Method for preparing protein-oligonucleotide complexes

Abstract

The present invention relates to methods of making protein-oligonucleotide complexes, and some aspects of the present disclosure relate to methods of purifying complexes comprising proteins (e.g., antibodies) covalently linked to oligonucleotides. In some embodiments, complexes comprising proteins covalently linked to oligonucleotides are purified and isolated from unlinked oligonucleotides using a mixed-mode resin (e.g., a hydroxyapatite resin) comprising a positively charged metal site and a negatively charged ionic site. In some embodiments, a complex comprising a protein covalently linked to an oligonucleotide is purified from a mixture comprising the complex, an unlinked protein, and an unlinked oligonucleotide using a purification step involving a hydrophobic interaction chromatography resin followed by a purification step involving a mixed mode resin.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of June 5, 2020, application number 202080040177.8, and invention name “Method for preparing protein-oligonucleotide complexes”.

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 858,964, filed on June 7, 2019, entitled “METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES,” and U.S. Provisional Application No. 62 / 992,187, filed on March 20, 2020, entitled “METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES,” the entire contents of each of which are incorporated herein by reference. Technical Field

[0003] The present application relates to methods of purifying complexes (eg, protein-oligonucleotide conjugates).

[0004] References to sequence listings

[0005] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a 56 kb file titled D082470010WO00-SEQ.txt created on June 5, 2020. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Background Art

[0006] In recent years, several oligonucleotides (e.g., antisense oligonucleotides) have been developed to combat tissue or cell-specific diseases (e.g., muscle-specific diseases such as various forms of muscular dystrophy). However, it has proven challenging to effectively deliver these oligonucleotides to their desired tissues or cells. Summary of the invention

[0007] The complex comprising tissue or cell specific proteins (e.g., antibodies) covalently linked to therapeutic oligonucleotides provides an excellent opportunity for delivering the therapeutic oligonucleotides. However, purification and separation of the complexes are challenging to keep them away from excessive proteins and oligonucleotides. Herein, the disclosure provides a method for preparing a complex comprising a protein covalently linked to an oligonucleotide, which separates unconjugated oligonucleotides (e.g., single-stranded oligonucleotides) and proteins (e.g., antibodies) from the complex.

[0008] According to some aspects, the present disclosure provides methods for treating (e.g., purifying) complexes (e.g., antibody-oligonucleotide conjugates). In some embodiments, the treated complexes target muscle cells to deliver molecular payloads (e.g., oligonucleotides) to those cells. In some embodiments, the treated complexes of the present disclosure promote muscle-specific delivery of molecular payloads (e.g., oligonucleotides) targeting muscle disease alleles. For example, in some embodiments, the treated complexes provided herein are particularly useful for delivering molecular payloads (e.g., oligonucleotides) that modulate the expression or activity of the gene in a subject suffering from or suspected of suffering from a muscle disease associated with a gene (e.g., the genes / diseases of Table 1).

[0009] Some aspects of the present disclosure provide methods of isolating a complex or a plurality of complexes, each complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:

[0010] (i) contacting a mixture comprising the complex and unlinked antibodies with a hydrophobic resin under conditions wherein the complex is adsorbed to the hydrophobic resin but the unlinked antibodies are not adsorbed to the hydrophobic resin, thereby separating the unlinked antibodies from the complex adsorbed to the hydrophobic resin; and

[0011] (ii) eluting the complex from the hydrophobic resin under conditions where the complex dissociates from the hydrophobic resin.

[0012] In some embodiments, the conditions in step (i) comprise a conductivity of at least 70 mS / cm, and / or the conditions in step (ii) comprise a conductivity of 10 to 70 mS / cm.

[0013] In some embodiments, the conditions in step (i) and or step (ii) are achieved using an anti-chaotropic salt, optionally wherein the anti-chaotropic salt is ammonium sulfate. In some embodiments, the mixture in step (i) further comprises at least 500 mM ammonium sulfate, optionally wherein the mixture in step (i) further comprises 500 mM to 1 M ammonium sulfate.

[0014] In some embodiments, the step includes washing the hydrophobic resin with a solution comprising at least 500 mM ammonium sulfate between step (i) and step (ii).

[0015] In some embodiments, step (ii) comprises applying an elution solution comprising up to 200 mM chloride ions and up to 100 mM ammonium sulfate to the hydrophobic resin to elute the complex. In some embodiments, the elution solution does not comprise ammonium sulfate. In some embodiments, the elution solution is PBS. In some embodiments, the elution solution comprises up to 25 mM chloride ions.

[0016] In some embodiments, step (ii) comprises applying a gradually decreasing concentration of ammonium sulfate to the hydrophobic resin to elute the complex, optionally wherein the concentration of ammonium sulfate is reduced from at least 500 mM to less than 100 mM. In some embodiments, the gradually decreasing concentration of ammonium sulfate is applied over 5 to 12 column volumes (CV), optionally 6 to 8 CV.

[0017] In some embodiments, the mixture in step (i) further comprises an unligated oligonucleotide, optionally wherein the oligonucleotide is adsorbed to a hydrophobic resin in step (i) and eluted with the complex in step (ii).

[0018] In some embodiments, the antibody is a full length IgG, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv fragment. In some embodiments, the antibody is an anti-transferrin receptor antibody.

[0019] In some embodiments, the oligonucleotide is single-stranded.In some embodiments, the oligonucleotide is an antisense oligonucleotide, optionally a spacer polymer or phosphorodiamidate morpholino oligomer (PMO).In some embodiments, the oligonucleotide is a chain of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is siRNA, and optionally wherein the chain is the sense strand of siRNA.In some embodiments, the oligonucleotide includes at least one modified internucleotide linkage (internucleotide linkage), optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.In some embodiments, the oligonucleotide includes one or more modified nucleotides, optionally wherein the modified nucleotide includes 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (lockednucleic acid, LNA), 2'-fluorine modification or morpholino modification.In some embodiments, the length of the oligonucleotide is 10 to 50 nucleotides, optionally 15 to 25 nucleotides in length.In some embodiments, the antibody is covalently linked to the 5' of the oligonucleotide. In some embodiments, the antibody is covalently linked to the 3' end of the oligonucleotide.

[0020] In some embodiments, the antibody is covalently linked to the oligonucleotide via a linker, optionally a Val-cit linker.

[0021] In some embodiments, the complex eluted in step (ii) comprises the antibody covalently linked to 1, 2, or 3 oligonucleotides.

[0022] In some embodiments, the hydrophobic resin comprises a hydrophobic moiety selected from butyl, tert-butyl, phenyl, ether, amide, or propyl.

[0023] In some embodiments, the hydrophobic resin is equilibrated prior to step (i), optionally with a solution comprising at least 500 mM ammonium sulfate.

[0024] In some embodiments, the eluate obtained from step (ii) comprises undetectable levels of unlinked antibody.

[0025] In some embodiments, the method further comprises separating the complex from the unligated oligonucleotide.

[0026] Still other aspects of the present disclosure provide methods of isolating a complex or a plurality of complexes, each complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:

[0027] (i) contacting a mixture comprising the complex and the unligated oligonucleotide with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions wherein the complex is adsorbed to the mixed mode resin, and

[0028] (ii) eluting the complex from the mixed mode resin under conditions where the complex dissociates from the mixed mode resin.

[0029] In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.

[0030] In some embodiments, the mixture in step (i) further comprises up to 20 mM phosphate ions and / or up to 30 mM chloride ions, optionally wherein the mixture in step (i) further comprises up to 10 mM phosphate ions and / or up to 25 mM chloride ions. In some embodiments, the unligated oligonucleotides are not adsorbed to the mixed mode resin in step (i).

[0031] In some embodiments, the mixture in step (i) further comprises up to 5 mM phosphate ions and / or up to 10 mM chloride ions, optionally wherein the mixture in step (i) further comprises up to 3 mM phosphate ions and / or up to 8 mM chloride ions. In some embodiments, some or all of the unattached oligonucleotides are adsorbed to the mixed mode resin in step (i). In some embodiments, the method further comprises washing the mixed mode resin between step (i) and step (ii) with a solution comprising up to 20 mM phosphate ions and / or up to 30 mM chloride ions, optionally wherein the solution comprises up to 10 mM phosphate ions and / or up to 25 mM chloride ions.

[0032] In some embodiments, step (ii) comprises applying an elution solution comprising at least 30 mM phosphate ions and / or at least 50 mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the elution solution comprises at least 100 mM phosphate ions and / or at least 100 mM chloride ions.

[0033] In some embodiments, the antibody is a full length IgG, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv fragment. In some embodiments, the antibody is an anti-transferrin receptor antibody.

[0034] In some embodiments, the oligonucleotide is single-stranded.In some embodiments, the oligonucleotide is an antisense oligonucleotide, optionally a spacer polymer or a phosphorodiamidate morpholino oligomer (PMO).In some embodiments, the oligonucleotide is a chain of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is siRNA, and optionally wherein the chain is the sense strand of siRNA.In some embodiments, the oligonucleotide includes at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.In some embodiments, the oligonucleotide includes one or more modified nucleotides, optionally wherein the modified nucleotide includes 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), 2'-fluorine modification or morpholino modification.In some embodiments, the length of the oligonucleotide is 10 to 50 nucleotides, optionally 15 to 25 nucleotides in length.In some embodiments, the antibody is covalently linked to the 5' of the oligonucleotide.In some embodiments, the antibody is covalently linked to the 3' of the oligonucleotide.

[0035] In some embodiments, the antibody is covalently linked to the oligonucleotide via a linker, optionally a Val-cit linker.

[0036] In some embodiments, the complex eluted in step (ii) comprises the antibody covalently linked to 1, 2, or 3 oligonucleotides.

[0037] In some embodiments, the eluate obtained from step (ii) comprises undetectable levels of unligated oligonucleotides.

[0038] In some embodiments, prior to step (i), the mixture in step (i) is separated from a hydrophobic interaction chromatography resin.

[0039] Also provided herein is a method of isolating a complex or a plurality of complexes, each complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:

[0040] (i) contacting a first mixture comprising a complex, an unlinked antibody, and an unlinked oligonucleotide with a hydrophobic resin under conditions wherein the complex and the unlinked oligonucleotide are adsorbed to the hydrophobic resin but the unlinked antibody is not adsorbed to the hydrophobic resin, thereby separating the unlinked antibody from the complex and the unlinked oligonucleotide adsorbed to the hydrophobic resin; and

[0041] (ii) obtaining a second mixture comprising the complex and the unligated oligonucleotide by eluting the complex and the unligated oligonucleotide from the hydrophobic resin under conditions in which the complex dissociates from the hydrophobic resin;

[0042] (iii) contacting the second mixture obtained in step (ii) with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions wherein the complex is adsorbed to the mixed mode resin, and

[0043] (iv) eluting the complex from the mixed mode resin under conditions where the complex dissociates from the mixed mode resin.

[0044] In some embodiments, the hydrophobic resin comprises a hydrophobic moiety selected from butyl, tert-butyl, phenyl, ether, amide, or propyl. In some embodiments, the mixed mode resin is an apatite resin, optionally wherein the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.

[0045] In some embodiments, the condition in step (i) includes a conductivity of at least 70mS / cm, and / or the condition in step (ii) includes a conductivity of 10 to 70mS / cm. In some embodiments, the condition in step (i) and or step (ii) is achieved using an anti-chaotropic salt, optionally wherein the anti-chaotropic salt is ammonium sulfate. In some embodiments, the hydrophobic resin is balanced before step (i), optionally with a solution balance comprising at least 500mM ammonium sulfate. In some embodiments, the mixture in step (i) also includes at least 500mM ammonium sulfate, optionally wherein the mixture in step (i) also includes 500mM to 1M ammonium sulfate. In some embodiments, the method also includes washing the hydrophobic resin with a solution comprising at least 500mM ammonium sulfate between step (i) and step (ii). In some embodiments, step (ii) includes applying a first elution solution comprising up to 200 mM chloride ions and up to 100 mM ammonium sulfate to elute the complex and unattached oligonucleotides to the hydrophobic resin, optionally wherein the first elution solution does not comprise ammonium sulfate. In some embodiments, the first elution solution is PBS, or comprises up to 25 mM chloride ions. In some embodiments, step (ii) includes applying a gradually decreasing concentration of ammonium sulfate to elute the complex and unattached oligonucleotides to the hydrophobic resin, optionally wherein the concentration of ammonium sulfate is reduced from at least 500 mM to less than 100 mM and / or the gradually decreasing concentration of ammonium sulfate is applied over 5 to 12 column volumes (CV), optionally 6 to 8 CV.

[0046] In some embodiments, the second mixture in step (iii) also comprises up to 20mM phosphate ions and / or up to 30mM chloride ions, optionally wherein the second mixture in step (iii) also comprises up to 10mM phosphate ions and / or up to 25mM chloride ions. In some embodiments, in step (iii), the unconnected oligonucleotide is not adsorbed to the mixed mode resin. In some embodiments, the second mixture in step (iii) also comprises up to 5mM phosphate ions and / or up to 10mM chloride ions, optionally wherein the second mixture in step (iii) also comprises up to 3mM phosphate ions and / or up to 8mM chloride ions. In some embodiments, in step (iii), some or all of the unconnected oligonucleotides are adsorbed to the mixed mode resin. In some embodiments, the method further comprises washing the mixed mode resin with a solution comprising up to 20 mM phosphate ions and / or up to 30 mM chloride ions between step (iii) and step (iv) to remove unattached oligonucleotides from the mixed mode resin, optionally wherein the solution comprises up to 10 mM phosphate ions and / or up to 25 mM chloride ions. In some embodiments, step (iv) comprises applying a second elution solution comprising at least 30 mM phosphate ions and / or at least 50 mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the second elution solution comprises at least 100 mM phosphate ions and / or at least 100 mM chloride ions.

[0047] In some embodiments, the antibody is a full length IgG, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv fragment. In some embodiments, the antibody is an anti-transferrin receptor antibody.

[0048] In some embodiments, the oligonucleotide is single-stranded.In some embodiments, the oligonucleotide is an antisense oligonucleotide, optionally a spacer polymer or a phosphorodiamidate morpholino oligomer (PMO).In some embodiments, the oligonucleotide is a chain of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is siRNA, and optionally wherein the chain is the sense strand of siRNA.In some embodiments, the oligonucleotide includes at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.In some embodiments, the oligonucleotide includes one or more modified nucleotides, optionally wherein the modified nucleotide includes 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), 2'-fluorine modification or morpholino modification.In some embodiments, the length of the oligonucleotide is 10 to 50 nucleotides, optionally 15 to 25 nucleotides in length.In some embodiments, the antibody is covalently linked to the 5' of the oligonucleotide.In some embodiments, the antibody is covalently linked to the 3' of the oligonucleotide.

[0049] In some embodiments, the antibody is covalently linked to the oligonucleotide via a linker, optionally a Val-cit linker.

[0050] In some embodiments, the complex eluted in step (iv) comprises the antibody covalently linked to 1, 2, or 3 oligonucleotides.

[0051] In some embodiments, the eluate obtained from step (iv) comprises undetectable levels of unligated oligonucleotide and / or comprises undetectable levels of unligated antibody.

[0052] In some aspects, a method of treating a complex comprising a protein covalently linked to one or more oligonucleotides comprises:

[0053] (i) separating the complex from the unligated oligonucleotide by contacting a mixture comprising the complex and the unligated oligonucleotide with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions wherein the complex is adsorbed to the mixed mode resin, and

[0054] (ii) The unligated oligonucleotide is eluted while the complex remains adsorbed to the mixed mode resin.

[0055] In some embodiments, the mixed mode resin is an apatite resin (e.g., a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin). In some embodiments, the mixture comprising the complex and the unattached oligonucleotide further comprises at least 1 mM of phosphate ions and / or at least 5 mM of chloride ions (e.g., 5 mM of phosphate ions and 25 mM of chloride ions). In some embodiments, the unattached oligonucleotide is eluted in step (ii) by adding a washing solution to the mixed mode resin, optionally wherein the washing solution comprises 1 to 50 mM of phosphate ions and / or at least 5 to 50 mM of chloride ions (e.g., 5 mM of phosphate ions and 25 mM of chloride ions).

[0056] In some embodiments, the method further comprises a step (iii) of eluting the plurality of complexes from the mixed mode resin after step (ii). In some embodiments, the plurality of complexes are eluted in step (iii) by adding an eluent solution (e.g., an eluent solution comprising at least 5 mM phosphate ions and / or at least 5 mM chloride ions (e.g., 100 mM phosphate ions and 100 mM chloride ions)) to the mixed mode resin.

[0057] In some aspects, a method of processing complexes, wherein each complex comprises a protein covalently linked to one or more oligonucleotides, the method comprising:

[0058] (i) contacting a first mixture comprising the complex, unligated oligonucleotide, and unligated protein with a hydrophobic interaction chromatography (HIC) resin under conditions wherein the complex and unligated oligonucleotide are adsorbed to the HIC resin;

[0059] (ii) elution of unbound proteins from the HIC resin;

[0060] (iii) after step (ii), eluting a second mixture comprising the complex and the unligated oligonucleotide from the HIC resin;

[0061] (iv) contacting the second mixture with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions wherein the complex adsorbs to the mixed mode resin;

[0062] (v) eluting the unligated oligonucleotide while the complex remains adsorbed to the mixed mode resin; and

[0063] (vi) after step (v), eluting the plurality of complexes from the mixed mode resin.

[0064] In some embodiments, the HIC resin comprises butyl, tert-butyl, methyl and / or ethyl functional groups. In some embodiments, the HIC resin is balanced prior to step (i), optionally with at least 500 mM ammonium sulfate. In some embodiments, unattached proteins are eluted in step (ii) by adding a HIC wash solution (e.g., a HIC wash solution comprising at least 500 mM ammonium sulfate) to the HIC resin. In some embodiments, complexes and unattached oligonucleotides are eluted from the HIC resin in step (iii) by adding a HIC eluent solution (e.g., a HIC eluent solution comprising less than 100 mM phosphate ions and / or 100 mM chloride ions (e.g., 5 mM phosphate ions and 25 mM chloride ions) to the HIC resin.

[0065] In some embodiments, the mixed mode resin is an apatite resin (e.g., a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin). In some embodiments, the mixture comprising the complex and the unattached oligonucleotide further comprises at least 1 mM of phosphate ions and / or at least 5 mM of chloride ions (e.g., 5 mM of phosphate ions and 25 mM of chloride ions). In some embodiments, the unattached oligonucleotide is eluted in step (ii) by adding a washing solution to the mixed mode resin, optionally wherein the washing solution comprises 1 to 50 mM of phosphate ions and / or at least 5 to 50 mM of chloride ions (e.g., 5 mM of phosphate ions and 25 mM of chloride ions).

[0066] In some embodiments, the method further comprises a step (iii) of eluting the plurality of complexes from the mixed mode resin after step (ii). In some embodiments, the plurality of complexes are eluted in step (iii) by adding an eluent solution (e.g., an eluent solution comprising at least 5 mM phosphate ions and / or at least 5 mM chloride ions (e.g., 100 mM phosphate ions and 100 mM chloride ions)) to the mixed mode resin.

[0067] In some embodiments, the protein of the complex to be treated is an antibody. The antibody can be a muscle targeting antibody, such as a muscle targeting antibody that specifically binds to the extracellular epitope of the transferrin receptor. In some embodiments, the muscle targeting antibody competes with the antibodies listed in Table 2 for specific binding to the transferrin receptor epitope. The antibody (e.g., muscle targeting antibody) can be in the form of a ScFv, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a Fv fragment.

[0068] In some embodiments, the oligonucleotide of the complex to be treated comprises at least one modified internucleotide linkage (e.g., a phosphorothioate linkage). The oligonucleotide may comprise one or more modified nucleotides. In some embodiments, the oligonucleotide is 10 to 50 nucleotides in length (e.g., 15 to 25 nucleotides in length). In some embodiments, the oligonucleotide comprises a region complementary to a gene listed in Table 1 or an mRNA encoded thereby.

[0069] In some embodiments, the complex (eg, processed as described herein) comprises undetectable levels of unligated protein and / or comprises undetectable levels of unligated oligonucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Provided is an image of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (non-reducing SDS-PAGE) gel. Lane 1 shows the recombinantly expressed anti-transferrin antibody DTX-A-001 Fab' after protein L chromatography purification; DTX-A-001 Fab' comprises a MW of approximately 49 kDa. Lane 2 shows a reference SeeBlue Plus2 ladder containing standard mass proteins.

[0071] Figure 2 Provided are images of SDS-PAGE gels containing a complex of DTX-A-001Fab' linked to an antisense oligonucleotide (DMPK ASO), a mixture of unlinked DMPK ASO and unlinked DTX-A-001Fab' before and after contact with a hydrophobic interaction chromatography (HIC) resin. The mixture before and after HIC purification is shown in the innermost lane; See Blue Plus2 ladder is shown in the outermost lane.

[0072] Figure 3 is an overlay of three analytical size exclusion chromatography (SEC) experiments demonstrating the purification of a complex comprising DTX-A-001 Fab' linked to an antisense oligonucleotide (DMPK ASO) from an unlinked DMPK ASO. An unpurified mixture of a complex comprising DTX-A-001 Fab' linked to an antisense oligonucleotide (DMPK ASO) and unlinked DMPK ASO before contact with hydroxyapatite (HA) resin ('HA load'); unlinked DMPK ASO that is not bound to the HA resin ('HA flow-through'); and a purified complex ('HA eluate').

[0073] Figure 4is a chromatogram showing the separation of a complex comprising DTX-P-060 covalently linked to DTX-A-012 from a mixture comprising the complex, unlinked DTX-P-060, and unlinked DTX-A-012 by hydrophobic interaction chromatography.

[0074] Figure 5 is a chromatogram showing the separation of a complex comprising DTX-P-060 covalently linked to DTX-A-012 from a mixture comprising the complex and unlinked DTX-P-060 by mixed mode resin (hydroxyapatite) chromatography.

[0075] Figure 6 is a chromatogram showing the separation of a complex comprising DTX-P-060 covalently linked to an anti-transferrin receptor antibody listed in Table 2 from a mixture comprising the complex, unlinked DTX-P-060 and unlinked antibody by hydrophobic interaction chromatography. The complex was eluted using a shallow gradient (gradient over 8 column volumes) of decreasing ammonium sulfate concentration, resulting in the separation of different DAR species (DAR 1 and DAR 2).

[0076] Figure 7 The use of analytical size exclusion chromatography (SEC) to Figure 6 The top panel shows an A280 nm overlay of the pooled fractions from the hydrophobic interaction chromatography. The middle panel shows the A280 nm of the flow-through from the hydrophobic interaction chromatography. The bottom panel shows the A280 nm of the shoulder peak from the hydrophobic interaction chromatography.

[0077] Figure 8 : is a chromatogram showing the separation of a complex comprising DTX-P-060 covalently linked to the Fab' of the anti-transferrin receptor antibody listed in Table 2 from a mixture comprising the complex, unlinked DTX-P-060 and unlinked antibody by hydrophobic interaction chromatography. The complex was eluted using a steep gradient (gradient over 6 column volumes) of decreasing ammonium sulfate concentration. Figure 6 Compared to the elution using a slow gradient, the DAR species were pooled.

[0078] Fig. 9 The use of analytical size exclusion chromatography (SEC) to Figure 8The upper graph shows the A280 nm of the merged peak with a conductivity of 41 mS / cm from the hydrophobic interaction chromatography, which contains >96% DAR1. The middle graph shows the A280 nm of the merged peak with a conductivity of 33 mS / cm from the hydrophobic interaction chromatography, which contains >55% DAR2. The lower graph shows the A280 nm of the merged peak with a conductivity of 22 mS / cm from the hydrophobic interaction chromatography, which contains >69% DAR 2 and some level of DAR 3.

[0079] Fig.10 Is from Figure 8 SDS-PAGE analysis of the pooled peaks.

[0080] Fig.11A and 11B Shown is the purification of a complex comprising an anti-transferrin receptor Fab' covalently linked to one or more oligonucleotides by hydrophobic interaction chromatography from a mixture comprising the complex, unlinked oligonucleotides, and unlinked antibodies. Fig.11A The purification chromatogram is shown. Fig. 11B Analysis of eluted fractions using SDS-PAGE is shown.

[0081] Fig. 12A and 12B Shown is the purification of a complex comprising an anti-transferrin receptor Fab' covalently linked to one or more oligonucleotides by hydrophobic interaction chromatography from a mixture comprising the complex, unlinked oligonucleotides, and unlinked antibodies. Fig. 12A The purification chromatogram is shown. Fig. 12B Analysis of eluted fractions using SDS-PAGE is shown.

[0082] Fig.13A and 13B Shown is the purification of a complex comprising the RI7217 antibody (full length IgG) covalently linked to DTX-P-60 from a mixture comprising the complex, unlinked DTX-P-60, and unlinked antibody by hydrophobic interaction chromatography. Fig.11A The purification chromatogram is shown. Fig. 11B Analysis of eluted fractions using SDS-PAGE is shown.

[0083] Fig.14 is a chromatogram showing the use of mixed mode resin (FA resin) Figures 13A to 13B The complexes described in are further separated from unligated oligonucleotides.

[0084] Fig.15 The analysis using analytical size exclusion chromatography (SEC) is shown. Fig.14The upper panel shows the dissociation of the unligated oligonucleotide from the mixed mode resin at a phosphate ion concentration of 10 mM. The lower panel shows the complex eluted at a phosphate ion concentration of 100 mM. DETAILED DESCRIPTION

[0085] As described herein, the present disclosure provides methods for purifying complexes, e.g., complexes comprising a protein, e.g., a muscle targeting agent (e.g., an antibody) covalently linked to a molecular payload (e.g., an oligonucleotide). In some embodiments, the complexes are purified from a mixture containing one or more complexes comprising a protein (e.g., an antibody) covalently linked to an oligonucleotide and unlinked (e.g., excess) protein using a hydrophobic resin. In some embodiments, the complexes are purified from a mixture containing one or more complexes comprising a protein (e.g., an antibody) covalently linked to an oligonucleotide and unlinked (e.g., excess) oligonucleotide using a mixed mode resin (e.g., hydroxyapatite resin, ceramic hydroxyapatite resin, hydroxyfluoroapatite resin, fluoroapatite resin, chloroapatite resin) comprising positively charged metal sites and negatively charged ionic sites. In some embodiments, the complexes are purified from a mixture containing one or more complexes comprising a protein (e.g., an antibody) covalently linked to an oligonucleotide, unlinked (e.g., excess) oligonucleotide, and unlinked (e.g., excess protein) using a first purification step involving a hydrophilic interaction chromatography resin and a second purification step involving a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites (e.g., hydroxyapatite resin, ceramic hydroxyapatite resin, hydroxyfluoroapatite resin, fluoroapatite resin, chloroapatite resin).

[0086] In some embodiments, the purified complexes are particularly useful for delivering molecular payloads (e.g., oligonucleotides) that modulate the expression or activity of a target gene in a muscle cell, such as in a subject suffering from or suspected of suffering from a muscle disease. For example, in some embodiments, the complexes can be used to treat subjects suffering from rare muscle diseases, including Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva, Friedreich's ataxia, or Duchenne muscular dystrophy. In some embodiments, different oligonucleotides can be used in such complexes, depending on the condition to be treated.

[0087] Other aspects of the disclosure are provided below, including descriptions of defined terms.

[0088] I. Definitions

[0089] Administration: As used herein, the term "administering" or "administration" means providing a complex to a subject in a physiologically and / or pharmacologically acceptable manner (eg, to treat a condition in a subject).

[0090] Approximately: As used herein, the term "approximately" or "about", as applied to one or more target values, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of a stated reference value, unless otherwise specified or otherwise apparent from the context (unless such a number exceeds 100% of possible values).

[0091] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant (e.g., a paratope that specifically binds to an antigen). In some embodiments, the antibody is a full-length antibody, such as a full-length IgG. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment, or a scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a double antibody. In some embodiments, the antibody comprises a framework with a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (referred to herein as VH) and / or a light (L) chain variable region (referred to herein as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains and their functional variations are known. With regard to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a specific embodiment, the antibody described herein comprises a human γ1CH1, CH2 and / or CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with any variable chain constant region provided herein. In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and / or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules.In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and / or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure connected to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues connected by a peptide bond and is used to connect one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). In addition, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, a marker peptide, and a C-terminal polyhistidine tag to prepare bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0092] CDR: As used herein, the term "CDR" refers to the complementarity determining region within an antibody variable sequence. There are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to the group of three CDRs that appear within a single variable region and are capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Subportions of the CDRs may be designated as L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)) have described other boundaries that define CDRs that overlap with the Kabat CDRs. Other CDR boundary definitions may not strictly follow one of the above systems, but may still be consistent with the Kabat CDRs. CDRs overlap, although they can be shortened or lengthened based on predictions or based on experimental findings that specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. Although preferred embodiments use CDRs defined by Kabat or Chothia, the methods used herein can utilize CDRs defined according to any of these systems.

[0093] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species but in which the sequence of one or more CDR regions of VH and / or VL is replaced by a CDR sequence from another species, such as an antibody having mouse heavy and light chain variable regions in which one or more mouse CDRs (e.g., CDR3) have been replaced by a human CDR sequence.

[0094] Chimeric antibody: The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0095] Complementary: As used herein, the term "complementary" refers to the ability to accurately pair between two nucleotides or two groups of nucleotides. In particular, complementarity is a term that characterizes the degree of binding between two nucleotides or two groups of nucleotides caused by hydrogen bond pairing. For example, if the base at one position of an oligonucleotide can hydrogen bond with the base at the corresponding position of a target nucleic acid (e.g., mRNA), the bases are considered to be complementary to each other at this position. Base pairing can include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, adenosine bases (A) are complementary to thymidine bases (T) or uracil bases (U), cytosine bases (C) are complementary to guanosine bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary. Inosine (I) is also considered in the art to be a universal base, and is considered complementary to any of A, C, U, or T.

[0096] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as those found in references compiling such methods: for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0097] Covalently linked: As used herein, the term "covalently linked" refers to the feature of two or more molecules being linked together by at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, such as a disulfide bond or a disulfide bridge, that acts as a linker between the molecules. However, in some embodiments, two or more molecules can be covalently linked together by a molecule that acts as a linker that links the two or more molecules together by multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0098] Cross-reactivity: As used herein and in the case of targeting agents (e.g., antibodies), the term "cross-reactivity" refers to the property of a substance being able to specifically bind to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity. For example, in some embodiments, antibodies cross-reactive to human and non-human primate antigens of similar types or classes (e.g., human transferrin receptor and non-human primate transfer receptor) are able to bind to human antigens and non-human primate antigens with similar affinity or avidity. In some embodiments, antibodies are cross-reactive to human antigens and rodent antigens of similar types or classes. In some embodiments, antibodies are cross-reactive to rodent antigens and non-human primate antigens of similar types or classes. In some embodiments, antibodies are cross-reactive to human antigens, non-human primate antigens, and rodent antigens of similar types or classes.

[0099] Disease allele: As used herein, the term "disease allele" refers to any alternative form (e.g., mutant form) of a gene in which the allele is associated with a disease and / or directly or indirectly contributes to or causes a disease. Relative to a wild-type (non-disease) allele, a disease allele may comprise a genetic alteration, including but not limited to insertions (e.g., disease-associated duplications described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, the disease allele has a loss-of-function mutation. In some embodiments, the disease allele has a gain-of-function mutation. In some embodiments, the disease allele encodes an activating mutation (e.g., encoding a constitutively active protein). In some embodiments, the disease allele is a recessive allele with a recessive phenotype. In some embodiments, the disease allele is a dominant allele with a dominant phenotype.

[0100] Disease-associated repeats: As used herein, the term "disease-associated repeats" refers to a repeated nucleotide sequence at a genomic position, wherein the number of units of the repeated nucleotide sequence is associated with a genetic disease and / or directly or indirectly contributes to or causes a genetic disease. The length of each repeat unit of the disease-associated repeat can be 2, 3, 4, 5 or more nucleotides. For example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, the disease-associated repeat comprises CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTG repeats, or any nucleotide complementary sequence thereof. In some embodiments, the disease-associated repeat is in the non-coding portion of the gene. However, in some embodiments, the disease-associated repeat is in the coding region of the gene. In some embodiments, the disease-associated repeat is amplified from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeat is in RNA (e.g., RNA transcript). In some embodiments, the disease-associated repeat is in DNA (e.g., chromosome, plasmid). In some embodiments, the disease-associated repeats are expanded in the chromosomes of germ cells. In some embodiments, the disease-associated repeats are expanded in the chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the congenital onset of the disease. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the childhood onset of the disease. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the adult onset of the disease.

[0101] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus CDR. Since the exact definition of CDR sequences can be determined by different systems, the meaning of the framework sequence has different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also distinguish the framework on the light chain and the heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. In the case where a specific subregion is not designated as FR1, FR2, FR3 or FR4, the framework region mentioned by others represents the combined FR in the variable region of a single natural immunoglobulin chain. As used herein, FR represents one of the four subregions, and FRs represents two or more of the four subregions constituting the framework region. Human heavy chain and light chain receptor sequences are known in the art. In one embodiment, receptor sequences known in the art may be used in the antibodies disclosed herein.

[0102] Hydrophobic interaction chromatography resin: As used herein, the term "hydrophobic interaction chromatography resin" or "HIC resin" refers to a chromatography resin or material that plays a role in the purification, separation, and / or isolation of molecules based on hydrophobic interactions between the resin and molecules (e.g., proteins). In some embodiments, the HIC resin comprises a hydrophobic functional group, such as silica bonded to the hydrophobic functional group. In some embodiments, the hydrophobic functional group of the HIC resin is a butyl, tert-butyl, phenyl, ether, amide, or propyl group. In some embodiments, the hydrophobic functional group of the HIC resin is a straight chain alkyl or aryl group.

[0103] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in CDR, particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0104] Humanized antibody: The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from non-human species (e.g., mice) but wherein at least a portion of the VH and / or VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining mouse anti-transferrin receptor monoclonal antibodies using traditional hybridoma technology and then humanizing using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO 2005 / 123126 A2 of Kasaian et al.

[0105] Internalized cell surface receptor: As used herein, the term "internalized cell surface receptor" refers to a cell surface receptor that is internalized by a cell under external stimulation (e.g., ligand binding to a receptor). In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, pits and raft-mediated uptake or constitutive clathrin-independent endocytosis. In some embodiments, the internalized cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or an extracellular domain, which may optionally further comprise a ligand binding domain. In some embodiments, the cell surface receptor is internalized by a cell after ligand binding. In some embodiments, the ligand may be a muscle targeting protein or a muscle targeting antibody. In some embodiments, the internalized cell surface receptor is a transferrin receptor.

[0106] Separated antibody: "Separated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., a separated antibody that specifically binds to transferrin receptor is substantially free of antibodies that specifically bind to antigens other than transferrin receptor). However, a separated antibody that specifically binds to transferrin receptor complex may have cross-reactivity with other antigens (e.g., transferrin receptor molecules from other species). In addition, the separated antibody may be substantially free of other cellular materials and / or chemicals.

[0107] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat notation" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or antigen-binding portion thereof that are more variable (i.e., hypervariable) than other amino acid residues (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 2001-0306). No.91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is from amino acids 31 to 35, the hypervariable region of CDR2 is from amino acids 50 to 65, and the hypervariable region of CDR3 is from amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is from amino acids 24 to 34, the hypervariable region of CDR2 is from amino acids 50 to 56, and the hypervariable region of CDR3 is from amino acids 89 to 97.

[0108] Mixed mode resin: As used herein, the term "mixed mode resin" refers to a chromatographic resin or material for purification, separation and / or isolation of biomolecules that contains positively charged metal sites and negatively charged ionic sites. In some embodiments, the metal sites contain calcium. In some embodiments, the negatively charged ionic sites contain phosphate, sulfate, fluoride, or chloride. In some embodiments, the metal sites contain calcium and the negatively charged ionic sites contain phosphate, and optionally sulfate, fluoride, or chloride. In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. In some embodiments, the apatite resin contains a mineral of the formula: Ca 10 (PO4)6(OH)2. In some embodiments, the apatite resin comprises a mineral of the formula: Ca 10 (PO4)6F2. In some embodiments, the apatite resin comprises a mineral of the formula: Ca 10 (PO4)6Cl2.

[0109] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or substance that functions to modulate a biological outcome. In some embodiments, the molecular payload is linked or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload functions to modulate transcription of a DNA sequence, modulate expression of a protein, or modulate activity of a protein. In some embodiments, the molecular payload is an oligonucleotide, such as an oligonucleotide comprising a strand having a region complementary to a target gene.

[0110] Muscle disease gene: As used herein, the term "muscle disease gene" refers to a gene having at least one disease allele associated with a muscle disease and / or directly or indirectly contributing to or causing a muscle disease. In some embodiments, the muscle disease is a rare disease, such as defined by the Genetic and Rare Diseases Information Center (GARD), which is a program of the National Center for Advancing Translational Sciences (NCATS). In some embodiments, the muscle disease is a rare disease characterized by affecting less than 200,000 people. In some embodiments, the muscle disease is a monogenic disease. In some embodiments, the muscle disease gene is a gene listed in Table 1.

[0111] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on the muscle cell can be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Generally speaking, the muscle targeting agent specifically binds to an antigen on the muscle cell, which helps to internalize the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on the muscle and can be internalized into the muscle cell by receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle targeting agent is connected to a molecular payload. In some embodiments, the muscle targeting agent is a muscle targeting protein (e.g., an antibody).

[0112] Muscle targeting antibody: As used herein, the term "muscle targeting antibody" refers to a muscle targeting agent that is an antibody that specifically binds to an antigen present in or on a muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an antigen on a muscle cell, which facilitates internalization of the muscle targeting antibody (and any associated molecular cargo) into the muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0113] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, spacer polymers, mixed polymers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, the oligonucleotide may include one or more modified nucleotides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, the oligonucleotide may include one or more modified internucleotide linkages. In some embodiments, the oligonucleotide may include one or more phosphorothioate linkages, which may be in Rp or Sp stereochemical conformations.

[0114] Recombinant antibody: As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail in this disclosure), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor, LD, et al. (1992) Nucl. Acids Res.20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgenic for human Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not be naturally present in the human antibody germline repertoire in vivo. One embodiment of the present disclosure provides fully human antibodies capable of binding to human transferrin receptor, which can be generated using techniques known in the art, such as, but not limited to, using human Ig phage libraries, such as those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.

[0115] Complementary region: As used herein, the term "complementary region" refers to a nucleotide sequence, such as an oligonucleotide, that is fully complementary to a homologous nucleotide sequence, such as a target nucleic acid, such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is completely complementary to the homologous nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the homologous nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the complementary region comprises 1, 2, 3, or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.

[0116] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity compared to an appropriate reference antigen or antigens that enables the antibody to be used to distinguish the specific antigen from other antigens, for example, to a degree that allows for preferential targeting of certain cells (e.g., muscle cells) by binding to an antigen as described herein. In some embodiments, if the K of an antibody binding to a target is D is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 - 12 M, 10 -13 M or less, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to the transferrin receptor (eg, an epitope of the apical domain of the transferrin receptor).

[0117] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a human patient suffering from or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of having a muscle disease (e.g., any of the diseases provided in Table 1).

[0118] Transferrin receptor: As used herein, the term "transferrin receptor" (also referred to as TFRC, CD71, p90 or TFR1) refers to a cell surface receptor that binds transferrin to promote internalization of iron uptake by endocytosis. In some embodiments, the transferrin receptor can be human-derived (NCBI gene ID 7037), non-human primate-derived (e.g., NCBI gene ID 711568 or NCBI gene ID 102136007), or rodent-derived (e.g., NCBI gene ID 22042). In addition, a variety of human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0119] Unligated oligonucleotide: As used herein, the term "unligated oligonucleotide" refers to free oligonucleotide or excess oligonucleotide present in solution, for example, after a coupling reaction used to generate a complex comprising a protein linked to an oligonucleotide.

[0120] Unlinked protein: As used herein, the term "unlinked protein" refers to free protein or excess protein (eg, free antibody or excess antibody) present in solution, such as after a coupling reaction used to generate a complex comprising the protein linked to an oligonucleotide.

[0121] II. Purification Methods

[0122] Provided herein is a method for purifying or separating a complex comprising a protein (e.g., an antibody) covalently attached to one or more oligonucleotides (e.g., a single-stranded oligonucleotide). In some aspects, the disclosure provides a method for separating the complex from a mixture of one or more complexes containing an antibody covalently attached to one or more oligonucleotides, unattached antibodies, and unattached oligonucleotides using one or more adsorption chromatography steps. In some embodiments, the adsorption chromatography step includes hydrophobic interaction chromatography (HIC) and mixed mode resin (e.g., apatite resin) chromatography. In some aspects, the disclosure provides a method for separating the complex from unattached antibodies, such as by hydrophobic interaction chromatography (HIC). In some aspects, the disclosure provides a method for separating the complex from unattached oligonucleotides, such as by mixed mode resin (e.g., apatite resin) chromatography. In some embodiments, the method for purifying one or more complexes described herein involves separating the complex from unattached oligonucleotides by removing unattached antibodies with hydrophobic interaction chromatography (HIC), and separating the complex from unattached oligonucleotides by mixed mode resin chromatography.

[0123] In some aspects, the methods of purifying one or more complexes described herein involve contacting a mixture comprising one or more complexes and unattached proteins (e.g., unattached antibodies) with a hydrophobic resin, removing unattached antibodies that do not adsorb to the hydrophobic resin, and eluting the adsorbed complexes from the hydrophobic resin. In some embodiments, the hydrophobic resin comprises a hydrophobic moiety selected from butyl, tert-butyl, phenyl, ether, amide, or propyl.

[0124] In some aspects, the methods of purifying one or more complexes described herein include contacting a mixture comprising one or more complexes and unattached oligonucleotides with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites, removing unattached oligonucleotides, and eluting adsorbed complexes from the mixed mode resin. In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.

[0125] In some embodiments, the complex is sufficiently purified to be free of unligated oligonucleotides and unligated proteins. In some embodiments, the composition of the complex after purification from unligated oligonucleotides and / or unligated proteins using the methods described herein does not contain any detectable levels of unligated oligonucleotides or unligated proteins.

[0126] In some embodiments, the methods described herein are suitable for separating complexes comprising antibodies covalently linked to one or more oligonucleotides. In some embodiments, the antibody can be a full-length IgG, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv fragment. The specific antibody sequence does not affect the purification outcome. In some embodiments, the antibody is an anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody listed in Table 2) or any antigen-binding fragment thereof (e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv fragment)

[0127] In some embodiments, oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide is a spacer polymer or a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the antibody is covalently linked to the 5' end of the single-stranded oligonucleotide. In some embodiments, the antibody is covalently linked to the 3' end of the single-stranded oligonucleotide. In some embodiments, the antibody is covalently linked to the 5' end of the antisense oligonucleotide (e.g., spacer polymer or PMO).

[0128] In some embodiments, the single-stranded oligonucleotide is a chain of a double-stranded oligonucleotide. The conditions of the hydrophobic interaction chromatography do not allow the two chains of the double-stranded oligonucleotide to anneal. However, a chain of the double-stranded oligonucleotide can be covalently conjugated to an antibody, and the complex can be separated using the methods described herein, and then the other chain of the double-stranded oligonucleotide is annealed. In some embodiments, the double-stranded oligonucleotide is siRNA, and the sense strand is covalently linked to the antibody (e.g., at the 3' end or the 5' end). In some embodiments, the complex purified using the methods described herein comprises an antibody covalently linked to the 3' end of the sense strand of the siRNA. The antisense strand of the siRNA can be annealed to the sense strand after purification.

[0129] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the modified nucleotide comprises 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), 2'-fluorine modification or morpholino modification.

[0130] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (e.g., an antisense oligonucleotide) containing a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), a locked nucleic acid (LNA), a 2'-fluoro modification, or a morpholino modification. In some embodiments, the antisense oligonucleotide is a spacer containing a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), a locked nucleic acid (LNA), or a 2'-fluoro modification. In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). The antisense oligonucleotide may contain more than one type of modification described herein, for example, with MOE and 2'-fluoro modification.

[0131] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (e.g., one strand of a double-stranded RNA (e.g., siRNA)) containing modified nucleotides comprising 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), or 2'-fluoro modifications. In some embodiments, the oligonucleotide is the sense strand of the siRNA, containing modified nucleotides comprising 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), or 2'-fluoro modifications. In some embodiments, the oligonucleotide is the antisense strand of the siRNA, containing modified nucleotides comprising 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), or 2'-fluoro modifications. The sense strand and antisense strand of the siRNA may contain the same type or different types of modifications described herein. One or both strands of the siRNA may contain more than one type of modification described herein, for example, with MOE and 2'-fluoro modifications.

[0132] In some embodiments, the oligonucleotide is 10 to 50 (e.g., 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 50, 20 to 40, 20 to 30, 30 to 50, 30 to 40, or 40 to 50 nucleotides) in length, and in some embodiments, the oligonucleotide is 15 to 25 (e.g., 10 to 25, 10 to 20, 10 to 25, 15 to 25, 15 to 20, or 20 to 25) nucleotides in length.

[0133] In some embodiments, the oligonucleotide is covalently linked to the antibody via a lysine or cysteine. In some embodiments, the oligonucleotide is covalently linked to the antibody via a linker (e.g., a linker comprising a Val-cit linker).

[0134] A. Removal of unbound proteins (e.g., antibodies) from a mixture of complexes and unbound oligonucleotides

[0135] It is shown herein that the use of HIC resin effectively purifies a mixture of complexes and unconnected oligonucleotides away from unconnected proteins (e.g., unconnected antibodies). In some aspects, the disclosure provides a method for separating a complex or multiple complexes, each of which comprises a protein (e.g., antibody) covalently linked to one or more oligonucleotides, the method comprising: (i) contacting a mixture comprising a complex and unconnected proteins (e.g., antibodies) with a hydrophobic resin under conditions (e.g., pH, ionic strength, conductivity) where the complex is adsorbed to the hydrophobic resin but the unconnected antibody is not adsorbed to the hydrophobic resin, thereby separating unconnected proteins (e.g., antibodies) from the complex adsorbed to the hydrophobic resin; and (ii) eluting the complex from the hydrophobic resin under conditions where the complex is dissociated from the hydrophobic resin. In some embodiments, the mixture in step (i) also comprises unconnected oligonucleotides. In some embodiments, under the conditions of step (i), unconnected oligonucleotides are also adsorbed to the hydrophobic resin, and under the conditions of step (ii), unconnected oligonucleotides are co-eluted with the complex.

[0136] In some embodiments, the conditions in step (i) that allow binding of the complex and optional unligated oligonucleotide but not binding of the unligated protein include a conductivity of at least 70 mS / cm (e.g., at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 mS / cm). In some embodiments, the conditions that allow the complex and optional unattached oligonucleotide to bind but the unattached protein to not bind in step (i) include a conductivity of 70 to 300 mS / cm (e.g., 70 to 300 mS / cm, 70 to 200 mS / cm, 70 to 100 mS / cm, 100 to 300 mS / cm, 100 to 200 mS / cm, or 200 to 300 mS / cm). In some embodiments, the conditions that allow the complex and optional unattached oligonucleotide to bind but the unattached protein to not bind in step (i) include a conductivity of 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mS / cm. In some embodiments, under conditions comprising a conductivity of at least 70 mS / cm, unattached antibodies (e.g., unattached full-length IgG or unattached Fab) are not adsorbed to the hydrophobic resin (i.e., in the flow-through), and thus the antibodies are separated from the complex and optional unattached oligonucleotide adsorbed to the hydrophobic resin. In some embodiments, the methods described herein further comprise washing the hydrophobic resin between steps (i) and (ii) with a solution having a conductivity that allows adsorption of the complex and optional unattached oligonucleotide but not adsorbing the unattached protein (e.g., unattached antibody) to remove loosely bound but unadsorbed unattached proteins (e.g., unattached antibodies).

[0137] In some embodiments, the conditions in steps (i) and (ii) are achieved using anti-chaotropic salts or molecular agents of varying concentrations. In some embodiments, anti-chaotropic salts or molecular agents are salts or molecular agents that allow water molecules to interact favorably and stabilize intramolecular interactions in macromolecules and biomolecules (e.g., proteins). Examples of anti-chaotropic salts and molecular agents include sulfates (e.g., ammonium sulfate or sodium sulfate), carbohydrates (e.g., trehalose and glucose), proline, and tert-butyl alcohol. In some embodiments, the anti-chaotropic salt used in the methods described herein is ammonium sulfate.

[0138] In some embodiments, the mixture in step (i) further comprises at least 500 mM of an anti-chaotropic salt (e.g., ammonium sulfate). For example, the mixture in step (i) may further comprise at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, at least 1.4 M, at least 1.5 M, at least 1.6 M, at least 1.7 M, at least 1.8 M, at least 1.9 M, or at least 2 M of an anti-chaotropic salt (e.g., ammonium sulfate). In some embodiments, the mixture in step (i) further comprises 500 mM to 1 M of an anti-chaotropic salt (e.g., ammonium sulfate). For example, the mixture in step (i) may further comprise a chaotropic salt (e.g., ammonium sulfate) at 500mM to 1M, 500mM to 900mM, 500mM to 800mM, 500mM to 700mM, 500mM to 600mM, 600mM to 1M, 600mM to 900mM, 600mM to 800mM, 600mM to 700mM, 700mM to 1M, 700mM to 900mM, 700mM to 800mM, 800mM to 1M, 800mM to 900mM, or 900mM to 1M. In some embodiments, the mixture in step (i) may further comprise a chaotropic salt (e.g., ammonium sulfate) at 500mM, 600mM, 700mM, 800mM, 900mM, or 1M. In some embodiments, the mixture in step (i) further comprises an anti-chaotropic salt (e.g., ammonium sulfate) at 600 mM, 800 mM, or 1 M. Under these conditions, unlinked antibody (e.g., unlinked full-length IgG, Fab', or (Fab')2) is not adsorbed to the hydrophobic resin (i.e., in the flow-through), while the complex and optionally unlinked oligonucleotide are adsorbed to the hydrophobic resin, thereby separating the antibody from the complex and optionally unlinked oligonucleotide.

[0139] In some embodiments, the hydrophobic resin is balanced with a solution before step (i). In some embodiments, the solution for rebalancing the hydrophobic resin has a conductivity (e.g., at least 70 mS / cm) that allows complexes and optional unconnected oligonucleotides to be adsorbed but unconnected proteins (e.g., unconnected antibodies) to not be adsorbed. In some embodiments, the solution for rebalancing the hydrophobic resin comprises at least 500 mM of anti-chaotropic salts (e.g., ammonium sulfate). In some embodiments, the solution for balancing the hydrophobic resin comprises at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, at least 1.4 M, at least 1.5 M, at least 1.6 M, at least 1.7 M, at least 1.8 M, at least 1.9 M or at least 2 M of anti-chaotropic salts (e.g., ammonium sulfate). In some embodiments, the solution used to equilibrate the hydrophobic resin comprises an anti-chaotropic salt (e.g., ammonium sulfate) of 500 mM to 1 M. For example, the solution used to equilibrate the hydrophobic resin may comprise an anti-chaotropic salt (e.g., ammonium sulfate) of 500 mM to 1 M, 500 mM to 900 mM, 500 mM to 800 mM, 500 mM to 700 mM, 500 mM to 600 mM, 600 mM to 1 M, 600 mM to 900 mM, 600 mM to 800 mM, 600 mM to 700 mM, 700 mM to 1 M, 700 mM to 900 mM, 700 mM to 800 mM, 800 mM to 1 M, 800 mM to 900 mM, or 900 mM to 1 M. In some embodiments, the solution used to equilibrate the hydrophobic resin comprises 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M of a chaotropic salt (e.g., ammonium sulfate). In some embodiments, the solution used to equilibrate the hydrophobic resin comprises 600 mM, 800 mM, or 1 M of a chaotropic salt (e.g., ammonium sulfate).

[0140] In some embodiments, the methods described herein further comprise washing the hydrophobic resin between step (i) and step (ii) with a solution having a conductivity (e.g., at least 70 mS / cm) that allows adsorption of the complex and optionally unattached oligonucleotides but not adsorbing unattached proteins (e.g., unattached antibodies). In some embodiments, the solution used to wash the hydrophobic resin between step (i) and step (ii) comprises at least 500 mM of an anti-chaotropic salt (e.g., ammonium sulfate) to remove loosely bound but unadsorbed unattached proteins (e.g., unattached antibodies). In some embodiments, the solution used to wash the hydrophobic resin between step (i) and step (ii) comprises at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, at least 1.4 M, at least 1.5 M, at least 1.6 M, at least 1.7 M, at least 1.8 M, at least 1.9 M, or at least 2 M of a chaotropic salt (e.g., ammonium sulfate). In some embodiments, the solution used to wash the hydrophobic resin between step (i) and step (ii) comprises 500 mM to 1 M of a chaotropic salt (e.g., ammonium sulfate). For example, the solution used to wash the hydrophobic resin between step (i) and step (ii) may contain 500mM to 1M, 500mM to 900mM, 500mM to 800mM, 500mM to 700mM, 500mM to 600mM, 600mM to 1M, 600mM to 900mM, 600mM to 800mM, 600mM to 700mM, 700mM to 1M, 700mM to 900mM, 700mM to 800mM, 800mM to 1M, 800mM to 900mM, or 900mM to 1M of an anti-chaotropic salt (e.g., ammonium sulfate). In some embodiments, the solution used to wash the hydrophobic resin between step (i) and step (ii) comprises 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M of a chaotropic salt (eg, ammonium sulfate).

[0141] In some embodiments, in order to elute the complex and optionally unattached oligonucleotide from the hydrophobic resin in step (ii), the hydrophobic resin and the adsorbed complex and optionally unattached oligonucleotide are subjected to conditions that allow the complex and optionally unattached oligonucleotide to dissociate from the hydrophobic resin. In some embodiments, the conditions that allow the complex and optionally unattached oligonucleotide to dissociate in step (ii) include a conductivity of 10 to 70 mS / cm. For example, the conditions allowing dissociation of the complex and, optionally, the unligated oligonucleotide in step (ii) may comprise a conductivity of 10 to 70 mS / cm, 10 to 60 mS / cm, 10 to 50 mS / cm, 10 to 40 mS / cm, 10 to 30 mS / cm, 10 to 20 mS / cm, 20 to 70 mS / cm, 20 to 60 mS / cm, 20 to 50 mS / cm, 20 to 40 mS / cm, 20 to 30 mS / cm, 30 to 70 mS / cm, 30 to 60 mS / cm, 30 to 50 mS / cm, 30 to 40 mS / cm, 40 to 70 mS / cm, 40 to 60 mS / cm, 40 to 50 mS / cm, 50 to 70 mS / cm, 50 to 60 mS / cm, or 60 to 70 mS / cm. In some embodiments, the conditions in step (ii) that allow dissociation of the complex and optional unligated oligonucleotide comprise a conductivity of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mS / cm.

[0142] In some embodiments, step (ii) comprises applying to the hydrophobic resin an elution solution comprising up to 200 mM (e.g., 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, or 200 mM) of chloride ions and up to 100 mM (e.g., 10 mM, 15 mM, 50 mM, or 100 mM) of a chaotropic salt (e.g., ammonium sulfate) to elute the complex. In some embodiments, step (ii) comprises applying to the hydrophobic resin an elution solution comprising 0 to 200 mM (e.g., 0 to 200 mM, 0 to 150 mM, 0 to 100 mM, 0 to 50 mM, 50 to 200 mM, 50 to 150 mM, 50 to 100 mM, 100 to 200 mM, 100 to 150 mM, or 150 to 200 mM) of chloride ions and 0 to 100 mM (e.g., 0 In some embodiments, the elution solution used in step (ii) does not include ammonium sulfate. In some embodiments, the elution solution is phosphate buffered saline (PBS, which includes 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4 and 1.8mM KH2PO4). In some embodiments, the elution solution comprises up to 25 mM (e.g., 5, 10, 15, 15, 20, or 25 mM) chloride ions and is free of chaotropic salts (e.g., ammonium sulfate), and optionally also comprises up to 10 mM (e.g., 2, 5, or 10 mM) phosphate ions. In some embodiments, the elution solution comprises 0 to 25 mM (e.g., 0 to 25 mM, 0 to 20 mM, 0 to 15 mM, 0 to 10 mM, 0 to 5 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 25 mM, 15 to 20 mM, or 20 to 25 mM) of chloride ions, and does not contain a chaotropic salt (e.g., ammonium sulfate), and optionally also comprises 0 to 10 mM (e.g., 0 to 10 mM, 0 to 5 mM, or 5 to 10 mM) of phosphate ions. In some embodiments, the elution solution comprises 25 mM of chloride and does not contain ammonium sulfate, and also comprises 10 mM of phosphate ions.

[0143] In some embodiments, the elution solution also comprises the counter ions of phosphate ion and chloride ion. In some embodiments, the counter ion of phosphate is calcium, sodium, magnesium, potassium or manganese. In some embodiments, the phosphate ion source is NaH2PO4, Na2HPO4 or Na3PO4. In some embodiments, the counter ion of chloride is calcium, sodium, magnesium, potassium or manganese. In some embodiments, the counter ion used in the method described herein is sodium. It will be readily appreciated by those skilled in the art that many other equivalent salts and ions can be used for the method described herein.

[0144] The elution step can be carried out using an elution solution containing a salt (e.g., an anti-chaotropic salt, such as ammonium sulfate) with a single / constant concentration, or using an elution solution with a reduced salt (e.g., ammonium sulfate) concentration gradient. During elution (step (ii)), using a reduced salt (e.g., ammonium sulfate) concentration gradient allows separation of complexes with different drug: antibody ratios (DAR) numbers. For example, as the salt (e.g., ammonium sulfate) concentration in the elution solution decreases, complexes with lower DAR are eluted first, followed by complexes with higher DAR. In some embodiments, a complex with a DAR of 1 (one oligonucleotide conjugated to one antibody) is eluted at a conductivity of about 35 to 45 mS / cm (e.g., 35 to 45 mS / cm, 35 to 40 mS / cm, or 40 to 45 mS / cm). In some embodiments, the complex with a DAR of 1 is eluted at a conductivity of about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mS / cm. In some embodiments, the complex with a DAR of 2 (two oligonucleotides conjugated to one antibody) is eluted at a conductivity of about 20 to 35 mS / cm (e.g., 20 to 35 mS / cm, 20 to 30 mS / cm, 20 to 25 mS / cm, 25 to 35 mS / cm, 25 to 30 mS / cm, or 30 to 35 mS / cm). In some embodiments, the complex with a DAR of 2 is eluted at a conductivity of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mS / cm. In some embodiments, the complex with a DAR of 3 (three oligonucleotides conjugated to one antibody) is eluted at a conductivity of about 15 to 25 mS / cm (e.g., 15 to 25 mS / cm, 15 to 20 mS / cm, or 20 to 25 mS / cm). In some embodiments, the complex with a DAR of 3 is eluted at a conductivity of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mS / cm.

[0145] In some embodiments, step (ii) comprises applying a gradually decreasing concentration of a chaotropic salt (e.g., ammonium sulfate) to the hydrophobic resin to elute the complex and optionally the unligated oligonucleotide. In some embodiments, the concentration of the chaotropic salt (e.g., ammonium sulfate) is reduced from at least 500 mM to less than 100 mM. For example, the concentration of the chaotropic salt (e.g., ammonium sulfate) throughout the gradient can be 1M to 0mM, 1M to 50mM, 1M to 100mM, 900mM to 0mM, 900mM to 50mM, 900mM to 100mM, 800mM to 0mM, 800mM to 50mM, 800mM to 100mM, 700mM to 0mM, 700mM to 50mM, 700mM to 100mM, 600mM to 0mM, 600mM to 50mM, 600mM to 100mM, 500mM to 0mM, 500mM to 50mM, or 500mM to 100mM. In some embodiments, the concentration of the chaotropic salt (e.g., ammonium sulfate) throughout the gradient is 600mM to 100mM. In some embodiments, the concentration of the anti-chaotropic salt (e.g., ammonium sulfate) throughout the gradient is 800 mM to 0 mM. In some embodiments, the anti-chaotropic salt (e.g., ammonium sulfate) concentration gradient can be applied over 5 to 12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) column volumes (CV). In some embodiments, the anti-chaotropic salt (e.g., ammonium sulfate) concentration gradient can be applied over 6 to 8 column volumes (CV).

[0146] In some embodiments, the washing solution and / or the eluent solution may further comprise a buffer to maintain a consistent pH. In some embodiments, the washing buffer and / or the eluent buffer comprises a neutral pH. In some embodiments, the washing buffer and / or the eluent buffer has a pH of about 6, about 6.5, about 7, about 7.5, about 8, or about 6 to 8. Examples of buffers used herein include ethylenediaminetetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, cacodylate, 2-(N-morpholino)-ethanesulfonic acid (MES), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-2-ethanesulfonic acid (PIPES), 2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO), N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)-propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (TAPSO) , 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-[tris(hydroxymethyl)-methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), tris(hydroxymethyl)aminomethane (Tris), and bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of the solutions for use herein will be apparent to those skilled in the art.

[0147] The methods described herein may include any hydrophobic interaction chromatography (HIC) resin. In some embodiments, the HIC resin comprises butyl, tert-butyl, methyl and / or ethyl functional groups. Typically, the HIC resin comprises hydrophobic functional groups that can interact with biomolecules using hydrophobic interactions.

[0148] In some embodiments, the HIC resin comprises one or more hydrophobic functional groups. In some embodiments, the HIC medium is HiTrap Butyl HP resin, CaptoPhenyl resin, Phenyl Sepharose TM 6 Resin, Phenyl Sepharose TM High performance resin, Octyl SepharoseTM High performance resin, Fractogel TM EMD Propyl resin, Fractogel TM EMD Phenyl resin, Macro-Prep TM Methyl resin, HiScreen Butyl FF, HiScreenOctyl FF or Tosoh Hexyl.

[0149] In some embodiments, the HIC resin can be equilibrated prior to contacting with the mixture of complexes, unligated oligonucleotides, and unligated proteins. In some embodiments, the HIC resin is equilibrated using a wash solution as described above. In some embodiments, the HIC resin is equilibrated such that the pH of the resin is neutral pH, a pH between 6 and 8, a pH of about 6.5, a pH of about 7.0, or a pH of about 7.5.

[0150] In some embodiments, the HIC resin is packed into a column (e.g., a vertical column). In some embodiments, the column can be used under pressure, optionally under pressure from top to bottom or from bottom to top. In some embodiments, the column can be used without external pressure, for example, using only gravity flow. In some embodiments, the HIC resin is used as a free resin, for example, using a batch process. In some embodiments, the batch process may also include a centrifugation and / or filtration step after the resin is contacted with the mixture of complexes, unligated oligonucleotides, and unligated proteins.

[0151] In some embodiments, the complex eluted in the step (ii) of hydrophobic chromatography described herein comprises an antibody covalently linked to 1, 2 or 3 oligonucleotides. In some embodiments, the complex of the oligonucleotides (e.g., 1, 2 or 3) with different numbers of connections is separated in different elution fractions, such as when eluting using a salt (e.g., ammonium sulfate) that reduces the concentration in step (ii). In some embodiments, the level of the unconnected antibody contained in the eluent obtained from step (ii) is undetectable. In some embodiments, the eluent obtained from step (ii) also comprises unconnected oligonucleotides. In some embodiments, the method described herein further comprises separating the complex from unconnected oligonucleotides.

[0152] B. Removal of unligated oligonucleotides from complexes using mixed mode resins

[0153] Purification of complexes comprising proteins covalently linked to oligonucleotides, especially on a large scale, has always been a challenge for scientists in the field. A major obstacle in the purification process is sufficient removal of unlinked oligonucleotides (e.g., excess oligonucleotides after a conjugation reaction between unlinked proteins and unlinked oligonucleotides) relative to the complexes comprising proteins covalently linked to oligonucleotides.

[0154] It is shown herein that the use of a mixed mode resin (e.g., apatite resin, e.g., hydroxyapatite resin) comprising a positively charged metal site and a negatively charged ionic site effectively purifies the complex away from unconnected oligonucleotides. This is a largely unexpected discovery, as no other purification strategy alternatives are able to remove substantially all unconnected oligonucleotides from a composition comprising a protein-oligonucleotide complex. In addition, the mixed mode resin purification method described herein is advantageous compared to other known methods for removing unconnected oligonucleotides and / or excess salt (desalting). One such known method is size exclusion chromatography (SEC). The mixed mode resin purification method is superior to SEC at least due to its scalability and higher recovery. Using the mixed mode resin method described herein, at least 90% of the complex recovery was achieved, while SEC could only achieve 20% to 30% of the complex recovery.

[0155] In some embodiments, the method of separating a complex or multiple complexes described herein, each complex comprising an antibody covalently linked to one or more oligonucleotides, comprises: (i) contacting a mixture comprising a complex and unattached oligonucleotides with a mixed mode resin comprising a positively charged metal site and a negatively charged ionic site under conditions where the complex is adsorbed to the mixed mode resin, and (ii) eluting the complex from the mixed mode resin under conditions where the complex dissociates from the mixed mode resin. In some embodiments, prior to step (i), the mixture in step (i) is separated from a hydrophobic interaction chromatography resin. As described herein, in some embodiments, the conditions under which the complex is adsorbed to the mixed mode resin in step (i) can be adjusted to allow or exclude adsorption of unattached oligonucleotides to the mixed mode resin.

[0156] In some embodiments, the conditions that make complex adsorbed to mixed mode resin in step (i) do not allow unconnected oligonucleotide to be adsorbed to mixed mode resin, so complex is separated from unconnected oligonucleotide.In some embodiments, the conditions are realized by including phosphate ions and / or chloride ions whose concentration allows complex adsorption to mixed mode resin but unconnected oligonucleotide is not adsorbed to mixed mode resin in the mixture in step (i).In some embodiments, the mixture comprising complex and unconnected oligonucleotide also comprises phosphate ions up to 20mM and / or chloride ions up to 30mM.In some embodiments, the mixture comprising complex and unconnected oligonucleotide also comprises phosphate ions up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM or up to 5mM). Additionally, in some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises up to 30 mM (e.g., up to 30 mM, up to 25 mM, up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM) chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 5 to 20 mM (e.g., 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 20 mM, 10 to 15 mM, or 15 to 20 mM) of phosphate ions and / or 5 to 30 mM of chloride ions (e.g., 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 30 mM, 20 to 25 mM, or 25 to 30 mM). In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises 20mM, 15mM, 10mM, 5mM or 1mM phosphate ions and / or 30mM, 25mM, 20mM, 15mM, 10mM or 5mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises 20mM phosphate ions and / or 30mM chloride ions, such as 20mM phosphate ions and 30mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises up to 10mM phosphate ions and / or up to 25mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises 5 to 10mM phosphate ions and / or 5 to 25mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 10 mM phosphate ions and / or 25 mM chloride ions, for example 10 mM phosphate ions and 25 mM chloride ions. Under these conditions, the unligated oligonucleotide is retained in the flow-through and is not adsorbed to the mixed mode resin.In some embodiments, the mixed mode resin may be further washed under these same conditions between step (i) and step (ii) to remove unligated oligonucleotides that are loosely bound but not adsorbed to the mixed mode resin.

[0157] In some embodiments, the conditions under which the complex is adsorbed to the mixed mode resin in step (i) also allow some or all (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the unattached oligonucleotides to be adsorbed to the mixed mode resin. In some embodiments, the conditions are achieved by including in the mixture of step (i) a concentration that allows both the complex and the unattached oligonucleotides to be adsorbed to the mixed mode resin with phosphate ions and / or chloride ions. In some embodiments, the mixture comprising the complex and the unattached oligonucleotides also comprises a maximum of 5 mM phosphate ions and / or a maximum of 10 mM chloride ions. In some embodiments, the mixture comprising the complex and the unattached oligonucleotides also comprises a maximum of 5 mM (e.g., a maximum of 5 mM, a maximum of 4 mM, a maximum of 3 mM, a maximum of 2 mM, or a maximum of 1 mM) phosphate ions. Additionally, in some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises chloride ions up to 10 mM (e.g., up to 10 mM, up to 9 mM, up to 8 mM, up to 7 mM, up to 6 mM, up to 5 mM, up to 4 mM, up to 3 mM, up to 2 mM, or up to 1 mM). In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 1 to 5 mM (e.g., 1 to 5 mM, 1 to 4 mM, 1 to 3 mM, 1 to 2 mM, 2 to 5 mM, 2 to 4 mM, 2 to 3 mM, 3 to 5 mM, 3 to 4 mM, or 4 to 5 mM) of phosphate ions and / or 1 to 10 mM (e.g., 1 to 10 mM, 1 to 8 mM, 1 to 6 mM, 1 to 4 mM, 1 to 2 mM, 2 to 10 mM, 2 to 8 mM, 2 to 6 mM, 2 to 4 mM, 4 to 10 mM, 4 to 8 mM, 4 to 6 mM, 6 to 10 mM, 6 to 8 mM, or 8 to 10 mM) of chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises 5mM, 4mM, 3mM, 2mM or 1mM phosphate ions and / or 10mM, 9mM, 8mM, 7mM, 6mM, 5mM, 4mM, 3mM, 2mM or 1mM chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises up to 3mM phosphate ions and / or up to 8mM chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises 1 to 3mM (e.g., 1, 2 or 3mM) phosphate ions and / or 1 to 8mM (e.g., 1, 2, 3, 4, 5, 6, 7 or 8mM) chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises 3mM phosphate ions and / or 8mM chloride ions, such as 3mM phosphate ions and 8mM chloride ions.Under these conditions, some of the total unligated oligonucleotides also adsorbed to the mixed mode resin.

[0158] In some embodiments, when some or all of the unconnected oligonucleotides are also adsorbed to the mixed mode resin, the method described herein also includes washing the mixed mode resin with a solution that allows the unconnected oligonucleotides to dissociate from the mixed mode resin but the complex does not dissociate from the mixed mode resin between step (i) and step (ii). In some embodiments, the solution for washing comprises a phosphate ion of up to 20mM and / or a chloride ion of up to 30mM, such as a phosphate ion of 20mM and a chloride ion of 30mM. In some embodiments, the solution for washing comprises a phosphate ion of up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM or up to 5mM). In addition, in some embodiments, the solution for washing comprises a chloride ion of up to 30mM (e.g., up to 30mM, up to 25mM, up to 20mM, up to 15mM, up to 10mM or up to 5mM). In some embodiments, the solution used for washing comprises 5 to 20 mM (e.g., 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 20 mM, 10 to 15 mM, or 15 to 20 mM) of phosphate ions and / or 5 to 30 mM of chloride ions (e.g., 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 30 mM, 20 to 25 mM, or 25 to 30 mM). In some embodiments, the solution for washing comprises 20mM, 15mM, 10mM, 5mM or 1mM phosphate ions and / or 30mM, 25mM, 20mM, 15mM, 10mM or 5mM chloride ions. In some embodiments, the solution for washing comprises 20mM phosphate ions and / or 30mM chloride ions, such as 20mM phosphate ions and 30mM chloride ions. In some embodiments, the solution for washing comprises up to 10mM phosphate ions and / or up to 25mM chloride ions, such as 10mM phosphate ions and up to 25mM chloride ions.

[0159] In some embodiments, in order to elute the complex from the mixed mode resin, in step (ii), the mixed mode and the combined complex are subjected to conditions that allow the complex to dissociate from the mixed mode resin. In some embodiments, the conditions that allow the complex to dissociate in step (ii) are achieved by applying an elution solution comprising a higher concentration of phosphate ions and / or chloride ions to the mixed mode resin. The elution step can be completed using an elution solution comprising a single phosphate ion concentration, or using an elution solution with an increased phosphate ion concentration gradient. Using an increased phosphate ion concentration gradient during elution (step (ii)) allows separation of complexes with different drug: antibody ratios (DAR) numbers. For example, as the phosphate ion concentration in the elution solution increases, complexes with lower DAR are eluted first, and then complexes with higher DAR are eluted.

[0160] In some embodiments, step (ii) comprises applying to the mixed mode resin an elution solution comprising at least 30 mM of phosphate ions and / or at least 50 mM of chloride ions to elute the complex. In some embodiments, the elution solution comprises at least 30 mM (e.g., at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, or at least 150 mM) of phosphate ions. In addition, in some embodiments, the elution solution comprises at least 50 mM (e.g., at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, or at least 200 mM) chloride ions. In some embodiments, the elution solution comprises at least 100 mM of phosphate ions and / or at least 100 mM of chloride ions. In some embodiments, the elution solution comprises 100 mM of phosphate ions and 100 mM of chloride ions.

[0161] In some embodiments, the mixture and solution used in the methods described herein also include counterions of phosphate ions and / or chloride ions. In some embodiments, the counterion of phosphate is calcium, sodium, magnesium, potassium or manganese. In some embodiments, the counterion used in the methods described herein is sodium. In some embodiments, the phosphate ion source is NaH2PO4, Na2HPO4 or Na3PO4. In some embodiments, the counterion of chloride is calcium, sodium, magnesium, potassium or manganese. In some embodiments, the chloride ion source is NaCl. It will be readily appreciated by those skilled in the art that many other equivalent salts and ions can be used for the methods described herein.

[0162] In some embodiments, the washing solution and / or the eluent solution may further comprise a buffer to maintain a consistent pH. In some embodiments, the washing buffer and / or the eluent buffer comprises a neutral pH. In some embodiments, the washing buffer and / or the eluent buffer has a pH of about 6, about 6.5, about 7, about 7.5, about 8, or about 6 to 8. Examples of buffers used herein include ethylenediaminetetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, cacodylate, 2-(N-morpholino)-ethanesulfonic acid (MES), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-2-ethanesulfonic acid (PIPES), 2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO), N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)-propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (TAPSO) , 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-[tris(hydroxymethyl)-methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), tris(hydroxymethyl)aminomethane (Tris), and bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of the solutions for use herein will be apparent to those skilled in the art.

[0163] According to the present disclosure, any mixed mode resin comprising a positively charged metal site and a negatively charged ionic site can be used. In some embodiments, the mixed mode resin used in the methods described herein is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. Apatite resins can comprise any form of apatite and are typically used as chromatographic solid phases in separation and purification of biomolecules (e.g., complexes described herein) using affinity, ion exchange, hydrophobic interactions, or a combination thereof.

[0164] In some embodiments, the hydroxyapatite resin is Bio-Gel HT resin, for example from Bio-Rad Laboratories, Inc. (Hercules, Calif., USA). In some embodiments, the ceramic hydroxyapatite resin is Bio-Scale Mini CHT resin, for example from Bio-Rad Laboratories, Inc. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) comprises spherical particles of apatite. In some embodiments, the diameter of the spherical particles of apatite is about 10 microns to about 100 microns, about 25 microns to about 50 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, or about 80 microns. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) is type I (medium porosity and high binding capacity) or type II (larger porosity and lower binding capacity). In some embodiments, the apatite particles can be mixed with another separation medium or support for use.

[0165] In some embodiments, the mixed mode resin can be balanced prior to contact with the mixture of complexes and unligated oligonucleotides. In some embodiments, the mixed mode resin is balanced using a wash solution as described above. In some embodiments, the mixed mode resin is balanced so that the pH of the resin is a neutral pH, a pH of 6 to 8, a pH of about 6.5, a pH of about 7.0, or a pH of about 7.5.

[0166] In some embodiments, the mixed mode resin is filled into a column (e.g., a vertical column). In some embodiments, the column can be used under pressure, optionally from top to bottom or from bottom to top. In some embodiments, the column can be used without external pressure, for example, using only gravity flow. In some embodiments, the mixed mode resin is used as a free resin, for example, using a batch process. In some embodiments, the batch process may also include a centrifugation and / or filtration step after the resin contacts the mixture of the complex and the unconnected oligonucleotide.

[0167] In some embodiments, the complex eluted in step (ii) of the mixed mode resin chromatography described herein comprises an antibody covalently linked to 1, 2 or 3 oligonucleotides. In some embodiments, complexes with different numbers of linked oligonucleotides (e.g., 1, 2 or 3) are separated in different elution fractions. In some embodiments, the eluate obtained from step (ii) comprises an undetectable level of unlinked oligonucleotides.

[0168] C. Removal of unbound proteins (e.g., antibodies) and unbound oligonucleotides relative to the complex using a hydrophobic resin followed by a mixed mode resin

[0169] In some aspects, the present disclosure provides a method for separating a complex or multiple complexes, each complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising: (i) contacting a first mixture comprising the complex, unlinked antibodies, and unlinked oligonucleotides with a hydrophobic resin under conditions wherein the complex and unlinked oligonucleotides are adsorbed to the hydrophobic resin but the unlinked antibodies are not adsorbed to the hydrophobic resin, thereby separating the unlinked antibodies from the complex and unlinked oligonucleotides adsorbed to the hydrophobic resin; and (ii) obtaining a second mixture comprising the complex and the unlinked oligonucleotides by eluting the complex and the unlinked oligonucleotides from the hydrophobic resin under conditions wherein the complex dissociates from the hydrophobic resin; (iii) contacting the second mixture obtained in step (ii) with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions wherein the complex is adsorbed to the mixed mode resin, and (iv) eluting the complex from the mixed mode resin under conditions wherein the complex dissociates from the mixed mode resin.

[0170] In some embodiments, the hydrophobic resin comprises a hydrophobic portion selected from butyl, tert-butyl, phenyl, ether, amide or propyl. In some embodiments, the mixed mode resin is an apatite resin, optionally wherein the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin or a chloroapatite resin. In some embodiments, the conditions in step (i) include a conductivity of at least 70 mS / cm (e.g., 70 to 300 mS / cm, 70 to 200 mS / cm, 70 to 100 mS / cm, 100 to 300 mS / cm, 100 to 200 mS / cm, or 200 to 300 mS / cm), and the conditions in step (ii) include a conductivity of 10 to 70 mS / cm (e.g., 10 to 70 mS / cm, 10 to 60 mS / cm, 10 to 50 mS / cm, 10 to 40 mS / cm, 10 to or 60 to 70 mS / cm).

[0171] In some embodiments, the conditions in step (i) and or step (ii) are achieved using an anti-chaotropic salt (e.g., ammonium sulfate). In some embodiments, the hydrophobic resin is balanced prior to step (i), for example, with a solution of ammonium sulfate comprising at least 500 mM (e.g., at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, at least 1.4 M, at least 1.5 M, at least 1.6 M, at least 1.7 M, at least 1.8 M, at least 1.9 M, or at least 2 M).

[0172] In some embodiments, the mixture in step (i) further comprises at least 500 mM (e.g., at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, at least 1.4 M, at least 1.5 M, at least 1.6 M, at least 1.7 M, at least 1.8 M, at least 1.9 M, or at least 2 M) of ammonium sulfate. In some embodiments, the mixture in step (i) further comprises 500mM to 1M (500mM to 1M, 500mM to 900mM, 500mM to 800mM, 500mM to 700mM, 500mM to 600mM, 600mM to 1M, 600mM to 900mM, 600mM to 800mM, 600mM to 700mM, 700mM to 1M, 700mM to 900mM, 700mM to 800mM, 800mM to 1M, 800mM to 900mM, or 900mM to 1M) ammonium sulfate.

[0173] In some embodiments, the method further comprises washing the hydrophobic resin between step (i) and step (ii) with ammonium sulfate comprising at least 500 mM (e.g., at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, at least 1.4 M, at least 1.5 M, at least 1.6 M, at least 1.7 M, at least 1.8 M, at least 1.9 M, or at least 2 M).

[0174] In some embodiments, step (ii) comprises applying a first elution solution to the hydrophobic resin to elute the complex and unligated oligonucleotide, wherein the first elution solution comprises up to 200 mM (e.g., 0 to 200 mM, 0 to 150 mM, 0 to 100 mM, 0 to 50 mM, 50 to 200 mM, 50 to 150 mM, 50 to 100 mM, 100 to 200 mM, 100 to 150 mM, or 150 mM). In some embodiments, the present invention provides an aqueous solution of at least 1 mM chloride ion, preferably at least 2 mM chloride ion, and preferably at least 2 mM ammonium sulfate. The aqueous solution may be present in an amount of at least 100 mM (e.g., 0 to 100 mM, 0 to 80 mM, 0 to 60 mM, 0 to 40 mM, 0 to 20 mM, 20 to 100 mM, 20 to 80 mM, 20 to 60 mM, 20 to 40 mM, 40 to 100 mM, 40 to 80 mM, 40 to 60 mM, 60 to 100 mM, 60 to 80 mM, or 80 to 100 mM).

[0175] In some embodiments, the first elution solution does not include ammonium sulfate. In some embodiments, the first elution solution is PBS. In some embodiments, the first elution solution includes up to 25mM chloride ions (e.g., 0 to 25mM, 0 to 20mM, 0 to 15mM, 0 to 10mM, 0 to 5mM, 5 to 25mM, 5 to 20mM, 5 to 15mM, 5 to 10mM, 10 to 25mM, 10 to 20mM, 10 to 15mM, 15 to 25mM, 15 to 20mM or 20 to 25mM) and does not contain ammonium sulfate.

[0176] In some embodiments, step (ii) comprises applying gradually decreasing concentrations of ammonium sulfate to the hydrophobic resin to elute the complex and unligated oligonucleotides, optionally wherein the concentration of ammonium sulfate is reduced from at least 500 mM to less than 100 mM and / or the gradually decreasing concentrations of ammonium sulfate are applied over 5 to 12 column volumes (CV), optionally 6 to 8 CV. For example, the ammonium sulfate concentration of the entire gradient can be 1M to 0mM, 1M to 50mM, 1M to 100mM, 900mM to 0mM, 900mM to 50mM, 900mM to 100mM, 800mM to 0mM, 800mM to 50mM, 800mM to 100mM, 700mM to 0mM, 700mM to 50mM, 700mM to 100mM, 600mM to 0mM, 600mM to 50mM, 600mM to 100mM, 500mM to 0mM, 500mM to 50mM, or 500mM to 100mM. In some embodiments, the ammonium sulfate concentration of the entire gradient is 600mM to 100mM. In some embodiments, the ammonium sulfate concentration of the entire gradient is 800mM to 0mM. In some embodiments, the anti-chaotropic salt (e.g., ammonium sulfate) concentration gradient can be applied over 5 to 12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) column volumes (CV). In some embodiments, the anti-chaotropic salt (e.g., ammonium sulfate) concentration gradient can be applied over 6 to 8 column volumes (CV).

[0177] In some embodiments, the second mixture in step (iii) further comprises up to 20 mM (e.g., 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 20 mM, 10 to 15 mM, or 15 to 20 mM) of phosphate ions and up to 30 mM (e.g., 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 30 mM, 20 to 25 mM, or 25 to 30 mM) of chloride ions. In some embodiments, the second mixture in step (iii) also includes up to 10 mM (e.g., 5 to 10 mM) of phosphate ions and up to 25 mM (e.g., 5 to 25 mM) of chloride ions. In some embodiments, the second mixture in step (iii) also includes 20 mM of phosphate ions and 30 mM of chloride ions. In some embodiments, the second mixture in step (iii) also includes 10 mM of phosphate ions and 25 mM of chloride ions. Under these conditions, the unattached oligonucleotide in step (iii) will not be adsorbed to the mixed mode resin.

[0178] In some embodiments, the second mixture in step (iii) also comprises up to 5mM (e.g., 1 to 5mM) of phosphate ions and / or up to 10mM (e.g., 5 to 10mM) of chloride ions, optionally wherein the second mixture in step (iii) also comprises up to 3mM (e.g., 1 to 3mM) of phosphate ions and / or up to 8mM (e.g., 5 to 8mM) of chloride ions. In some embodiments, the second mixture in step (iii) also comprises up to 5mM of phosphate ions and 10mM of chloride ions. In some embodiments, the second mixture in step (iii) also comprises up to 3mM of phosphate ions and 8mM of chloride ions. Under these conditions, some or all of the unattached oligonucleotides in step (iii) are adsorbed to the mixed mode resin.

[0179] In some embodiments, when some or all of the unligated oligonucleotides are also adsorbed to the mixed mode resin, the method further comprises washing the mixed mode resin between step (iii) and step (iv) with a solution comprising up to 20 mM (e.g., 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 20 mM, 10 to 15 mM, or 15 to 20 mM) of phosphate ions and up to 30 mM (e.g., 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 30 mM, 20 to 25 mM, or 25 to 30 mM) of chloride ions to remove the unligated oligonucleotides from the mixed mode resin. In some embodiments, the solution used for washing comprises up to 10 mM (eg, 5 to 10 mM) of phosphate ions and up to 25 mM (eg, 5 to 25 mM) of chloride ions.

[0180] In some embodiments, step (iv) comprises applying a second elution solution to the mixed mode resin to elute the complex, wherein the second elution solution comprises at least 30 mM (e.g., at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, or at least 150 mM) of phosphate ions and at least 50 mM (e.g., at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, or at least 200 mM) of chloride ions. In some embodiments, the second elution solution comprises 100 mM phosphate ions and 100 mM chloride ions.

[0181] In some embodiments, the mixture and solution used in the methods described herein also include counterions of phosphate ions and / or chloride ions. In some embodiments, the counterion of phosphate is calcium, sodium, magnesium, potassium or manganese. In some embodiments, the counterion used in the methods described herein is sodium. In some embodiments, the phosphate ion source is NaH2PO4, Na2HPO4 or Na3PO4. In some embodiments, the counterion of chloride is calcium, sodium, magnesium, potassium or manganese. In some embodiments, the chloride ion source is NaCl. It will be readily appreciated by those skilled in the art that many other equivalent salts and ions can be used for the methods described herein.

[0182] In some embodiments, the washing solution and / or the eluent solution may further comprise a buffer to maintain a consistent pH. In some embodiments, the washing buffer and / or the eluent buffer comprises a neutral pH. In some embodiments, the washing buffer and / or the eluent buffer has a pH of about 6, about 6.5, about 7, about 7.5, about 8, or about 6 to 8. Examples of buffers used herein include ethylenediaminetetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, cacodylate, 2-(N-morpholino)-ethanesulfonic acid (MES), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-2-ethanesulfonic acid (PIPES), 2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO), N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)-propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (TAPSO) , 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-[tris(hydroxymethyl)-methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), tris(hydroxymethyl)aminomethane (Tris), and bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of the solutions for use herein will be apparent to those skilled in the art.

[0183] D. Composition of the Purified Complex

[0184] The methods described herein can produce substantially pure complexes, wherein the composition of the purified complex does not contain detectable amounts of unattached oligonucleotides or unattached proteins. In some embodiments, the composition of the purified complex comprises at least 9: 1, at least 95: 5, 96: 4, 97: 3, 98: 2, 99: 1, 99.5: 5 or more molar or weight ratios of complex: unattached oligonucleotides. In some embodiments, the composition of the purified complex comprises at least 9: 1, at least 95: 5, 96: 4, 97: 3, 98: 2, 99: 1, 99.5: 5 or more molar or weight ratios of complex: unattached proteins.

[0185] In some embodiments, the composition of the purified complexes does not include detectable levels (e.g., detectable amounts) of unligated proteins. In some embodiments, the composition of the purified complexes does not include detectable levels (e.g., detectable amounts) of unligated oligonucleotides.

[0186] In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unattached protein (e.g., antibody) by molar ratio. In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unattached oligonucleotide by molar ratio. In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% unattached protein (e.g., antibody) on a molar basis and less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% unattached oligonucleotide on a molar basis.

[0187] In some embodiments, the composition of the purified complex comprises at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%) of the complex (i.e., protein (e.g., antibody) linked to one or more oligonucleotides) on a molar basis.

[0188] In some embodiments, the composition of the purified complexes comprises a protein (e.g., an antibody) linked to one oligonucleotide, two oligonucleotides, three oligonucleotides, and / or more oligonucleotides. In some embodiments, in the composition of the purified complexes, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) or more of the complexes comprise a protein (e.g., an antibody) (DAR1) linked to one oligonucleotide. In some embodiments, in the composition of the purified complexes, about 50%, 60%, 70%, 80%, 90%, or 95% of the complexes comprise a protein (e.g., an antibody) (DAR1) linked to one oligonucleotide.

[0189] In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30% or more of the complexes in the purified composition of complexes comprise one protein (e.g., antibody) linked to two oligonucleotides (DAR2). In some embodiments, about 1%, 2%, 3%, 5%, 7%, 10%, 20% or more of the complexes in the purified composition of complexes comprise one protein (e.g., antibody) linked to three or more oligonucleotides (DAR3+).

[0190] III. Complex

[0191] Provided herein are complexes comprising a targeting agent (e.g., an antibody) covalently linked to a molecular payload (e.g., an oligonucleotide). In some embodiments, the complex comprises a muscle targeting antibody covalently linked to an oligonucleotide. The complex may comprise an antibody that specifically binds to a single antigenic site or binds to at least two antigenic sites that may be present on the same or different antigens. The complex can be used to regulate the activity or function of at least one gene, protein, and / or nucleic acid. In some embodiments, the molecular payload present with the complex is responsible for the regulation of genes, proteins, and / or nucleic acids. The molecular payload can be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity that can regulate the activity or function of a gene, protein, and / or nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets a muscle disease allele in a muscle cell.

[0192] In some embodiments, the complex comprises a muscle targeting agent, such as an anti-transferrin receptor antibody, covalently linked to a molecular payload, such as an antisense oligonucleotide that targets a muscle disease allele.

[0193] In some embodiments, the complex can be used to treat muscle diseases, wherein the molecular payload affects the activity of the corresponding gene provided in Table 1. For example, depending on the condition, the molecular payload can modulate (e.g., reduce, increase) the transcription or expression of the gene, modulate the expression of the protein encoded by the gene, or modulate the activity of the encoded protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to the target gene provided in Table 1.

[0194] Table 1. List of muscle diseases and corresponding genes.

[0195]

[0196]

[0197]

[0198] A. Cell Targeting Agents

[0199] Some aspects of the present disclosure provide cell targeting agents (e.g., muscle targeting proteins) for, e.g., delivering oligonucleotides to muscle cells. In some embodiments, such cell targeting proteins are capable of binding to specific cells (e.g., by specifically binding to an antigen on the cell) and delivering the associated oligonucleotide to the cell. In some embodiments, the oligonucleotide is bound to the cell targeting agent (e.g., covalently bound), and the oligonucleotide is internalized into the cell (e.g., by endocytosis) after the cell targeting agent binds to an antigen on the cell.

[0200] Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, by specifically binding to an antigen on a muscle cell, and deliver the associated molecular payload to the muscle cell. In some embodiments, the molecular payload is bound to the muscle targeting agent (e.g., covalently bound), and after the muscle targeting agent binds to an antigen on a muscle cell, it is internalized into the muscle cell, for example, by endocytosis. Exemplary muscle targeting agents are described in further detail herein, however, it should be understood that the exemplary muscle targeting agents provided herein are not meant to be limiting.

[0201] Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscles (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, any muscle targeting agent provided herein binds to (e.g., specifically binds to) antigens on skeletal muscle cells, smooth muscle cells, and / or cardiac muscle cells.

[0202] By interacting with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters can be used to deliver molecular payloads (e.g., oligonucleotides) into muscle tissue. Binding to muscle surface recognition elements is followed by endocytosis, which allows even macromolecules (e.g., antibodies) to enter muscle cells. As another example, oligonucleotides conjugated to transferrin or anti-transferrin receptor antibodies can be taken up by muscle cells by binding to transferrin receptors, and can then be internalized, for example, by clathrin-mediated endocytosis.

[0203] The use of muscle targeting agents can be used to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells compared to another cell type within the subject. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells (e.g., skeletal muscle, smooth muscle, or cardiac muscle cells) in an amount up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times less than in non-muscle cells (e.g., liver, neurons, blood, or adipocytes). In some embodiments, when the molecular payload is delivered to a subject in conjunction with a muscle targeting agent, its toxicity in the subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%.

[0204] In some embodiments, a muscle recognition element (e.g., a myocyte antigen) may be required to achieve muscle selectivity. As an example, a muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, a muscle targeting agent may be an antibody that enters myocytes via transporter-mediated endocytosis. As another example, a muscle targeting agent may be a ligand that binds to a cell surface receptor on myocytes. It should be understood that while transporter-based approaches provide a direct route for cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0205] Myocytes encompassed by the present disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiomyocytes, myoblasts, and myocytes.

[0206] i. Muscle-targeted antibodies

[0207] In some embodiments, the muscle targeting agent is an antibody. Generally speaking, the high specificity of antibodies to their target antigens provides the potential for selective targeting of muscle cells (e.g., skeletal muscle, smooth muscle, and / or cardiomyocytes). This specificity can also limit off-target toxicity. Examples of antibodies that can target muscle cell surface antigens have been reported and are within the scope of the present disclosure. For example, antibodies targeting the surface of muscle cells are described in: Arahata K., et al. "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988; 333: 861-3; Song KS, et al. "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996; 271: 15160-5; and Weisbart RH et al., "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar, 39(13): 78309; the entire contents of each of which are incorporated herein by reference.

[0208] a. Anti-transferrin receptor antibodies

[0209] Some aspects of the present disclosure are based on such recognition: substances (e.g., anti-transferrin receptor antibodies) that bind to transferrin receptors can target myocytes. Transferrin receptors are internalized cell surface receptors that transfer transferrin through the cell membrane and participate in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-binding proteins that can bind to transferrin receptors. Therefore, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, the binding proteins that bind to transferrin receptors are internalized into myocytes together with any bound molecular payloads (e.g., oligonucleotides). As used herein, antibodies that bind to transferrin receptors can be referred to as anti-transferrin receptor antibodies. Antibodies that bind to transferrin receptors (e.g., specifically bind) can be internalized into cells after binding to transferrin receptors, for example, by receptor-mediated endocytosis.

[0210] It should be understood that several known methods (e.g., library design using phage display) can be used to produce, synthesize and / or derive anti-transferrin receptor antibodies. Exemplary methods have been characterized in the art and incorporated by reference (Díez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134: 2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies." 1985, Springer). In other embodiments, anti-transferrin antibodies have been previously characterized or disclosed.Antibodies that specifically bind to transferrin receptor are known in the art (see, e.g., U.S. Patent No. 4,364,934, filed December 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Patent No. 8,409,573, filed June 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Patent No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; US 9,611,323, filed December 19, 2014, "Low affinity bloodbrain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed December 24, 2014, "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al. al. "Structural features of the cellsurface receptor for transferrin that is recognized by the monoclonalantibody OKT9." J Biol Chem.1982,257:14,8516-8522.; Lee et al. "Targeting RatAnti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-BrainBarrier in Mouse" 2000,J Pharmacol. Exp. Ther., 292:1048-1052).

[0211] Any suitable anti-transferrin receptor antibody can be used in the complex disclosed herein. Examples of anti-transferrin receptor antibodies are listed in Table 2, including relevant references and binding epitopes. In some embodiments, the anti-transferrin receptor antibody comprises the complementary determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of any anti-transferrin receptor antibody provided herein (e.g., the anti-transferrin receptor antibody listed in Table 2).

[0212] Table 2 - List of anti-transferrin receptor antibody clones, including relevant references and binding epitope information.

[0213]

[0214]

[0215]

[0216] In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody. In some embodiments, the anti-transferrin receptor antibody specifically binds to a transferrin protein having an amino acid sequence as disclosed herein. In some embodiments, the anti-transferrin receptor antibody can specifically bind to any extracellular epitope of the transferrin receptor or an epitope that begins to be exposed to the antibody (including the apical domain, the transferrin binding domain, and the protease-like domain). In some embodiments, the anti-transferrin receptor antibody binds to an amino acid fragment of a human or non-human primate transferrin receptor (as provided in SEQ ID No. 1 to 3) within the range of amino acids C89 to F760. In some embodiments, the transferrin receptor antibody is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The anti-transferrin receptor antibodies used herein may be able to compete for binding with other anti-transferrin receptor antibodies (e.g., OKT9, 8D3) that bind with an affinity of 10 M or less. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M or less binds to the transferrin receptor.

[0217] An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, Homo sapiens) is as follows:

[0218]

[0219] An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) is as follows:

[0220]

[0221] An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, cynomolgus monkey) is as follows:

[0222]

[0223] An exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows:

[0224]

[0225] In some embodiments, the anti-transferrin receptor antibody binds to the following receptor amino acid fragments:

[0226] And does not inhibit the binding interaction between transferrin receptor and transferrin and / or human hemochromatin (also known as HFE).

[0227] Appropriate methods can be used to obtain and / or produce antibodies, antibody fragments or antigen binding agents, for example, by using recombinant DNA protocols. In some embodiments, antibodies can also be produced by the production of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secretingantibody of predefined specificity" Nature, 1975, 256: 495-497). The target antigen can be used as an immunogen in any form or entity (e.g., recombinant or natural form or entity). Hybridomas are screened using standard methods (e.g., ELISA screening) to find at least one hybridoma that produces antibodies targeting specific antigens. Antibodies can also be produced by screening protein expression libraries (e.g., phage display libraries) expressing antibodies. In some embodiments, phage display library design can also be used (see, e.g., U.S. Patent No 5,223,409, filed March 1, 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed April 10, 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed May 1, 1991, "Recombinant library screening methods"; WO 1992 / 20791, filed May 15, 1992, "Methods for producing members of specific binding pairs"; WO 1992 / 15679, filed February 28, 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest can be used to immunize non-human animals, such as rodents or goats. In some embodiments, antibodies are then obtained from non-human animals and optionally modified using a variety of methods (e.g., using recombinant DNA technology). Other examples of antibody production and methods are known in the art (see, e.g., Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988.).

[0228] In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and / or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and / or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3 or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack enzymes in the N- or O-glycosylation pathway, such as glycosyltransferases. In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules, as described in International Patent Application Publication WO2014065661 entitled "Modified antibody, antibody-conjugate and process for the preparation thereof" published on May 1, 2014.

[0229] Some aspects of the present disclosure provide proteins that are combined with transferrin receptor (e.g., the extracellular part of transferrin receptor). In some embodiments, the transferrin receptor antibody provided herein specifically binds to transferrin receptor (e.g., human transferrin receptor). Transferrin receptor is an internalized cell surface receptor that transfers transferrin through the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. In some embodiments, the transferrin receptor antibody provided herein specifically binds to transferrin receptor from people, non-human primates, mice, rats, etc. In some embodiments, the transferrin receptor antibody provided herein binds to human transferrin receptor. In some embodiments, the transferrin receptor antibody provided herein specifically binds to human transferrin receptor. In some embodiments, the transferrin receptor antibody provided herein binds to the apical domain of human transferrin receptor. In some embodiments, the transferrin receptor antibody provided herein specifically binds to the apical domain of human transferrin receptor.

[0230] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences selected from any one of the anti-transferrin receptor antibodies of Table 2. In some embodiments, the transferrin receptor antibodies comprise CDR-H1, CDR-H2, and CDR-H3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the transferrin receptor antibodies comprise CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the transferrin receptor antibodies comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. The present disclosure also includes any nucleic acid sequence encoding a molecule comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 provided for any anti-transferrin receptor antibody selected from Table 2. In some embodiments, antibody heavy chain and light chain CDR3 domains may play a particularly important role in the binding specificity / affinity of the antibody to the antigen. Therefore, the anti-transferrin receptor antibody of the present disclosure may at least comprise a heavy chain and / or light chain CDR3 of any anti-transferrin receptor antibody selected from Table 2.

[0231] In some examples, any anti-transferrin receptor antibody of the present disclosure has one or more CDRs (e.g., CDR-H or CDR-L) that are substantially similar to any CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 sequence of an anti-transferrin receptor antibody selected from Table 2. In some embodiments, the position of one or more CDRs of the antibodies described herein along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions can be changed by one, two, three, four, five, or six amino acid positions, as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). For example, in some embodiments, the positions of the CDRs defining any of the antibodies described herein can be changed by moving the N-terminal and / or C-terminal boundaries of the CDRs relative to the positions of the CDRs of any of the antibodies described herein by one, two, three, four, five, or six amino acids, so long as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived). In another embodiment, the length of one or more CDRs of the antibodies described herein along the VH (e.g., CDR-H1, CDR-H2 or CDR-H3) and / or VL (e.g., CDR-L1, CDR-L2 or CDR-L3) can be changed (e.g., made shorter or longer) by one, two, three, four, five or more amino acids, as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).

[0232] Thus, in some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3 described herein may be shorter than one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2) by one, two, three, four, five or more amino acids, as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3 described herein may be one, two, three, four, five or more amino acids longer than one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., a CDR of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., a CDR of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., a CDR of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2), as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). Any method can be used to determine whether immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained, such as using binding assays and conditions described in the art.

[0233] In some examples, any anti-transferrin receptor antibody of the present disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences that are substantially similar to any anti-transferrin receptor antibody selected from Table 2. For example, the antibody may include one or more CDR sequences of any anti-transferrin receptor antibody selected from Table 2, which contain up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR region of any CDR provided herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) relative to the binding of the original antibody from which it is derived. In some embodiments, any amino acid variation in any CDR provided herein may be a conservative variation. Conservative variation can be introduced into CDR at a position (e.g., as determined based on a crystal structure) where the residue is unlikely to participate in interacting with a transferrin receptor protein (e.g., a human transferrin receptor protein). Some aspects of the present disclosure provide transferrin receptor antibodies comprising one or more heavy chain variable (VH) and / or light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein comprises one or more CDR-H sequences (e.g., CDR-H1, CDR-H2, and CDR-H3) provided herein, such as any CDR-H sequence provided in any anti-transferrin receptor antibody selected from Table 2. In some embodiments, any VL domain provided herein comprises one or more CDR-L sequences (e.g., CDR-L1, CDR-L2, and CDR-L3) provided herein, such as any CDR-L sequence provided in any anti-transferrin receptor antibody selected from Table 2.

[0234] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibodies comprising a heavy chain variable domain and / or a light chain variable domain of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibodies comprising a heavy chain variable and a light chain variable pair of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2).

[0235] Some aspects of the present disclosure provide anti-transferrin receptor antibodies, which have any homologous heavy chain variable (VH) and / or light chain variable (VL) domain amino acid sequences described herein. In some embodiments, anti-transferrin receptor antibodies include a heavy chain variable sequence and / or light chain variable sequence with at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identity with any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, homologous heavy chain variable and / or light chain variable amino acid sequences do not change in any CDR sequence provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) may occur in the heavy chain variable and / or light chain variable sequence provided herein that does not include any CDR sequence. In some embodiments, any anti-transferrin receptor antibody provided herein comprises a heavy chain variable sequence and a light chain variable sequence comprising a framework region sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the framework region sequence of any anti-transferrin receptor antibody (e.g., any one of the anti-transferrin receptor antibodies selected from Table 2).

[0236] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising any CDR-L domain (CDR-L1, CDR-L2, and CDR-L3) of any anti-transferrin receptor antibody selected from Table 2, or a CDR-L domain variant provided herein. In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence, which comprises one, two, three or four framework regions of a light chain variable region sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibody comprises one, two, three or four framework regions of a light chain variable region sequence, which has at least 75%, 80%, 85%, 90%, 95% or 100% identity with one, two, three or four framework regions of a light chain variable region sequence of any transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, but there are up to 10 amino acid substitutions, deletions and / or insertions, preferably up to 10 amino acid substitutions. In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues are substituted for amino acids present in analogous positions in the corresponding non-human primate or human light chain variable framework region.

[0237] In some embodiments, the anti-transferrin receptor antibody that specifically binds to the transferrin receptor comprises CDR-L1, CDR-L2 and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the antibody further comprises one, two, three or all four VL framework regions of the VL derived from humans or primates. The primate or human light chain framework region of the antibody selected for the light chain CDR sequence described herein may have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98% or at least 99%) identity with the light chain framework region of the non-human parent antibody. The amino acid numbering of the selected primate or human antibody in its light chain complementary determining region may be identical or substantially identical to the amino acid numbering in the light chain complementary determining region of any antibody provided herein (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the primate or human light chain framework region amino acid residues are from a natural primate or human antibody light chain framework region, which has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity with the light chain framework region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three or all four VL framework regions derived from a human light chain variable κ subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three or all four VL framework regions derived from a human light chain variable λ subfamily.

[0238] In some embodiments, any anti-transferrin receptor antibody provided herein comprises a light chain variable domain, which further comprises a light chain constant region. In some embodiments, the light chain constant region is a κ or λ light chain constant region. In some embodiments, the κ or λ light chain constant region is from a mammal, such as from a human, monkey, rat or mouse. In some embodiments, the light chain constant region is a human κ light chain constant region. In some embodiments, the light chain constant region is a human λ light chain constant region. It should be understood that any light chain constant region provided herein can be a variant of any light chain constant region provided herein. In some embodiments, the light chain constant region comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with any light chain constant region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2).

[0239] In some embodiments, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2.

[0240] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2), and wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises any VL domain or VL domain variant, and any VH domain or VH domain variant, wherein the VL and VH domains or variants thereof are from the same antibody clone, and wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule. Non-limiting examples of human constant regions are described in the art, see, e.g., Kabat EA et al., (1991), supra.

[0241] In some embodiments, the antibodies of the present disclosure can bind to a target antigen (e.g., transferrin receptor) with relatively high affinity, such as K D Less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less. For example, anti-transferrin receptor antibodies can be combined with transferrin receptor protein (for example, human transferrin receptor) with an affinity of 5pM to 500nM, for example 50pM to 100nM, for example 500pM to 50nM. The disclosure also includes antibodies that compete with any antibody described herein for the combination of transferrin receptor protein (for example, human transferrin receptor) and have an affinity of 50nM or less (for example, 20nM or less, 10nM or less, 500pM or less, 50pM or less or 5pM or less). Any suitable method can be used to test the affinity and binding kinetics of anti-transferrin receptor antibodies, including but not limited to biosensor technology (for example, OCTET or BIACORE).

[0242] In some embodiments, the antibodies of the present disclosure can bind to a target antigen (e.g., transferrin receptor) with relatively high affinity, such as K D Less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9M, 10 -10 M, 10 -11 M or less. For example, anti-transferrin receptor antibodies can be combined with transferrin receptor protein (for example, human transferrin receptor) with an affinity of 5pM to 500nM, for example 50pM to 100nM, for example 500pM to 50nM. The disclosure also includes antibodies that compete with any antibody described herein for the combination of transferrin receptor protein (for example, human transferrin receptor) and have an affinity of 50nM or less (for example, 20nM or less, 10nM or less, 500pM or less, 50pM or less or 5pM or less). Any suitable method can be used to test the affinity and binding kinetics of anti-transferrin receptor antibodies, including but not limited to biosensor technology (for example, OCTET or BIACORE).

[0243] In some embodiments, the muscle targeting agent is a transferrin receptor antibody (eg, an antibody and variants thereof as described in International Application Publication No. WO2016 / 081643, incorporated herein by reference).

[0244] Heavy and light chain CDRs of antibodies according to different definition systems are provided in Table 3. Different definition systems have been described, such as the Kabat definition, the Chothia definition and / or the contact definition. See, e.g., (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0245] Table 3 Heavy and light chain CDRs of mouse transferrin receptor antibodies

[0246]

[0247] Also provided are heavy chain variable domain (VH) and light chain variable domain sequences:

[0248] VH

[0249]

[0250] V L

[0251]

[0252] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2 and CDR-H3 identical to those shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise CDR-L1, CDR-L2 and CDR-L3 identical to those shown in Table 3.

[0253] In some embodiments, the transferrin receptor antibody of the present disclosure comprises CDR-H1, CDR-H2 and CDR-H3, which together comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variations) compared to the CDR-H1, CDR-H2 and CDR-H3 shown in Table 3. "Common" means that the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or in addition, the transferrin receptor antibody of the present disclosure may comprise CDR-L1, CDR-L2 and CDR-L3, which together comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variations) compared to the CDR-L1, CDR-L2 and CDR-L3 shown in Table 3.

[0254] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding heavy chain CDR shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding light chain CDR shown in Table 3.

[0255] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 comprising no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the CDR-L3 shown in Table 3. In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 comprising 1 amino acid variation compared to the CDR-L3 shown in Table 3. In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system). In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are identical to the CDR-H1, CDR-H2, and CDR-H3 shown in Table 3, and comprise a CDR-L3 of QHFAGTPLT (SEQ ID NO:31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO:32, according to the Contact definition system).

[0256] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise heavy chain CDRs that collectively have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise light chain CDRs that collectively have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the light chain CDRs shown in Table 3.

[0257] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 33. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:34.

[0258] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH shown in SEQ ID NO: 33. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO: 34.

[0259] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VH shown in SEQ ID NO: 33. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VL shown in SEQ ID NO: 34.

[0260] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3, and comprise humanized heavy chain variable regions and / or humanized light chain variable regions.

[0261] Humanized antibodies are human immunoglobulins (acceptor antibodies), wherein the residues from the complementary determining regions (CDRs) of the acceptor are replaced by residues from non-human species (donor antibodies) such as mice, rats or rabbits with desired specificity, affinity and capacity. In some embodiments, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may be included in the acceptor antibodies or the CDRs or framework sequences imported, but are not found but are included to further improve and optimize antibody performance. Generally speaking, humanized antibodies will include at least one and generally all of two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin consensus sequences. Humanized antibodies will also optimally include at least a portion of immunoglobulin constant regions or domains (Fc) (typically those of human immunoglobulins). Antibodies may have Fc regions modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, which is also referred to as one or more CDRs derived from one or more CDRs from the original antibody.Humanized antibodies may also involve affinity maturation.

[0262] In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 3) into IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL shown in SEQ ID NO: 34, and / or comprising one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID NO: 33. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL shown in SEQ ID NO:34, and / or comprising amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID NO:33.

[0263] In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized antibody and comprises residues at positions 43 and 48 of VL as shown in SEQ ID NO: 34. Alternatively or additionally, the transferrin receptor antibody of the present disclosure is a humanized antibody and comprises residues at positions 48, 67, 69, 71, and 73 of VH as shown in SEQ ID NO: 33.

[0264] The VH and VL amino acid sequences of exemplary humanized antibodies that can be used according to the present disclosure are provided:

[0265] Humanized VH

[0266]

[0267] Humanized VL

[0268]

[0269] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:36.

[0270] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH shown in SEQ ID NO: 35. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO: 36.

[0271] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VH shown in SEQ ID NO: 35. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VL shown in SEQ ID NO: 36.

[0272] In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising an amino acid substitution at one or more of positions 43 and 48 compared to the VL shown in SEQ ID NO: 34, and / or an amino acid substitution at one or more of positions 48, 67, 69, 71, and 73 compared to the VH shown in SEQ ID NO: 33. In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising an S43A and / or V48L mutation compared to the VL shown in SEQ ID NO: 34, and / or comprising one or more of A67V, L69I, V71R, and K73T mutations compared to the VH shown in SEQ ID NO: 33.

[0273] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising an amino acid substitution at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to the VL shown in SEQ ID NO:34, and / or an amino acid substitution at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID NO:33.

[0274] In some embodiments, the transferrin receptor antibody of the present disclosure is a chimeric antibody, which may include a heavy constant region and a light constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region or a part of a variable region from a first species and a constant region from a second species. Generally speaking, in these chimeric antibodies, the variable regions of both light and heavy chains simulate the variable regions of antibodies derived from a mammal (e.g., non-human mammals, such as mice, rabbits and rats), while the constant portion is homologous to the sequence in an antibody derived from another mammal (e.g., people). In some embodiments, amino acid modifications may be performed in the variable region and / or constant region.

[0275] In some embodiments, the transferrin receptor antibody described herein is a chimeric antibody, which may include a heavy constant region and a light constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Generally speaking, in these chimeric antibodies, the variable regions of both light and heavy chains simulate the variable regions of antibodies derived from a mammal (e.g., non-human mammals, such as mice, rabbits and rats), while the constant portion is homologous to the sequence in an antibody derived from another mammal (e.g., human). In some embodiments, amino acid modifications may be performed in the variable region and / or constant region.

[0276] In some embodiments, the heavy chain of any transferrin receptor antibody described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). Exemplary human IgG1 constant regions are given below:

[0277]

[0278] In some embodiments, the light chain of any transferrin receptor antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some instances, CL is a kappa light chain. In other instances, CL is a lambda light chain. In some embodiments, CL is a kappa light chain, the sequence of which is provided below:

[0279]

[0280] Additional antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0281] Exemplary heavy and light chain amino acid sequences of the transferrin receptor antibodies are provided below:

[0282] Heavy chain (VH + human IgG1 constant region)

[0283]

[0284] Light chain (VL+κ light chain)

[0285]

[0286] Heavy chain (humanized VH + human IgG1 constant region)

[0287]

[0288] Light chain (humanized VL + kappa light chain)

[0289]

[0290] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to SEQ ID NO: 39. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to SEQ ID NO: 40. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 39. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0291] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a heavy chain comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain shown in SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a light chain comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain shown in SEQ ID NO: 40.

[0292] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity with SEQ ID NO: 41. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity with SEQ ID NO: 42. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0293] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a heavy chain comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain of the humanized antibody shown in SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a light chain comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain of the humanized antibody shown in SEQ ID NO: 40.

[0294] In some embodiments, the transferrin receptor antibody is an antigen binding fragment (FAB) of a complete antibody (full-length antibody). The antigen binding fragment of a complete antibody (full-length antibody) can be prepared by conventional methods. For example, a F(ab')2 fragment can be produced by pepsin digestion of an antibody molecule, and a Fab fragment can be produced by reducing the disulfide bonds of the F(ab')2 fragment. An exemplary FAB amino acid sequence of a transferrin receptor antibody described herein is provided below:

[0295] Heavy chain FAB (VH + part of the human IgG1 constant region)

[0296]

[0297] Heavy chain FAB (humanized VH + part of human IgG1 constant region)

[0298]

[0299] The transferrin receptor antibodies described herein can be in any antibody form, including but not limited to complete (i.e., full-length) antibodies, antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the transferrin receptor antibodies described herein are scFv. In some embodiments, the transferrin receptor antibodies described herein are scFv-Fab (e.g., scFv fused to a portion of a constant region). In some embodiments, the transferrin receptor antibodies described herein are scFv fused to a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO:39).

[0300] b. Other muscle-targeted antibodies

[0301] In some embodiments, the muscle targeting antibody is an antibody that specifically binds hemojuvelin, caveolin-3, dystrophin, myosin Iib or CD63. In some embodiments, the muscle targeting antibody is an antibody that specifically binds myogenic precursor protein. Exemplary myogenic precursor proteins include but are not limited to ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin α7, integrin α7β1, MYF-5, MyoD, myogenic protein, NCAM-1 / CD56, Pax3, Pax7 and Pax9. In some embodiments, the muscle targeting antibody is an antibody that specifically binds skeletal muscle protein. Exemplary skeletal muscle proteins include, but are not limited to, alpha-Sarcoglycan, beta-Sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-Sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, but are not limited to, α-smooth muscle actin, VE-cadherin, calmodulin binding protein / CALD1, calmodulin 1, desmin, histamine H2 R, motilin R / GPR38, transglutamin / TAGLN, and vimentin. However, it should be understood that antibodies to other targets are within the scope of the present disclosure, and the exemplary list of targets provided herein is not intended to be limiting.

[0302] c. Antibody function / change

[0303] In some embodiments, conservative mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in interacting with the target antigen (e.g., transferrin receptor) (e.g., as determined based on the crystal structure). In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or the CH3 domain (residues 341 to 447 of human IgG1) and / or the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular toxicity.

[0304] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

[0305] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or the CH3 domain (residues 341 to 447 of human IgG1) and / or the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Examples of mutations in an antibody Fc receptor that can be made to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0306] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain or an FcRn binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that will alter (e.g., reduce or increase) the half-life of an antibody in vivo.

[0307] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231 to 340 of human IgG1) and / or the third constant (CH3) domain (residues 341 to 447 of human IgG1) (numbered according to the EU index in Kabat (Kabat EA et al., (1991) supra)). In some embodiments, the constant region of the IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, the positions being numbered according to the EU index in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to increase half-life by 4 times compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, as numbered according to the EU index as in Kabat.

[0308] In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change one or more effector functions of the anti-transferrin receptor antibody. The effector ligand that changes affinity can be, for example, an Fc receptor or the C1 component of complement. The method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domain (by point mutation or other means) can reduce the Fc receptor binding of circulating antibodies, thereby improving tumor localization. For descriptions of mutations that delete or inactivate the constant domain and thereby improve tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions can be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604).

[0309] In some embodiments, one or more amino groups in the constant region of the muscle-targeted antibodies described herein can be replaced with different amino acid residues so that the antibody has altered Clq binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibodies described herein are changed to change the ability of the antibody to fix complement. This method is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibodies described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase the affinity of the antibody for Fcγ receptors. This method is further described in International Publication No. WO 00 / 42072.

[0310] In some embodiments, the heavy chain and / or light chain variable domain sequences of the antibodies provided herein can be used to produce, for example, CDR grafted, chimeric, humanized or composite human antibodies or antigen-binding fragments, as described elsewhere herein. As understood by those of ordinary skill in the art, any variant (CDR grafted, chimeric, humanized or composite antibody) derived from any antibody provided herein can be used in the compositions and methods described herein, and will maintain the ability to specifically bind to transferrin receptor, so that relative to the original antibody from which it is derived, the variant (CDR grafted, chimeric, humanized or composite antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor.

[0311] In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to known Fab arm exchange with natural IgG4 mAb, the antibodies provided herein may include stability 'Adair' mutations (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), wherein the 228th (EU numbering, according to Kabat numbering, the 241st residue) serine is converted to proline, resulting in an IgG1-like hinge sequence. Therefore, any antibody may include stability 'Adair' mutations.

[0312] As provided herein, the antibodies of the present disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be connected to a light chain constant domain, such as Cκ or Cλ, at its C-terminus. Similarly, the VH domain or a portion thereof may be connected to all or a portion of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of the present disclosure may include VH and VL domains or antigen binding portions thereof in combination with any suitable constant region.

[0313] ii. Muscle Targeting Peptides

[0314] Some aspects of the present disclosure provide muscle targeting peptides as muscle targeting agents.Short peptide sequences (eg, peptide sequences 5 to 20 amino acids in length) that bind to specific cell types have been described. For example, cell targeting peptides have been described in: Vines E., et al., A. "Cell-penetrating and cell-targeting peptides in drug delivery" Biochim Biophys Acta 2008, 1786: 126-38; Jarver P., et al., "Invivo biodistribution and efficacy of peptide mediated delivery" Trends Pharmacol Sci 2010; 31: 528-35; Samoylova TI, et al., "Elucidation of muscle-binding peptides by phage display screening" Muscle Nerve 1999; 22: 460-6; ​​U.S. Patent No. 6,329,501, which was issued on December 11, 2001, and is entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM, et al., "Recognition of cell-specific binding of Phage display derived peptides using an acoustic wavesensor." Biomol Eng 2002; 18: 269-72; the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for desired tissues such as muscle can be achieved. Skeletal muscle targeting has been studied and is capable of delivering a range of molecular payloads. These methods can be highly selective for muscle tissue without many of the practical disadvantages of large antibodies or viral particles. Therefore, in some embodiments, the muscle targeting agent is a muscle targeting peptide of 4 to 50 amino acids in length. In some embodiments, the muscle targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.Muscle targeting peptides can be produced using any of several methods, such as phage display.

[0315] In some embodiments, the muscle targeting peptide can bind to an internalized cell surface receptor that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells, such as the transferrin receptor. In some embodiments, the muscle targeting peptide can target the transferrin receptor (e.g., bind to it). In some embodiments, the peptide targeting the transferrin receptor can comprise a fragment of a natural ligand (e.g., transferrin). In some embodiments, the peptide targeting the transferrin receptor is as described in U.S. Patent No. 6,743,893, filed on November 30, 2000, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THATBIND THE HUMAN TRANSFERRIN RECEPTOR". In some embodiments, the peptide targeting the transferrin receptor is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011Aug18; 11: 359. In some embodiments, the peptide targeting transferrin receptor is as described in U.S. Patent No. 8,399,653, filed May 20, 2011, “TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNADELIVERY.”

[0316] As described above, some examples of muscle targeting peptides have been reported. For example, muscle-specific peptides were identified using a phage display library presenting surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 6) binds to C2C12 mouse myotubes in vitro and binds to mouse muscle tissue in vivo. Therefore, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 6). The peptide exhibits increased specificity for binding to cardiac and skeletal muscle tissue after intravenous injection in mice, as well as reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, a 12 amino acid peptide was identified by a phage display library for muscle targeting in the context of DMD treatment. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats." Int J Pharm 2002; 231: 177-84; the entire contents of which are incorporated herein by reference. Here, a 12 amino acid peptide with the sequence SKTFNTHPQSTP (SEQ ID NO:7) was identified and this muscle-targeted peptide showed improved binding to C2C12 cells relative to the ASSLNIA (SEQ ID NO:6) peptide.

[0317] Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types includes in vitro selection, which has been described in Ghosh D., et al., "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting" J Virol 2005; 79: 13667-72; the entire contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After several rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 8) appeared most frequently. Therefore, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 8).

[0318] Muscle targeting agents can be amino acid-containing molecules or peptides. Muscle targeting peptides can correspond to protein sequences that preferentially bind to protein receptors found in muscle cells. In some embodiments, muscle targeting peptides contain highly propensity hydrophobic amino acids, such as valine, so that the peptide preferentially targets muscle cells. In some embodiments, muscle targeting peptides have not been characterized or disclosed previously. Any of several methods (e.g., phage display peptide libraries, single bead single compound peptide libraries, or position scanning synthetic peptide combinatorial libraries) can be used to conceive, produce, synthesize and / or derive these peptides. Exemplary methods have been characterized in the art and incorporated by reference (Gray, BPand Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114: 2, 1020–1081.; Samoylova, TIand Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22: 4.460-6.). In some embodiments, muscle targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004; 12: 185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006, 24: 3, 191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005, 112: 11, 1601-11.; McGuire, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004, 342: 1, 171-82.).Exemplary muscle targeting peptides include amino acid sequences of the following groups: CQAQGQLVC (SEQ ID NO: 9), CSERSMNFC (SEQ ID NO: 10), CPKTRRVPC (SEQ ID NO: 11), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 12), ASSLNIA (SEQ ID NO: 6), CMQHSMRVC (SEQ ID NO: 13), and DDTRHWG (SEQ ID NO: 14). In some embodiments, the muscle targeting peptide may include about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The muscle targeting peptide may include natural amino acids such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-natural amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art. In some embodiments, the muscle targeting peptide can be linear; in other embodiments, the muscle targeting peptide can be cyclic, such as bicyclic (see, e.g., Silvana, M G et al. Mol. Therapy, 2018, 26: 1, 132–147.). The muscle targeting agent can be an aptamer, such as a peptide aptamer, which preferentially targets muscle cells relative to other cell types.

[0319] iii. Muscle-targeted receptor ligands

[0320] The muscle targeting agent can be a ligand, such as a ligand that binds to a receptor protein. The muscle targeting ligand can be a protein, such as transferrin that binds to an internalized cell surface receptor expressed by a muscle cell. Therefore, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to a transferrin receptor. The muscle targeting ligand can alternatively be a small molecule, such as a lipophilic small molecule that preferentially targets muscle cells relative to other cell types. Some exemplary lipophilic small molecules that can target muscle cells include compounds comprising: cholesterol, cholesterol radicals, stearic acid, palmitic acid, oleic acid, oleyl, linolene, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, trityl groups, and alkoxy acids.

[0321] iv. Other muscle targeting agents

[0322] One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates of muscle transporter proteins (e.g., transporter proteins expressed on the sarcolemma). In some embodiments, a muscle targeting agent is a substrate of an influx transporter that is specific for muscle tissue. In some embodiments, the influx transporter is specific for skeletal muscle tissue. Two major classes of transporters are expressed on the skeletal muscle sarcolemma: (1) the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue and (2) the solute carrier (SLC) superfamily, which facilitates substrate influx into skeletal muscle. In some embodiments, a muscle targeting agent is a substrate that binds to the ABC superfamily or the SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a natural substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-natural substrate, for example, a synthetic derivative that binds to the ABC or SLC superfamily of transporters.

[0323] In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. SLC transporters are equilibrative, or use a proton or sodium ion gradient generated across the membrane to drive the transport of substrates. Exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can facilitate substrate influx into skeletal muscle, providing an opportunity for muscle targeting.

[0324] In some embodiments, the muscle targeting agent is a substrate of the equilibrative nucleoside transporter 2 (ENT2) transporter. ENT2 has one of the highest mRNA expressions in skeletal muscle relative to other transporters. Although human ENT2 (hENT2) is expressed in most body organs such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, it is particularly abundant in skeletal muscle. Human ENT2 promotes the absorption of its substrate according to its concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleoside bases. The hENT2 transporter has a low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. Therefore, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2', 3'-dideoxyinosine, and calofarabine. In some embodiments, any muscle targeting agent provided herein is associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular cargo. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular cargo.

[0325] In some embodiments, the muscle targeting agent is a substrate of the organic cation / carnitine transporter (OCTN2), which is a sodium ion-dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, meldonate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, meldonate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload). The muscle targeting agent can be a protein that is present in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein can be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), which is a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin can be full length or a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack N-terminal signaling, and / or lack a C-terminal anchoring domain. In some embodiments, hemojuvelin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be understood that hemojuvelin may be of human, non-human primate, or rodent origin.

[0326] B. Molecular Loading

[0327] Some aspects of the present disclosure provide molecular payloads, for example, for regulating biological outcomes, such as transcription of a DNA sequence, expression of a protein, or activity of a protein. In some embodiments, the molecular payload is connected to or otherwise associated with a muscle targeting agent. It should be understood that various types of muscle targeting agents can be used according to the present disclosure. For example, the molecular payload may comprise or consist of an oligonucleotide (e.g., an antisense oligonucleotide), a peptide (e.g., a peptide that binds to a disease-associated nucleic acid or protein in a muscle cell), a protein (e.g., a protein that binds to a disease-associated nucleic acid or protein in a muscle cell), or a small molecule (e.g., a small molecule that regulates the function of a disease-associated nucleic acid or protein in a muscle cell). In some embodiments, such a molecular payload is capable of targeting a muscle cell, for example, by specifically binding to a nucleic acid or protein in a muscle cell after being delivered to the muscle cell by a relevant muscle targeting agent. In some embodiments, the molecular payload is an oligonucleotide comprising a chain having a region complementary to the gene provided in Table 1. Exemplary molecular payloads are described in further detail herein, however, it should be understood that the exemplary molecular payloads provided herein are not meant to be limiting.

[0328] In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) molecular payloads (e.g., oligonucleotides) are linked to a muscle targeting agent. In some embodiments, all molecular payloads linked to a muscle targeting agent are identical, e.g., target the same gene. In some embodiments, all molecular payloads linked to a muscle targeting agent are different, e.g., the molecular payloads can target different parts of the same target gene, or the molecular payloads can target at least two different target genes. In some embodiments, a muscle targeting agent can be linked to some of the same molecular payloads or some different molecular payloads.

[0329] The present disclosure also provides compositions comprising a plurality of complexes, wherein at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the complexes comprise a molecular targeting agent linked to the same number of molecular payloads (e.g., oligonucleotides).

[0330] i. Oligonucleotides

[0331] As described herein, any suitable oligonucleotide can be used as a molecular payload. In some embodiments, oligonucleotides can be designed to cause degradation of mRNA (for example, oligonucleotides can be spaced polymers, siRNA, ribozymes or aptamers that cause degradation). In some embodiments, oligonucleotides can be designed to block translation of mRNA (for example, oligonucleotides can be mixed polymers, siRNA or aptamers that block translation). In some embodiments, oligonucleotides can be designed to cause degradation of mRNA and block translation. In some embodiments, oligonucleotides can be guide nucleic acids (for example, guide RNA) for guiding enzyme (for example, gene editing enzyme) activity. Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, by incorporating a functional sequence (for example, an antisense strand sequence) from one format into another format, an oligonucleotide of one format (for example, an antisense oligonucleotide) can be appropriately adapted to another format (for example, an siRNA oligonucleotide).

[0332] In some embodiments, an oligonucleotide may comprise a region complementary to a target gene provided in Table 1. Further non-limiting examples for selected genes of Table 1 are provided below.

[0333] DMPK / DM1

[0334] In some embodiments, some examples of oligonucleotides that can be used to target DMPK (e.g., for the treatment of DM1) are provided in the following: U.S. Patent Application Publication 20100016215A1, published on January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication 20130237585A1, published on July 19, 2010, Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication 20150064181A1, published on March 5, 2015, entitled “Antisense Conjugates For Decreasing Expression Of Dmpk”; U.S. Patent Application Publication 20150238627A1, published on August 27, 2015, entitled “Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy"; Pandey, S Ketal. "Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for theTreatment of Myotonic Dystrophy Type 1" J. of Pharmacol Exp Ther, 2015, 355:329-340.; Langlois, M. et al. "Cytoplasmic and Nuclear Retained DMPK mRNAs AreTargets for RNA Interference in Myotonic Dystrophy Cells" J. Biological Chemistry, 2005, 280: 17, 16949-16954.; Jauvin, D. et al. "Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice", Mol.Ther: Nucleic Acids, 2017, 7: 465-474.; Mulders, SA et al. "Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy" PNAS, 2009, 106: 33, 13915-13920.; Wheeler, TM et al., "Targeting nuclear RNA for in vivo correction of myotonic dystrophy" Nature, 2012, 488 (7409): 111-115.; and U.S. Patent Application Publication No. 20160304877A1, published on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression", the contents of each of which are incorporated herein by reference in their entirety. .

[0335] Some examples of oligonucleotides for promoting DMPK gene editing include U.S. Patent Application Publication No. 20170088819A1, published on March 3, 2017, entitled “Genetic Correction Of Myotonic Dystrophy Type 1”; and International Patent Application Publication No. WO18002812A1, published on April 1, 2018, entitled “Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM1) And Other Related Disorders”, the contents of each of which are incorporated herein by reference in their entirety.

[0336] In some embodiments, the oligonucleotide may have a region complementary to a mutant form of DMPK, for example, as reported in: Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients." J Med Genet. 2008 Oct; 45(10): 639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review." Muscle Nerve. 2005 Jul; 32(1): 1-18.; the contents of each of which are incorporated herein by reference in their entirety.

[0337] In some embodiments, the oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK. In some embodiments, the oligonucleotide targeted is any antisense oligonucleotide targeting DMPK (e.g., spaced polymers), as described in U.S. Patent Application Publication US20160304877A1, which is disclosed on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," which is incorporated herein by reference). In some embodiments, the DMPK targeting oligonucleotide targets the region of the DMPK gene sequence as shown in Genbank Accession No.NM_001081560.2 or as shown in Genbank Accession No.NG_009784.1.

[0338] In some embodiments, the DMPK targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region, the target region being at least 10 consecutive nucleotides (e.g., at least 10, at least 12, at least 14, at least 16, or more consecutive nucleotides) in Genbank Accession No. NM_001081560.2.

[0339] In some embodiments, the DMPK targeting oligonucleotide comprises a spacer polymer motif. "Spacer polymer" means a chimeric antisense compound in which an internal region having a plurality of nucleotides supporting RNA enzyme H cleavage is located between an external region having one or more nucleotides, wherein the nucleotides comprising the internal region are chemically different from one or more nucleotides comprising the external region. The internal region may be referred to as a "spacer segment" and the external region may be referred to as a "wing segment". In some embodiments, the DMPK targeting oligonucleotide comprises one or more modified nucleotides, and / or one or more modified internucleotide linkages. In some embodiments, the internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA spacer polymer (e.g., 3-10-3; cET-DNA-cET) with a cET end. In some embodiments, the DMPK targeting oligonucleotide comprises one or more 6'-(S)-CH3 biological cyclic nucleotides, one or more β-D-2'-deoxyribonucleotides and / or one or more 5-methylcytosine nucleotides.

[0340] DUX4 / FSHD

[0341] In some embodiments, examples of oligonucleotides that can be used to target DUX4 (e.g., for the treatment of FSHD) are provided in the following: U.S. Patent No. 9,988,628, which was published on February 2, 2017, and is entitled “AGENTS USEFUL INTREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; U.S. Patent No. 9,469,851, which was published on October 30, 2014, and is entitled “RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4”; U.S. Patent Application Publication No. 20120225034, which was published on September 6, 2012, and is entitled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; PCT Patent Application Publication No. WO 2013 / 120038, published on August 15, 2013, entitled "MORPHOLINO TARGETING DUX4 FOR TREATING FSHD"; Chen et al., "Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics," Molecular Therapy, 2016, 24:8, 1405-1411.; and Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes, 2017, 8, 93., the contents of each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA or other nucleotide that hybridizes to the target DUX4 gene or mRNA.

[0342] In some embodiments, for example for the treatment of FSHD, the oligonucleotide may have a region complementary to the hypomethylated compact D4Z4 repeats, as described in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," published in Curr Opin Genet Dev in 2015, Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817–4828, the contents of each of which are incorporated herein in their entirety.

[0343] DNM2 / CNM

[0344] In some embodiments, examples of oligonucleotides that can be used to target DNM2 (e.g., for the treatment of CNM) are provided in U.S. Patent Application Publication No. 20180142008, published on May 24, 2018, entitled "DYNAMIN2 INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY," and PCT Application Publication No. WO 2018 / 100010A1, published on June 7, 2018, entitled "ALLELE-SPECIFIC SILENCING THERAPY FOR DYNAMIN 2-RELATED DISEASES." For example, in some embodiments, the oligonucleotide is an RNAi, antisense nucleic acid, siRNA, or ribozyme that specifically interferes with the expression of DNM2. Other examples of oligonucleotides that can be used to target DNM2 are provided in: Tasfaout, et al., "Single Intramuscular Injection of AAV-shRNAReduces DNM2 and Prevents Myotubular Myopathy in Mice," published in Mol. Ther. on April 4, 2018, and Tasfaout, et al., "Antisense oligonucleotide-mediated Dnm2knockdown prevents and reverts myotubular myopathy in mice," Nature Communications volume 8, Article number: 15661 (2017). In some embodiments, the oligonucleotide is an shRNA or morpholino that effectively targets DNM2 mRNA. In some embodiments, the oligonucleotide encodes a wild-type DNM2 that is resistant to miR-133 activity, as described in Todaka, et al. "Overexpression of NF90-NF45 Represses Myogenic MicroRNA Biogenesis, Resulting in Development of Skeletal Muscle Atrophy and Centronuclear Muscle Fibers," published in Mol. Cell Biol., July 2015.Additional examples of oligonucleotides that can be used to target DNM2 are provided in Gibbs, et al., “Two Dynamin-2 Genes are Required for Normal Zebrafish Development,” published in PLoS One in 2013, the contents of each of which are incorporated herein in their entirety.

[0345] In some embodiments, for example for the treatment of CNM, the oligonucleotide may have a region complementary to a mutant in DNM2 associated with CNM, as described below: et al, “Mutation Spectrumin the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy,” as published in Hum. Mutat., 2012, the contents of which are incorporated herein in their entirety.

[0346] Pompe disease

[0347] In some embodiments, for example for the treatment of Pompe disease, the oligonucleotide mediates the inclusion of exon 2 in the GAA disease allele, as described in van der Wal, et al., "GAA Deficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells," Mol Ther Nucleic Acids. 2017 Jun 16; 7: 101–115, the contents of which are incorporated herein by reference. Thus, in some embodiments, the oligonucleotide may have a region complementary to the GAA disease allele.

[0348] In some embodiments, for example, for the treatment of Pompe disease, oligonucleotides (e.g., RNAi or antisense oligonucleotides) are used to inhibit the expression of wild-type GYS1 in muscle cells, as reported, for example, in Clayton, et al., “Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease,” published in Mol Ther Nucleic Acids in 2017, or U.S. Patent Application Publication No. 2017182189, published on June 29, 2017, entitled “INHIBITING OR DOWNREGULATING GLYCOGEN SYNTHASE BY CREATING PREMATURE STOP CODONS USING ANTISENSE OLIGONUCLEOTIDES,” the contents of which are incorporated herein by reference. Thus, in some embodiments, an oligonucleotide can have an antisense strand having a region complementary to a sequence corresponding to the human GYS1 sequence of RefSeq No. NM_002103.4 and / or a mouse GYS1 sequence corresponding to RefSeq No. NM_030678.3.

[0349] ACVR1 / FOP

[0350] In some embodiments, examples of oligonucleotides that can be used to target ACVR1 (e.g., for the treatment of FOP) are provided in the following: U.S. Patent Application 2009 / 0253132, published on October 8, 2009, "Mutated ACVR1 for diagnosis and treatment of fibrodyplasia ossificans progressiva (FOP)"; WO2015 / 152183, published on October 8, 2015, "Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive"; Lowery, JW et al, "Allele-specific RNA Interference in FOP-Silencing the FOP gene", GENE THERAPY, vol. 19, 2012, pages 701-702; Takahashi, M. et al. "Disease-causing allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva" Gene Therapy (2012) 19, 781–785; Shi, S. et al. “Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor ThatIs Overactive in Fibrodysplasia Ossificans Progressiva” Plos One, July 2013, Vol8:7, e69096.; U.S. Patent Application 2017 / 0159056, published on June 8, 2017, “Antisense oligonucleotides and methods of use thereof”; U.S. Patent No.8,859,752, granted on October 4, 2014, "SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)"; WO2004 / 094636, published on November 4, 2004, "Effective siRNA knock-down constructs", the contents of each of which are incorporated herein in their entirety. .

[0351] FXN / Friedreich's Ataxia

[0352] In some embodiments, examples of oligonucleotides that can be used to target FXN and / or otherwise compensate for frataxin deficiency (e.g., for the treatment of Friedreich's ataxia) are provided in the following: Li, L. et al "Activating frataxin expression by repeat-targeted nucleic acids" Nat. Comm. 2016, 7: 10606.; WO 2016 / 094374, published on June 16, 2016, "Compositions and methods for treatment of offriedreich's ataxia."; WO 2015 / 020993, published on February 12, 2015, "RNAi COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA"; WO 2017 / 186815, published on November 2, 2017, "Antisense oligonucleotides for enhanced expression of frataxin"; WO 2008 / 018795, published on February 14, 2008, "Methods and means for treating DNA repeat instability associated genetic disorders"; U.S. patent application 2018 / 0028557, published on February 1, 2018, "Hybrid oligonucleotides and uses thereof"; WO 2015 / 023975, published on February 19, 2015, "Compositions and methods for modulating RNA"; WO 2015 / 023939, published on February 19, 2015, "Compositions and methods for modulating expression of frataxin"; U.S. patent application 2017 / 0281643, published on October 5, 2017, "Compounds and methods for modulating frataxin expression"; Li L. et al., "Activating frataxin expression by repeat-targeted nucleic acids" Nature Communications, published on February 4, 2016; and Li L. et al. "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat" Nucleic Acid Ther. 2018 Feb; 28(1): 23-33., the contents of each of which are incorporated herein in their entirety. .

[0353] In some embodiments, the oligonucleotide payload is configured (e.g., as a spacer or RNAi oligonucleotide) to inhibit expression of a natural antisense transcript that inhibits FXN expression, such as disclosed in U.S. Pat. No. 9,593,330, filed June 9, 2011, “Treatment of frataxin (FXN) related diseases by inhibition of natural antisense transcript to FXN,” the contents of which are incorporated herein by reference in their entirety.

[0354] Examples of oligonucleotides for facilitating FXN gene editing include WO 2016 / 094845, published on June 16, 2016, “Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides”; WO 2015 / 089354, published on June 18, 2015, “Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders”; WO 2015 / 139139, published on September 24, 2015, “CRISPR-based methods and products for increasing frataxin levels and uses thereof”, and WO 2018 / 002783, published on January 4, 2018, “Materials and methods for treatment of Friedreich ataxia and other related disorders”, the contents of each of which are incorporated herein in their entirety.

[0355] Examples of oligonucleotides for promoting FXN gene expression by targeting non-FXN genes (eg, epigenetic regulators of FXN) include WO 2015 / 023938, published February 19, 2015, "Epigenetic regulators of frataxin," the contents of which are incorporated herein in their entirety.

[0356] In some embodiments, the oligonucleotide may have a region complementary to a sequence such as the FXN gene from human (Gene ID 2395; NC 000009.12) and / or the FXN gene from mouse (Gene ID 14297; NC_000085.6). In some embodiments, the oligonucleotide may have a region complementary to a mutant form of FXN, such as reported in, for example, Montemini, L. et al. "The Friedreich ataxia GAA triplet repeat: premutation and normal alleles." Hum. Molec. Genet., 1997, 6: 1261-1266.; Filla, A. et al. "The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia." Am. J. Hum. Genet. 1996, 59: 554-560.; Pandolfo, M. Friedreich ataxia: the clinical picture. J. Neurol. 2009, 256, 3–8., the contents of each of which are incorporated herein by reference in their entirety.

[0357] DMD / Dystrophinopathy

[0358] Examples of oligonucleotides that can be used to target DMD are provided in the following: U.S. Patent Application Publication US20100130591A1, which was published on May 27, 2010, and is entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD”; U.S. Patent No. 8,361,979, which was issued on January 29, 2013, and is entitled “MEANS AND METHOD FOR INDUCING EXON-SKIPPING”; U.S. Patent Application Publication 20120059042, which was published on March 8, 2012, and is entitled “METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULARDYSTROPHY AND ASSOCIATED MEANS; U.S. Patent Application Publication 20140329881, which was published on November 6, 2014, and is entitled “EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY”; U.S. Patent No. 8,232,384, issued on July 31, 2012, entitled “ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF”; U.S. Patent Application Publication 20120022134A1, published on January 26, 2012, entitled “METHODS AND MEANS FOR EFFICIENTSKIPPING OF EXON 45IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA”; U.S. Patent Application Publication 20120077860, published on March 29, 2012, entitled “ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING ADISPENSABLE DOMAN PROTEIN”; U.S. Patent No. 8,324,371, issued on December 4, 2012, entitled “OLIGOMERS”; U.S. Patent No. 9,078,911, issued on July 14, 2015, entitled “ANTISENSE OLIGONUCLEOTIDES”; U.S. Patent No. 9,079,934, issued on July 14, 2015, entitled “ANTISENSE NUCLEIC ACIDS”; U.S. Patent No.9,034,838, issued on May 19, 2015, entitled “MIR-31 IN DUCHENNE MUSCULAR DYSTROPHY THERAPY”; and International Patent Publication WO2017062862A3, issued on April 13, 2017, entitled “OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF”; the contents of each of which are incorporated herein in their entirety. .

[0359] Examples of oligonucleotides for promoting DMD gene editing include International Patent Publication WO2018053632A1, published on March 29, 2018, entitled “METHODS OF MODIFYING THE DYSTROPHIN GENE ANDRESTORING DYSTROPHIN EXPRESSION AND USES THEREOF”; International Patent Publication WO2017049407A1, published on March 30, 2017, entitled “MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF”; International Patent Publication WO2016161380A1, published on October 6, 2016, entitled “CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY”; International Patent Publication WO2017095967, which was published on June 8, 2017, and is entitled “THERAPEUTIC TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE”; International Patent Publication WO2017072590A1, which was published on May 4, 2017, and is entitled “MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULARDYSTROPHY”; International Patent Publication WO2018098480A1, which was published on May 31, 2018, and is entitled “PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE EDITING”; U.S. Patent Application Publication US20170266320A1, published on September 21, 2017, entitled “RNA-Guided Systems for In Vivo Gene Editing”; International Patent Publication WO2016025469A1, published on February 18, 2016, entitled “PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CAS9-MEDIATED GENE EDITING”;U.S. Patent Application Publication 2016 / 0201089, published on July 14, 2016, entitled "RNA-GUIDED GENE EDITING AND GENE REGULATION"; and U.S. Patent Application Publication 2013 / 0145487, published on June 6, 2013, entitled "MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF", the contents of each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotides may have regions complementary to DMD gene sequences of multiple species (e.g., selected from human, mouse, and non-human species). ;

[0360] In some embodiments, the oligonucleotide may have a region complementary to a mutant DMD allele, such as a DMD allele having at least one mutation in any one of exons 1 to 79 of human DMD that results in a frameshift and improper RNA splicing / processing.

[0361] MYH7 / Hypertrophic Cardiomyopathy

[0362] Examples of oligonucleotides that can be used as payloads (e.g., for targeting MYH7) are provided in the following: U.S. Patent Application Publication 20180094262, published on April 5, 2018, entitled Inhibitors of MYH7B and Uses Thereof; U.S. Patent Application Publication 20160348103, published on December 1, 2016, entitled Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference; U.S. Patent Application Publication 20160237430, published on August 18, 2016, entitled “Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy”; U.S. Patent Application Publication 20160032286, published on February 4, 2016, entitled “Inhibitors of MYH7B and Uses Thereof”. Thereof”; U.S. Patent Application Publication 20140187603, published on July 3, 2014, entitled “MicroRNA Inhibitors Comprising Locked Nucleotides”; U.S. Patent Application Publication 20140179764, published on June 26, 2014, entitled “Dual Targeting of miR-208and miR-499in the Treatment of Cardiac Disorders”; U.S. Patent Application Publication 20120114744, published on May 10, 2012, entitled “Compositions and Methods to Treat Muscular and Cardiovascular Disorders”; the contents of each of which are incorporated herein in their entirety.

[0363] In some embodiments, oligonucleotides can target lncRNA or mRNA, for example, for degradation. In some embodiments, oligonucleotides can target (e.g., for degradation) nucleic acids encoding proteins (e.g., MSH2, MutLalpha, MutSbeta, MutLalpha) involved in mismatch repair pathways. Proteins involved in mismatch repair pathways (wherein mRNA encoding such proteins can be targeted by oligonucleotides described herein) are described in the following: Iyer, RRet al., "DNAtriplet repeat expansion and mismatch repair" Annu Rev Biochem. 2015; 84: 199-226.; and Schmidt MHand Pearson CE, "Disease-associated repeat instabilityand mismatch repair" DNA Repair (Amst). 2016Feb; 38: 117-26.

[0364] a. Oligonucleotide size / sequence

[0365] Oligonucleotide can have multiple different lengths, for example, depending on format. In some embodiments, the length of oligonucleotide is 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,75 or more nucleotides. In some embodiments, the length of oligonucleotide is 8 to 50 nucleotides, the length is 8 to 40 nucleotides, the length is 8 to 30 nucleotides, the length is 10 to 15 nucleotides, the length is 10 to 20 nucleotides, the length is 15 to 25 nucleotides, the length is 21 to 23 nucleotides, etc.

[0366] In some embodiments, when the binding of the complementary nucleic acid sequence of the oligonucleotide to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) resulting in a lack of activity (e.g., inhibition of translation) or expression (e.g., degradation of the target mRNA), and has a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-targets under the following conditions, for the purposes of the present disclosure, the complementary nucleic acid sequence of the oligonucleotide can specifically hybridize with the target nucleic acid or have specificity for the target nucleic acid: under conditions where it is desired to avoid non-specific binding, such as under physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays, under conditions where the assay is performed under suitable stringent conditions. Thus, in some embodiments, the oligonucleotide can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the contiguous nucleotides of the target nucleic acid. In some embodiments, a complementary nucleotide sequence need not be 100% complementary to its targeted sequence to specifically hybridize or be specific to a target nucleic acid.

[0367] In some embodiments, the oligonucleotide comprises a region complementary to the target nucleic acid, and the length of the region is 8 to 15, 8 to 30, 8 to 40 or 10 to 50, or 5 to 50 or 5 to 40 nucleotides. In some embodiments, the length of the complementary region of the oligonucleotide and the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In some embodiments, the complementary region is complementary to at least 8 continuous nucleotides of the target nucleic acid. In some embodiments, compared with the continuous nucleotide portion of the target nucleic acid, the oligonucleotide can include 1, 2 or 3 base mispairings. In some embodiments, an oligonucleotide can have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0368] b. Oligonucleotide modification:

[0369] The oligonucleotides described herein may be modified, for example, to include a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide, and / or a combination thereof. In addition, in some embodiments, the oligonucleotide may exhibit one or more of the following properties: not mediating alternative splicing; not immunostimulatory; having nuclease resistance; having increased cellular uptake compared to unmodified oligonucleotides; being nontoxic to cells or mammals; increasing internal expulsion of endosomes in cells; minimizing TLR stimulation; or avoiding pattern recognition receptors. Any modified chemistry or format of the oligonucleotides described herein may be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included in the same oligonucleotide.

[0370] In some embodiments, certain nucleotide modifications may be used that render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact longer than unmodified oligonucleotides. Some specific examples of modified oligonucleotides include those containing modified backbones, such as modified internucleoside linkages, such as phosphorothioate linkages, phosphotriester linkages, methylphosphonate linkages, short-chain alkyl linkages or cycloalkyl sugar inter-linkages or short-chain heteroatom linkages or heterocyclic sugar inter-linkages. Thus, the oligonucleotides of the present disclosure may be stabilized, for example, by incorporating modifications such as nucleotide modifications, to resist nucleolytic degradation.

[0371] In some embodiments, the length of the oligonucleotide can be up to 50 or up to 100 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45 or more nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide can be 8 to 30 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide can be 8 to 15 nucleotides, wherein 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides. Optionally, the oligonucleotide can have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified nucleotides. Oligonucleotide modifications are further described herein.

[0372] c. Modified nucleotides

[0373] In some embodiments, the oligonucleotide comprises 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O--N-methylacetamido (2'-O--NMA).

[0374] In some embodiments, the oligonucleotide may include at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all nucleotides include 2'-O-methyl modifications. In some embodiments, the oligonucleotide includes a modified nucleotide in which the ribose ring includes a bridge portion connecting two atoms in the ring, such as a 2'-O atom and a 4'-C atom. In some embodiments, the oligonucleotide is "locked", for example, comprising a modified nucleotide in which the ribose ring is "locked" by a methylene bridge connecting a 2'-O atom and a 4'-C atom. Some examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, which was published on April 17, 2008, and is entitled "RNA Antagonist Compounds For The Modulation Of PCSK9", the contents of which are incorporated herein by reference in their entirety.

[0375] Other modifications that can be used for the oligonucleotides disclosed herein include ethylene bridged nucleic acids (ENAs). ENAs include, but are not limited to, 2'-O, 4'-C-ethylene bridged nucleic acids. Some examples of ENAs are provided in: International Patent Publication No. WO2005 / 042777, published on May 12, 2005, and entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1: 241-242, 2001; Surono et al., Hum. Gene Ther., 15: 749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49: 171-172, 2005; the disclosures of which are incorporated herein by reference in their entirety.

[0376] In some embodiments, an oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. In some embodiments, the oligonucleotide comprises a modified nucleotide disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, which was issued on July 15, 2008, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,741,457, which was issued on June 22, 2010, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 8,022,193, which was issued on September 20, 2011, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,569,686, which was issued on August 4, 2009, and is entitled “Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,335,765, which was issued on February 26, 2008, and is entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,314,923, issued on January 1, 2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,816,333, issued on October 19, 2010, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same” and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued on February 17, 2015, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same”, the entire contents of each of which are incorporated herein by reference for all purposes.

[0377] In some embodiments, the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2'O-methyl modifications on the ribose of a pyrimidine, abasic residue or inverted base at the 3' end of the RNA.

[0378] In some embodiments, the oligonucleotide may have at least one modified nucleotide that results in an increase in the Tm of the oligonucleotide by 1° C., 2° C., 3° ​​C., 4° C., or 5° C. compared to an oligonucleotide that does not have at least one modified nucleotide. The oligonucleotide may have multiple modified nucleotides that result in an overall increase in the Tm of the oligonucleotide by 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or more compared to an oligonucleotide that does not have a modified nucleotide.

[0379] The oligonucleotide may comprise different types of alternative nucleotides. For example, the oligonucleotide may comprise alternative deoxyribonucleotides or ribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternative deoxyribonucleotides or ribonucleotides and 2'-O-methyl nucleotides. The oligonucleotide may comprise alternative 2'-fluoro nucleotides and 2'-O-methyl nucleotides. The oligonucleotide may comprise alternative bridging nucleotides and 2'-fluoro or 2'-O-methyl nucleotides.

[0380] d. Internucleotide linkage / backbone

[0381] In some embodiments, the oligonucleotide may comprise a phosphorothioate linkage or other modified internucleotide linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage between all nucleotides. For example, in some embodiments, the oligonucleotide comprises a modified internucleotide linkage at the first, second and / or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.

[0382] Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3' alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' phosphoramidates and aminoalkylphosphoramidates, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with inverted polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. Nos. 3,687,808; 4, and 5,625,050.

[0383] In some embodiments, the oligonucleotide may have a heteroatom backbone, such as a methylene (methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28: 366-374); a morpholino backbone (see Summerton and Weller, U.S. Pat. No. 5,034,506); or a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotides are directly or indirectly bound to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0384] e. Stereospecific oligonucleotides

[0385] In some embodiments, the internucleotide phosphorus atom of the oligonucleotide is chiral, and the characteristics of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, appropriate methods can be used to synthesize P-chiral oligonucleotide analogs in a stereo controlled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorusatoms. Chem Soc Rev. 2011 Dec; 40 (12): 5829-43). In some embodiments, oligonucleotides containing thiophosphates are provided, which include nucleoside units connected together by substantially all Sp or substantially all Rp thiophosphate sugar inter-linkages. In some embodiments, such thiophosphate oligonucleotides with substantially chiral pure sugar inter-linkages are prepared by enzyme or chemical synthesis, as described in, for example, U.S. Patent No. 5,587,261, authorized on December 12, 1996, the contents of which are incorporated herein by reference as a whole. In some embodiments, the oligonucleotides controlled by chirality provide a selective cleavage pattern of a target nucleic acid. For example, in some embodiments, chirality-controlled oligonucleotides provide single-site cleavage within the complementary sequence of a nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399A1, published on February 2, 2017, entitled “CHIRAL DESIGN,” the contents of which are incorporated herein by reference in their entirety.

[0386] f. Morpholino

[0387] In some embodiments, the oligonucleotide can be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41 (14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).

[0388] g. Peptide nucleic acid (PNA)

[0389] In some embodiments, both the sugar and the internucleoside linkage (main chain) of the nucleotide unit of the oligonucleotide are replaced by new groups. In some embodiments, the base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound (an oligonucleotide mimetic that has been shown to have excellent hybridization properties) is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-main chain of the oligonucleotide is replaced by an amide-containing main chain (e.g., an aminoethylglycine main chain). The core base is retained and is directly or indirectly bound to the aza nitrogen atom of the main chain amide portion. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patents no. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0390] h. Gap polymer

[0391] In some embodiments, oligonucleotide is a spacer. Spacer oligonucleotides generally have the formula 5'-XYZ-3', wherein X and Z are used as flanking regions around spacer Y. In some embodiments, the Y region is a continuous extension of nucleotides, for example, a region of at least 6 DNA nucleotides, which can recruit RNA enzymes (such as RNA enzyme H). In some embodiments, the spacer is combined with the target nucleic acid, at which point the RNA enzyme is recruited and can subsequently cut the target nucleic acid. In some embodiments, the flanks of both 5' and 3' of the Y region are X and Z regions comprising high-affinity modified nucleotides, for example, 1 to 6 modified nucleotides. Some examples of modified nucleotides include but are not limited to 2'MOE or 2'OMe or locked nucleic acid bases (LNA). In some embodiments, the length of the flanking sequences X and Z can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides. Flanking sequences X and Z can have similar lengths or different lengths. In some embodiments, the spacer segment Y can be a nucleotide sequence of 5 to 20 nucleotides, with a length size of 12 nucleotides or 6 to 10 nucleotides.

[0392] In some embodiments, in addition to DNA nucleotides, the spacer of the spacer oligonucleotide may include modified nucleotides known to be acceptable for effective RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabinose (arabino) configuration nucleotides. In some embodiments, the spacer comprises one or more unmodified internucleosides. In some embodiments, one or two flanking regions each independently comprise one or more thiophosphate nucleoside interlinkages (e.g., thiophosphate nucleoside interlinkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the spacer and two flanking regions each independently comprise modified nucleoside interlinkages (e.g., thiophosphate nucleoside interlinkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

[0393] The gapmers may be produced using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,65 2,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. Patent Publication Nos. US20090286969, US20100197762, and US20110112170; and PCT Publication Nos. WO2008049085 and WO2009090182, each of which is incorporated herein by reference in its entirety.

[0394] i. Mixed polymers

[0395] In some embodiments, oligonucleotides described herein can be mixed polymers or comprise mixed polymer sequence patterns. Generally speaking, mixed polymers are oligonucleotides comprising both natural and non-natural nucleotides or oligonucleotides comprising two different types of non-natural nucleotides with alternative patterns. Mixed polymers have higher binding affinity than unmodified oligonucleotides usually, and can be used for specific binding target molecules, for example, to block the binding site on the target molecule. Generally speaking, mixed polymers do not raise RNA enzymes to the target molecule and therefore do not promote the cutting of the target molecule. Such oligonucleotides that can not raise RNA enzyme H have been described, for example, referring to WO2007 / 112754 or WO2007 / 112753.

[0396] In some embodiments, the mixed polymer comprises or consists of a repeating pattern of nucleotide analogs and natural nucleotides, or one type of nucleotide analogs and a second type of nucleotide analogs. However, the mixed polymer need not comprise a repeating pattern, and may alternatively comprise any arrangement of modified nucleotides and naturally occurring nucleotides, or any arrangement of one modified nucleotide and a second modified nucleotide. The repeating pattern may be, for example, that every second or every third nucleotide is a modified nucleotide (e.g., LNA), and the remaining nucleotides are natural nucleotides (e.g., DNA) or are 2' substituted nucleotide analogs, such as 2'MOE or 2' fluorine analogs, or any other modified nucleotides described herein. It is recognized that a repeating pattern of modified nucleotides, such as LNA units, may be combined with modified nucleotides at fixed positions, such as at the 5' or 3' end.

[0397] In some embodiments, the mixed polymer does not include a region of more than 5, more than 4, more than 3, or more than 2 consecutive natural nucleotides (e.g., DNA nucleotides). In some embodiments, the mixed polymer includes at least one region consisting of at least two consecutive modified nucleotides, such as at least two consecutive LNAs. In some embodiments, the mixed polymer includes at least one region consisting of at least three consecutive modified nucleotide units, such as at least three consecutive LNAs.

[0398] In some embodiments, the mixed polymer does not contain regions of more than 7, more than 6, more than 5, more than 4, more than 3 or more than 2 consecutive nucleotide analogs such as LNA. In some embodiments, the LNA units may be replaced by other nucleotide analogs such as those mentioned herein.

[0399] Mixed polymers can be designed to include a mixture of modified nucleotides with enhanced affinity (e.g., LNA nucleotides and 2'-O-methyl nucleotides in a non-limiting example). In some embodiments, the mixed polymers include modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

[0400] Any suitable method may be used to produce the mixed polymers. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixed polymers include U.S. Patent Publication Nos. US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851 and U.S. Patent No. 7,687,617.

[0401] In some embodiments, the mixed polymer comprises one or more morpholino nucleotides. For example, in some embodiments, the mixed polymer may comprise morpholino nucleotides mixed (e.g., mixed in an alternating manner) with one or more other nucleotides (e.g., DNA, RNA nucleotides) or modified nucleotides (e.g., LNA, 2'-O-methyl nucleotides).

[0402] In some embodiments, the mixed polymer can be used for splicing correction or exon skipping, for example, as reported in: Touznik A., et al., LNA / DNA mixmer-based antisense oligonucleotides correctalternative splicing of the SMN2 gene and restore SMN protein expression intype 1SMA fibroblasts Scientific Reports, volume 7, Article number: 3672 (2017), Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA) -Uridine Phosphoramidite, and Exon Skipping Using MNA / 2′-O-Methyl Mixmer Antisense Oligonucleotide, Molecules 2016, 21, 1582, the contents of each of which are incorporated herein by reference.

[0403] j. RNA interference (RNAi)

[0404] In some embodiments, the oligonucleotides provided herein can be in the form of small interfering RNA (small interfering RNA, siRNA, also referred to as short interfering RNA or silencing RNA). siRNA is a type of double-stranded RNA molecule, usually about 20 to 25 base pairs in length, which targets nucleic acids (e.g., mRNA) for degradation by RNA interference (RNAi) pathways in cells. The specificity of siRNA molecules can be determined by the binding of the antisense strand of the molecule to its target RNA. Although longer siRNAs may also be effective, the length of effective siRNA molecules is usually less than 30 to 35 base pairs to prevent nonspecific RNA interference pathways in cells from being triggered by interferon responses.

[0405] After selecting an appropriate target RNA sequence, an appropriate method can be used to design and prepare siRNA molecules comprising a nucleotide sequence complementary to all or part of the target sequence (ie, an antisense sequence) (see, e.g., PCT Publication No. WO 2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791).

[0406] The siRNA molecule can be double-stranded (i.e., a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single-stranded (i.e., an ssRNA molecule comprising only an antisense strand). The siRNA molecule can comprise a duplex, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure having a self-complementary sense strand and an antisense strand.

[0407] Double-stranded siRNA can comprise RNA chains of the same length or different lengths. Double-stranded siRNA molecules can also be assembled into stem-loop structures by single oligonucleotides, wherein the self-complementary sense and antisense regions of siRNA molecules are connected by the following: one or more nucleic acid-based or non-nucleic acid-based joints, and circular single-stranded RNA, which has two or more loop structures and a stem comprising self-complementary sense strands and antisense strands, wherein circular RNA can be processed in vivo or in vitro to produce active siRNA molecules that can mediate RNAi. Therefore, small hairpin RNA (small hairpin RNA, shRNA) molecules are also considered herein. Except the reverse complementary (sense) sequence separated by spacer or loop sequence usually, these molecules also comprise specific antisense sequences. The cutting of spacer or loop provides single-stranded RNA molecules and their reverse complements, so that they can be annealed to form dsRNA molecules (optionally, with the other processing steps that can cause the 3' end and / or 5' end of any one or two chains to add or remove one, two, three or more nucleotides). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally, subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3' end and / or 5' end of either or both strands). The spacer sequence may be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, when annealed into a double-stranded nucleic acid, comprises the shRNA.

[0408] The total length of the siRNA molecule can vary from about 14 to about 100 nucleotides depending on the type of siRNA molecule being designed. Generally speaking, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double-stranded siRNA or a single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, and when the siRNA is a shRNA or a circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.

[0409] The siRNA molecule may include a 3' overhang at one end of the molecule, and the other end may be a flat end or also have an overhang (5' or 3'). When the siRNA molecule includes overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one embodiment, the siRNA molecule of the present disclosure includes a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule.

[0410] k. MicroRNA (miRNA)

[0411] In some embodiments, oligonucleotides can be microRNA (miRNA). MicroRNA (referred to as "miRNA") is a small non-coding RNA, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Generally speaking, miRNA is produced by large RNA precursors (referred to as primary miRNA (pri-miRNA)), which are processed into about 70 nucleotide precursor miRNAs in the nucleus, and the precursor miRNAs are folded into imperfect stem-loop structures. These precursor miRNAs are usually subjected to additional processing steps in the cytoplasm, and the mature miRNAs of 18 to 25 nucleotides in length are cut off from one side of the precursor miRNA hairpin by the RNase III enzyme Dicer.

[0412] The miRNA used herein includes a fragment of a variant thereof of primary miRNA, precursor miRNA, mature miRNA or the biological activity of a mature miRNA. In one embodiment, the size range of miRNA can be 21 nucleotides to 170 nucleotides. In one embodiment, the size range of miRNA is a length of 70 to 170 nucleotides. In another embodiment, a mature miRNA of 21 to 25 nucleotides can be used.

[0413] 1. Aptamers

[0414] In some embodiments, the oligonucleotide provided herein can be in the form of an aptamer. Generally speaking, in the case of molecular load, an aptamer is any nucleic acid that specifically binds to a target (e.g., a small molecule, a protein, a nucleic acid in a cell). In some embodiments, an aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, a nucleic acid aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA). It should be understood that a single-stranded nucleic acid aptamer can form a spiral and / or ring structure. The nucleic acid forming a nucleic acid aptamer can include natural nucleotides, modified nucleotides, natural nucleotides with hydrocarbon joints (e.g., alkylene) or polyether joints (e.g., PEG joints) inserted between one or more nucleotides, modified nucleotides with hydrocarbons or PEG joints inserted between one or more nucleotides, or a combination thereof. Exemplary publications and patents describing aptamers and methods of making aptamers include, for example, Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; ​​5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT Application WO 99 / 31275, each of which is incorporated herein by reference.

[0415] m. Ribozyme

[0416] In some embodiments, the oligonucleotides provided herein can be in the form of ribozymes. Ribozymes (ribonucleases) are molecules that can perform specific biochemical reactions, similar to the action of protein enzymes, typically RNA molecules. Ribozymes are molecules with catalytic activity, including the ability to cut at specific phosphodiester bonds in RNA molecules (e.g., mRNA, RNA-containing substrates, lncRNA, and ribozymes themselves) hybridized therewith.

[0417] Ribozymes can adopt one of several physical structures, one of which is called "hammerhead". Hammerhead ribozymes consist of a catalytic core containing 9 conserved bases, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the catalytic core of the target RNA flanking regions. The flanking regions enable the ribozyme to specifically bind to the target RNA by forming double-stranded stems I and III. Cleavage occurs next to a specific ribonucleotide triplet in cis (i.e., cleaving the same RNA molecule containing the hammerhead motif) or in trans (cleaving RNA substrates other than the RNA substrate containing the ribozyme) by transesterification of 3',5'-phosphodiester to 2',3'-cyclic phosphodiester. Without wishing to be bound by theory, it is believed that this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.

[0418] Modification in ribozyme structure also comprises multiple non-core parts of replacement or substitution molecule with non-nucleotide molecule.For example, Benseler et al. (J.Am.Chem.Soc. (1993) 115:8483-8484) disclose hammerhead-like molecule, wherein two base pairs of stem II and all four nucleotides of ring II are replaced by non-nucleoside joints based on hexaethylene glycol, propylene glycol, bis(triethylene glycol) phosphate, tri(propylene glycol) diphosphate or bis(propylene glycol) phosphate.Ma et al. (Biochem. (1993) 32:1751-1758; Nucleic Acids Res. (1993) 21:2585-2589) replaced six nucleotide rings of TAR ribozyme hairpin with the relevant joint of ethylene glycol of non-nucleotide. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear non-nucleotidic linkers of 13, 17, and 19 atoms in length.

[0419] Ribozyme oligonucleotides can be prepared using known methods (see, e.g., PCT Publications WO9118624; WO9413688; WO9201806; and WO 92 / 07065; and U.S. Pat. Nos. 5,436,143 and 5,650,502), or can be purchased from commercial sources (e.g., USBiochemicals), and if desired, nucleotide analogs can be incorporated to increase the resistance of the oligonucleotide to degradation by nucleases in cells. Ribozymes can be synthesized in any known manner, such as by using commercially available synthesizers produced by, for example, Applied Biosystems, Inc. or Milligen. Ribozymes can also be produced in recombinant vectors by conventional means. See Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (current edition). Ribozyme RNA sequences can be conventionally synthesized, for example, by using RNA polymerases such as T7 or SP6.

[0420] n.Guide nucleic acid

[0421] In some embodiments, the oligonucleotide is a guide nucleic acid, for example, a guide RNA (gRNA) molecule. Generally speaking, a guide RNA is a short synthetic RNA consisting of: (1) a scaffold sequence that binds to a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., Cas9), and (2) a nucleotide spacer portion that defines a DNA target sequence (e.g., a genomic DNA target) that binds to the gRNA to direct the nucleic acid programmable DNA binding protein to a proximity DNA target sequence. In some embodiments, the napDNAbp is a nucleic acid programmable protein that forms a complex (e.g., binds or associates) with one or more RNAs, and the RNA targets the nucleic acid programmable protein to a target DNA sequence (e.g., a target genomic DNA sequence). In some embodiments, when compounded with RNA, a nucleic acid programmable nuclease may be referred to as a nuclease: RNA complex. The guide RNA may be present as a complex of two or more RNAs, or as a single RNA molecule.

[0422] A guide RNA (gRNA) that exists as a single RNA molecule may be referred to as a single-guide RNA (sgRNA), although gRNA is also used to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Generally speaking, a gRNA that exists as a single RNA species comprises two domains: (1) a domain that shares homology with the target nucleic acid (i.e., guides the binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence referred to as tracrRNA and comprises a stem-loop structure. In some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012) (the entire contents of which are incorporated herein by reference).

[0423] In some embodiments, the gRNA comprises two or more of domains (1) and (2) and may be referred to as an extended gRNA. For example, as described herein, an extended gRNA will bind to two or more Cas9 proteins and bind to the target nucleic acid at two or more different regions. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease: RNA complex.In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, such as Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai H.S., Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada Z.A., Eckert MR, Vogel J., Charpentier E., Nature 471: 602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337: 816-821 (2012), the entire contents of each of which are incorporated herein by reference.

[0424] o. Splice-altering oligonucleotides

[0425] In some embodiments, oligonucleotides of the present disclosure (e.g., antisense oligonucleotides comprising morpholinos) target splicing. In some embodiments, oligonucleotides target splicing by inducing exon skipping and restoring reading frames in genes. As non-limiting examples, oligonucleotides can induce exon skipping of exons encoding frameshift mutations and / or exons encoding premature stop codons. In some embodiments, oligonucleotides can induce exon skipping by blocking the recognition of spliceosomes to splice sites. In some embodiments, compared with reference proteins, exon skipping leads to truncated but functional proteins (e.g., truncated but functional DMD proteins as described below). In some embodiments, oligonucleotides promote the inclusion of specific exons (e.g., exon 7 of SMN2 genes as described below). In some embodiments, oligonucleotides can induce the inclusion of exons by targeting splice site inhibitory sequences. RNA splicing is related to muscle diseases, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).

[0426] Changes (e.g., deletions, point mutations, and duplications) in the gene encoding dystrophin (DMD) cause DMD. These changes can result in frameshift mutations and / or nonsense mutations. In some embodiments, the oligonucleotides of the present disclosure promote the skipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and / or exon 55) and produce functional truncated proteins. See, e.g., U.S. Patent No. 8,486,907, published on July 16, 2013, and US20140275212, published on September 18, 2014.

[0427] In SMA, there is a loss of functional SMN1. Although the SMN2 gene is a paralog of SMN1, alternative splicing of the SMN2 gene primarily results in the skipping of exon 7 and the subsequent production of a truncated SMN protein that cannot compensate for the loss of SMN1. In some embodiments, the oligonucleotides of the present disclosure promote the inclusion of SMN2 exon 7. In some embodiments, the oligonucleotide is an antisense oligonucleotide targeted to an SMN2 splice site inhibitory sequence (see, e.g., U.S. Pat. No. 7,838,657, published Nov. 23, 2010).

[0428] p. Polymer

[0429] In some embodiments, the molecular payload may comprise a polymer of two or more oligonucleotides connected by a linker (e.g., a concatemer). In some embodiments, in this manner, the oligonucleotide loading of the complex / conjugate may be increased to exceed the available attachment sites on the targeting agent (e.g., available thiol sites on an antibody), or otherwise adjusted to achieve a specific payload loading. The oligonucleotides in a polymer may be the same or different (e.g., targeting different genes or different sites on the same gene or its products).

[0430] In some embodiments, the polymer comprises 2 or more oligonucleotides connected together by a cleavable joint. However, in some embodiments, the polymer comprises 2 or more oligonucleotides connected together by a non-cleavable joint. In some embodiments, the polymer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides connected together. In some embodiments, the polymer comprises 2 to 5, 2 to 10 or 4 to 20 oligonucleotides connected together.

[0431] In some embodiments, a polymer comprises 2 or more oligonucleotides connected end to end (in a linear arrangement). In some embodiments, a polymer comprises 2 or more oligonucleotides connected end to end by a joint based on oligonucleotides (e.g., a poly-dT joint, a baseless joint). In some embodiments, a polymer comprises the 5' end of an oligonucleotide, which is connected to the 3' end of another oligonucleotide. In some embodiments, a polymer comprises the 3' end of an oligonucleotide, which is connected to the 3' end of another oligonucleotide. In some embodiments, a polymer comprises the 5' end of an oligonucleotide, which is connected to the 5' end of another oligonucleotide. Nevertheless, in some embodiments, a polymer may comprise a branched structure comprising a plurality of oligonucleotides connected together by a branched joint.

[0432] Other examples of multimers that can be used in the complexes provided herein are disclosed in, for example, U.S. Patent Application No. 2015 / 0315588A1, entitled Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers, published on November 5, 2015; U.S. Patent Application No. 2015 / 0247141A1, entitled Multimeric Oligonucleotide Compounds, published on September 3, 2015; U.S. Patent Application No. US2011 / 0158937A1, entitled Immunostimulatory Oligonucleotide Multimers, published on June 30, 2011; and U.S. Patent No. 5,693,773, entitled Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And Pyrimidines, issued December 2, 1997, the contents of each of which are incorporated herein by reference in their entirety.

[0433] C. Connector

[0434] The complexes described herein typically comprise a linker connecting a muscle targeting agent (e.g., a muscle targeting protein) to a molecular payload (e.g., an oligonucleotide). The linker comprises at least one covalent bond. In some embodiments, the linker can be a single bond connecting the muscle targeting agent to the molecular payload, such as a disulfide bond or a disulfide bridge. However, in some embodiments, the linker can connect the muscle targeting agent to the molecular payload via multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker. The linker is generally stable in vitro and in vivo, and can be stable in certain cellular environments. In addition, generally speaking, the linker does not negatively affect the functional properties of the muscle targeting agent or the molecular payload. Examples and methods of linker synthesis are known in the art (see, e.g., Kline, T. et al. "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32: 11, 3480-3493.; Jain, N. et al. "Current ADC Linker Chemistry" Pharm Res. 2015, 32: 11, 3526-3540.; McCombs, JR and Owen, SC "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry" AAPS J. 2015, 17: 2, 339-351.).

[0435] The precursor of the joint will generally contain two different reactive species that allow connection to both the muscle targeting agent and the molecular payload. In some embodiments, the two different reactive species can be nucleophiles and / or electrophiles. In some embodiments, the joint is connected to the muscle targeting agent by conjugating to a lysine residue or a cysteine ​​residue of the muscle targeting agent. In some embodiments, the joint is connected to the cysteine ​​residue of the muscle targeting agent by a maleimide-containing joint, wherein optionally, the maleimide-containing joint comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the joint is connected to the cysteine ​​residue of the muscle targeting agent or a sulfhydryl-functionalized molecular payload by a 3-arylpropionitrile functional group. In some embodiments, the joint is connected to the muscle targeting agent and / or the molecular payload by an amide bond, a hydrazide, a triazole, a thioether, or a disulfide bond.

[0436] i. Cleavable Linkers

[0437] The cleavable linker can be a protease-sensitive linker, a pH-sensitive linker or a glutathione-sensitive linker. These linkers are usually only cleavable intracellularly and are preferably stable in an extracellular environment, such as outside a muscle cell.

[0438] Protease-sensitive joints can be cut by protease enzyme activity. These joints usually contain peptide sequences, and the length can be 2 to 10 amino acids, about 2 to 5 amino acids, about 5 to 10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids or about 2 amino acids. In some embodiments, the peptide sequence may include natural amino acids such as cysteine, alanine or non-naturally occurring or modified amino acids. Non-natural amino acids include β-amino acids, high amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids and other amino acids known in the art. In some embodiments, protease-sensitive joints include valine-citrulline or alanine-citrulline dipeptide sequences. In some embodiments, protease-sensitive joints can be cut by lysosomal proteases (e.g., cathepsin B (cathepsin B)) and / or endosomal proteases.

[0439] A pH sensitive linker is a covalent linkage that is easily degraded in a high or low pH environment. In some embodiments, the pH sensitive linker can be cleaved at a pH of 4 to 6. In some embodiments, the pH sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH sensitive linker is cleaved in an endosome or lysosome.

[0440] In some embodiments, the glutathione-sensitive linker comprises a disulfide portion. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with an intracellular glutathione substance. In some embodiments, the disulfide portion further comprises at least one amino acid, such as a cysteine ​​residue. In some embodiments, the cleavable linker is a valine-citrulline linker.

[0441] In some embodiments, the linker is a Val-cit linker (e.g., as described in U.S. Pat. No. 6,214,345, which is incorporated herein by reference). In some embodiments, prior to conjugation, the val-cit linker has the following structure:

[0442]

[0443] In some embodiments, after conjugation, the val-cit linker has the following structure:

[0444]

[0445] In some embodiments, prior to conjugation, the val-cit linker has the following structure:

[0446]

[0447] Where n is any number from 0 to 10.

[0448] In some embodiments, prior to conjugation, the val-cit linker has the following structure:

[0449]

[0450] In some embodiments, prior to conjugation, the val-cit linker has the following structure:

[0451]

[0452] Where n is any number from 0 to 10.

[0453] In some embodiments, after conjugation to the oligonucleotide, the val-cit linker has the following structure:

[0454]

[0455] Where n is any number from 0 to 10.

[0456] In some embodiments, after conjugation to the oligonucleotide, the val-cit linker has the following structure:

[0457]

[0458] Where n is any number from 0 to 10.

[0459] ii. Non-cleavable linker

[0460] In some embodiments, a non-cleavable linker may be used. Generally, a non-cleavable linker is not easily degraded in a cell or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitution may include halogens, hydroxyls, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl group, an optionally substituted alkylene group, an optionally substituted arylene group, a heteroarylene group, a peptide sequence comprising at least one non-natural amino acid, a truncated polysaccharide, one or more sugars that cannot be degraded by an enzyme, an azide, an alkyne-azide, a peptide sequence comprising the LPXT sequence (SEQ ID NO: 15), a thioether, biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an ester, an amide, a sulfonamide, and / or an alkoxy-amine linker. In some embodiments, a sortase-mediated connection will be used to covalently link a muscle targeting agent comprising the LPXT sequence to a protein comprising (G) nIn some embodiments, the linker comprises a LPXTG sequence (SEQ ID NO: 16), wherein X is any amino acid.

[0461] In some embodiments, the linker can comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino group, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide.

[0462] iii. Linker conjugation

[0463] In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload via a phosphate, thioether, ether, carbon-carbon bond, or amide bond. In some embodiments, the linker is connected to the oligonucleotide via a phosphate or phosphorothioate group, such as a terminal phosphate of the oligonucleotide backbone. In some embodiments, the linker is connected to the muscle targeting agent via a lysine or cysteine ​​residue present on the muscle targeting agent (e.g., an antibody).

[0464] In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload to form a triazole by a cycloaddition reaction between an azide and an alkyne, wherein the azide and the alkyne may be located on the muscle targeting agent, the molecular payload, or the linker. In some embodiments, the alkyne may be a cycloalkyne, such as a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also referred to as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, which was published on November 3, 2011, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions". In some embodiments, the azide may be a sugar or carbohydrate molecule comprising an azide. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the sugar or carbohydrate molecule comprising an azide is as described in International Patent Application Publication No. WO2016170186, published on October 27, 2016, entitled “Process For The Modification Of AGlycoprotein Using AGlycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”. In some embodiments, a cycloaddition reaction is performed between an azide and an alkyne to form a triazole, wherein the azide and the alkyne can be located on a muscle targeting agent, a molecular payload, or a linker, as described in International Patent Application Publication No. WO2014065661, published on May 1, 2014, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”; or International Patent Application Publication No. WO2016170186, published on October 27, 2016, entitled “Process For The Modification OfAGlycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”.

[0465] In some embodiments, the linker further comprises a spacer, such as a polyethylene glycol spacer or an acyl / carbamoylsulfonamide spacer, such as HydraSpace TM Spacer. In some embodiments, the spacer is as described in Verkade, JMMetal., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.

[0466] In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload via a Diels-Alder reaction between a dienophile and a diene / heterodiene, wherein the dienophile and the diene / heterodiene can be located on the muscle targeting agent, the molecular payload, or the linker. In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload via other pericyclic reactions, such as ene reactions. In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload via an amide, thioamide, or sulfonamide bonding reaction. In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload via a condensation reaction to form an oxime, hydrazone, or semicarbazide group present between the linker and the muscle targeting agent and / or the molecular payload.

[0467] In some embodiments, the linker is connected to the muscle targeting agent and / or molecular payload by a conjugate addition reaction between a nucleophile (e.g., an amine or hydroxyl) and an electrophile (e.g., a carboxylic acid or aldehyde). In some embodiments, the nucleophile may be present on the linker and the electrophile may be present on the muscle targeting agent or molecular payload prior to the reaction between the linker and the muscle targeting agent or molecular payload. In some embodiments, the electrophile may be present on the linker and the nucleophile may be present on the muscle targeting agent or molecular payload prior to the reaction between the linker and the muscle targeting agent or molecular payload. In some embodiments, the electrophile may be azide, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center and / or activated sulfur center. In some embodiments, the nucleophile can be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl, an amino, an alkylamino, an anilino, or a thiol group.

[0468] D. Some Examples of Antibody-Molecular Loading Complexes

[0469] Other aspects of the present disclosure provide a complex comprising any of the muscle targeting agents described herein (e.g., transferrin receptor antibodies) covalently linked to any molecular payload described herein (e.g., oligonucleotides). In some embodiments, the muscle targeting agent (e.g., transferrin receptor antibodies) is covalently linked to the molecular payload (e.g., oligonucleotides) via a linker. Any linker described herein can be used. In some embodiments, the linker is connected to the 5' end, 3' end, or internally of the oligonucleotide. In some embodiments, the linker is connected to the antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0470] An exemplary structure of a complex comprising a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker is provided below:

[0471]

[0472] Wherein the linker is attached to the 5' end, the 3' end, or internally of the oligonucleotide, and wherein the linker is attached to the antibody via a thiol-reactive linkage (eg, through a cysteine ​​in the antibody).

[0473] In some embodiments, the following provides a complex comprising a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker:

[0474]

[0475] Wherein the linker is attached to the 5' end, the 3' end, or internally of the oligonucleotide, and wherein the linker is attached to the antibody via an amine-reactive linkage (eg, through a lysine in the antibody).

[0476] It should be understood that antibodies can be connected to oligonucleotides with different stoichiometries, and this property can be referred to as drug-antibody ratio (DAR), where the "drug" is an oligonucleotide. In some embodiments, one oligonucleotide is connected to one antibody (DAR=1). In some embodiments, two oligonucleotides are connected to one antibody (DAR=2). In some embodiments, three oligonucleotides are connected to one antibody (DAR=3). In some embodiments, four oligonucleotides are connected to one antibody (DAR=4). In some embodiments, a mixture of different complexes is provided, each complex having a different DAR. In some embodiments, the average DAR of the complex in such a mixture can be in the range of 1 to 3, 1 to 4, 1 to 5 or more. DAR can be increased by conjugating oligonucleotides to different sites on the antibody and / or by conjugating a polymer to one or more sites on the antibody. For example, DAR of 2 can be achieved by conjugating a single oligonucleotide to two different sites on the antibody or by conjugating a dimer oligonucleotide to a single site of the antibody.

[0477] In some embodiments, the complex described herein comprises a transferrin receptor antibody (e.g., an antibody as described herein or any variant thereof) covalently linked to an oligonucleotide. In some embodiments, the complex described herein comprises a transferrin receptor antibody (e.g., an antibody as described herein or any variant thereof) covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker). In some embodiments, a linker (e.g., a Val-cit linker) is connected to the 5' end, 3' end or internal end of an oligonucleotide. In some embodiments, a linker (e.g., a Val-cit linker) is connected to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in an antibody).

[0478] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to CDR-H1, CDR-H2, and CDR-H3 shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to CDR-L1, CDR-L2, and CDR-L3 shown in Table 3.

[0479] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36.

[0480] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42.

[0481] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3.

[0482] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36.

[0483] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42.

[0484] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3, and wherein the complex comprises the following structure:

[0485]

[0486] wherein the linker Val-cit linker is linked to the 5' end, the 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine ​​in the antibody).

[0487] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34, and wherein the complex comprises the following structure:

[0488]

[0489] wherein the linker Val-cit linker is linked to the 5' end, the 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine ​​in the antibody).

[0490] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36, and wherein the complex comprises the following structure:

[0491]

[0492] wherein the linker Val-cit linker is linked to the 5' end, the 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine ​​in the antibody).

[0493] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40, and wherein the complex comprises the following structure:

[0494]

[0495] wherein the linker Val-cit linker is linked to the 5' end, the 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine ​​in the antibody).

[0496] In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42, and wherein the complex comprises the following structure:

[0497]

[0498] wherein the linker Val-cit linker is linked to the 5' end, the 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine ​​in the antibody).

[0499] In some embodiments, the complex describe...

Claims

1. A method of isolating a complex or a plurality of complexes, each of which comprises an antibody covalently linked to one or more oligonucleotides, wherein the one or more oligonucleotides comprise at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage, the method comprising: (i) contacting a mixture comprising the complex and unlinked antibodies with a hydrophobic resin under conditions where the complex is adsorbed to the hydrophobic resin but the unlinked antibodies are not adsorbed to the hydrophobic resin, thereby separating the unlinked antibodies from the complex adsorbed to the hydrophobic resin; and (ii) eluting the complex from the hydrophobic resin under conditions wherein the complex dissociates from the hydrophobic resin.

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