327. The guide strand of the siRNA consists of the sequence of SEQ ID NO: 413, the passenger strand of the siRNA consists of the sequence of SEQ ID NO: 146, and the anti-transferrin receptor antibody binds to the 5' end of the passenger strand via the linker. Conjugate.

24. The conjugate according to claim 23, wherein the linker is a severable linker or a non-severable linker.

25. The conjugate according to claim 23, wherein the linker is a homobifunctional linker or a heterobifunctional linker.

26. A conjugate comprising (i) an anti-transferrin receptor antibody, (ii) siRNA including a guide strand and a passenger strand, and (iii) a linker. The anti-transferrin receptor antibody comprises two heavy chains each containing the sequence of SEQ ID NO: 312, and two light chains each containing the sequence of SEQ ID NO:

327. The guide strand of the siRNA consists of the sequence of SEQ ID NO: 413, the passenger strand of the siRNA consists of the sequence of SEQ ID NO: 146, and the anti-transferrin receptor antibody binds to the 5' end of the passenger strand via the linker. Conjugate.

27. The conjugate according to claim 26, wherein the linker is a severable linker or a non-severable linker.

28. The conjugate according to claim 26, wherein the linker is a homobifunctional linker or a heterobifunctional linker.

29. A conjugate comprising (i) an anti-transferrin receptor antibody or its antigen-binding fragment, (ii) siRNA including a guide strand and a passenger strand, and (iii) a linker. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region including HCDR1 containing the sequence of SEQ ID NO: 281, HCDR2 containing the sequence of SEQ ID NO: 284, and HCDR3 containing the sequence of SEQ ID NO:

283. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable light chain (VL) region including LCDR1 containing the sequence of SEQ ID NO: 286, LCDR2 containing the sequence of SEQ ID NO: 287, and LCDR3 containing the sequence of SEQ ID NO:

288. The guide strand of the siRNA consists of the sequence of SEQ ID NO: 416, the passenger strand of the siRNA consists of the sequence of SEQ ID NO: 201, and the anti-transferrin receptor antibody or its antigen-binding fragment binds to the end of the guide strand or the passenger strand via the linker. Conjugate.

30. The use of the conjugate according to any one of claims 1 to 29 in the manufacture of a drug for treating muscular dystrophy in a subject requiring treatment for muscular dystrophy, The conjugate reduces the level of the human DUX4 mRNA transcript by mediating RNA interference with the human DUX4 mRNA transcript, thereby treating the muscular dystrophy in the subject. use.

31. The use according to claim 30, wherein the RNA interference reduces the level of human DUX4 mRNA transcript by at least 50%, at least 60%, or at least 70% compared to the level of human DUX4 mRNA transcript in untreated cells.

32. The use according to claim 30, wherein the RNA interference reduces 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.

33. The use according to claim 32, wherein reducing the expression includes reducing the expression of the marker gene in the cells by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or more.

34. The use according to claim 30, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

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