Tissue-specific imaging and therapeutic agents targeting proteins expressed on the muscle cell surface

Abstract

Disclosed herein are muscle-targeting agents for tissue-specific delivery of therapeutic and diagnostic agents. Also disclosed herein are methods for delivering agents in a tissue-specific manner, particularly to muscle tissue, by targeting proteins expressed on the cell surface of muscle tissue. The methods can be used for detection, imaging and / or treatment, as well as diagnosis of pathologies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 297,245, filed January 7, 2022, for all subject matter common to the Therapeutic Applications, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification supersedes and / or takes precedence over any such conflicting material. [Background technology]

[0003] Currently, there is no cure for disorders involving various muscles, and treatment options are limited. For example, current treatments for muscular dystrophies include Eteplirsen, Viltolarsen, Golodirsen, and Casimersen, all of which are non-targeted antisense oligonucleotides. Eteplirsen is administered intravenously at 30 mg / kg weekly. Two-thirds of the dose is lost within 24 hours of administration due to renal clearance. Viltolarsen is also administered intravenously at 80 mg / kg. The recommended dose of Casimersen is 30 mg / kg, which is administered once weekly as a 35-60 minute intravenous infusion through an in-line 0.2 micron filter. The recommended dose of Golodirsen is 30 mg / kg, which is administered once weekly as a 35-60 minute intravenous infusion through an in-line 0.2 micron filter.

[0004] It would be beneficial to be able to selectively target therapeutic agents to muscle tissue using therapeutic targeting agents.One example of therapeutic targeting agents is antibody-drug conjugates (ADCs).ADCs have proven useful in oncological and other disorders to improve delivery to target tissues, increasing the efficacy and therapeutic index of conjugated drugs and allowing the safe use of many compounds.

[0005] Of particular interest is the targeted delivery of oligonucleotides to muscle tissue via conjugation to antibodies against muscle-specific or muscle-enriched surface antigens. This targeted delivery should create a better dose-to-toxicity ratio, increase therapeutic half-life, reduce losses due to renal clearance, and minimize off-target effects. This new strategy would significantly expand the therapeutic landscape to treat a wide range of muscle-related diseases, such as Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), diabetes, or cardiomyopathies. The field has been hampered by the lack of muscle-specific receptors stably expressed in diseased tissues that would enable antibody-targeted delivery. Furthermore, many muscle-related diseases are rare disorders with small patient populations. As antibody therapeutics can cost thousands to hundreds of millions of US dollars to develop, the development of treatments for these rare diseases would be greatly enhanced by the availability of one or more muscle-specific antibodies that can be used in ADCs across multiple disorders. There is therefore a need to develop muscle-specific therapeutic targeting agents against muscle-specific receptors stably expressed in diseased tissues. Summary of the Invention [Means for solving the problem]

[0006] Disclosed herein are methods of delivering agents to muscle tissue in a tissue-specific manner in vivo, which may include contacting the surface of muscle cells with an agent that specifically binds to a target protein expressed on the cell surface of the muscle tissue. In some embodiments, the target protein may be enriched in muscle tissue relative to other tissues. In some embodiments, the target protein may have stable or increased expression in diseased tissue relative to normal tissue. In some embodiments, the target protein may be internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0007] In some embodiments, the target protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1. In some embodiments, the agent is a specific binding agent for the target protein. In some embodiments, the specific binding agent may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor may include the extracellular domain of the receptor. In some embodiments, the soluble receptor may be an Fc fusion protein.

[0008] In some embodiments, the agent may be an antibody or an antigen-binding fragment thereof, which may be selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, a VNAR, and a NANOBODY® molecule.

[0009] Also disclosed herein are methods of treating a condition in an individual, which may include administering to the individual a therapeutic targeting agent that specifically binds to a target protein expressed on the muscle tissue cell surface. In some embodiments, the target protein may be enriched in muscle tissue relative to other tissues. In some embodiments, the target protein may have stable or increased expression in diseased tissue relative to normal tissue. In some embodiments, the target protein may be internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0010] In some embodiments, a therapeutic targeting agent can be an agent that includes an active agent component and a targeting agent component. In some embodiments, the active agent component may be selected from the group consisting of a radionuclide, a chemotherapeutic agent, an immunostimulant, an anti-tumor agent, an anti-inflammatory agent, a pro-inflammatory agent, a pro-apoptotic agent, a pro-coagulant, a toxin, an antibiotic, a hormone, an enzyme, a protein, a carrier protein, a lytic agent, a small molecule, an aptamer, a cell, a vaccine-induced cell or other immune cell, a nanoparticle, transferrin, an immunoglobulin, a multivalent antibody, a lipid, a lipoprotein, a liposome, a modified natural ligand, a gene or a nucleic acid, an oligonucleotide, an RNA, an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a miximer, a double-stranded RNA (dsRNA), a single-stranded RNAi (ssRNAi), a DNA-dependent RNA interference (ddRNAi), an RNA activating oligonucleotide (RNAa), an aptamer, an exon skipping oligonucleotide, an miRNA, an miRNA mimic, an mRNA, a guide RNA, a viral or non-viral gene delivery vector, a prodrug, and a promolecule. In some embodiments, the targeting agent component is capable of specifically binding to a targeted protein.

[0011] In some embodiments, the targeting agent component may comprise a specific binding agent for the protein to be targeted. In some embodiments, the specific binding agent may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor may comprise the extracellular domain of the receptor. In some embodiments, the soluble receptor may be an Fc fusion protein. In some embodiments, the targeting agent component may comprise an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof may be selected from the group consisting of monoclonal antibodies, bispecific antibodies, immunoglobulin single variable domains (ISVs), such as Fab, Fab-Fc, Fv, single chain Fv (scFv), diabodies, minibodies, VNARs, and NANOBODY® molecules. In some embodiments, the active agent component may be an oligonucleotide. In some embodiments, the targeting agent component may be an antibody or an antigen-binding fragment thereof. In some embodiments, the active agent component may be conjugated to the targeting agent component. In some embodiments, the oligonucleotide may target a disease gene expressed in muscle tissue. In some embodiments, the targeted protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0012] In some embodiments, the targeted protein is KLHL41. In some embodiments, the targeted protein is LMOD2. In some embodiments, the targeted protein is ENO3. In some embodiments, the targeted protein is FABP3. In some embodiments, the targeted protein is CHRNA1. In some embodiments, the targeted protein is SEMA6C. In some embodiments, the targeted protein is XIRP2. In some embodiments, the targeted protein is XIRP1. In some embodiments, the targeted protein is CAVIN4. In some embodiments, the targeted protein is CFL2. In some embodiments, the targeted protein is SVIL. In some embodiments, the targeted protein is MUSK. In some embodiments, the targeted protein is ART1. In some embodiments, the targeted protein is CACNA1S. In some embodiments, the targeted protein is CDH15. In some embodiments, the targeted protein is CLCN1. In some embodiments, the targeted protein is CLCN1. In some embodiments, the targeted protein is MYMX. In some embodiments, the targeted protein is ACTA1.

[0013] Also disclosed herein is a method of delivering an imaging agent to muscle tissue in a tissue-specific manner, which may include contacting a surface of the muscle tissue with an imaging agent, which may include an imaging agent component and a targeting agent component. In some embodiments, the targeting agent component may specifically bind to a targeted protein expressed on the cell surface of the tissue. In some embodiments, the targeted protein may be enriched in muscle tissue compared to other tissues. In some embodiments, the targeted protein may have stable or increased expression in diseased tissue compared to normal tissue. In some embodiments, the target protein may be internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0014] In some embodiments, the targeted protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1. In some embodiments, the targeting agent component may be a specific binding agent for the targeted protein. In some embodiments, the specific binding agent may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor may include the extracellular domain of the receptor. In some embodiments, the soluble receptor may be an Fc fusion protein.

[0015] In some embodiments, the targeting agent moiety may be an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof may be selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, a VNAR, and a NANOBODY® molecule. In some embodiments, the imaging agent moiety may be selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus that induces a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0016] Also disclosed herein is a method of delivering an imaging agent to a tissue sample in a tissue-specific manner, which may include contacting the tissue sample with an imaging agent, which may include an imaging agent component and a targeting agent component. In some embodiments, the targeting agent component may specifically bind to a targeted protein expressed on the muscle cell surface of the tissue. In some embodiments, the targeted protein may be enriched in muscle tissue compared to other tissues. In some embodiments, the targeted protein may have stable or increased expression in diseased tissue compared to normal tissue. In some embodiments, the target protein may be internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0017] In some embodiments, the targeted protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1. In some embodiments, the targeting agent component may be a specific binding agent for the targeted protein. In some embodiments, the specific binding agent may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor may include the extracellular domain of the receptor. In some embodiments, the soluble receptor may be an Fc fusion protein.

[0018] In some embodiments, the targeting agent moiety may be an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, a VNAR, and a NANOBODY® molecule. In some embodiments, the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus that induces a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0019] Also disclosed herein is a method of performing physical imaging of muscle tissue of an individual, which may include administering to the individual an imaging agent, which may include a targeting agent component and an imaging agent component. In some embodiments, the targeting agent component may specifically bind to a targeted protein expressed on the cell surface of muscle tissue. In some embodiments, the targeted protein may be enriched in muscle tissue compared to other tissues. In some embodiments, the targeted protein may have stable or increased expression in diseased tissue compared to normal tissue. In some embodiments, the target protein may be internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0020] In some embodiments, the targeted protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1. In some embodiments, the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0021] Also disclosed herein is a method of assessing an individual for the presence or absence of a muscle tissue pathology, which may include administering to the individual an imaging agent, which may include an imaging agent component and a targeting agent component, and assessing the individual for the presence or absence of a concentration of the imaging agent. In some embodiments, the targeting agent component specifically binds to a targeted protein expressed on the cell surface of muscle tissue. In some embodiments, the presence or absence of a concentration of the imaging agent may indicate the presence of a pathology. In some embodiments, the targeting agent component may be a specific binding agent for the targeted protein. In some embodiments, the specific binding agent may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor may include an extracellular domain of the receptor. In some embodiments, the soluble receptor may be an Fc fusion protein.

[0022] In some embodiments, the targeting agent component may be an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof may be selected from the group consisting of monoclonal antibodies, bispecific antibodies, Fab, Fab-Fc, Fv, single-chain Fv (scFv), diabodies, minibodies, VNAR, and immunoglobulin single variable domains (ISVs) such as NANOBODY® molecules. In some embodiments, the targeted protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1. In some embodiments, the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0023] Also disclosed is a method of evaluating the response of muscle tissue from an individual to treatment with a therapeutic targeting agent, which may include evaluating the level of a target protein in a sample from the individual before treatment with the therapeutic targeting agent, evaluating the level of the target protein in a sample from the individual during or after treatment with the therapeutic targeting agent, and then comparing the pre-treatment level to the during or after treatment level. In some embodiments, the therapeutic targeting agent may specifically bind to a target protein expressed on the cell surface of muscle tissue. In some embodiments, a level of the target protein during or after treatment that is significantly lower than the level of the target protein before treatment may indicate the effectiveness of treatment with the therapeutic targeting agent. In some embodiments, the therapeutic targeting agent may specifically bind to a target protein expressed on the cell surface of muscle tissue. In some embodiments, a level of the target protein during or after treatment that is lower than the level of the target protein before treatment may indicate the effectiveness of treatment with the therapeutic targeting agent. In some embodiments, the target protein may be enriched in muscle tissue compared to other tissues. In some embodiments, the target protein may have stable or increased expression in diseased tissue compared to normal tissue. In some embodiments, the target protein may be internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0024] In some embodiments, the targeted protein may be selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0025] The novel features of the exemplary embodiments are set forth with particularity in the appended claims. A better understanding of the features and advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the exemplary embodiments are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows an outline of the analytical strategy that was implemented to identify muscle-specific targets stably expressed across muscle-related diseases suitable for therapeutic targeting agents. [Diagram 2] Fluorescence microscopy images of immunofluorescence validation of muscle membrane receptor expression and accessibility to antibody binding in live differentiated human myotubes are shown. [Diagram 3] Fluorescence microscopy images of immunofluorescence validation of muscle membrane receptor expression and accessibility to antibody binding in live differentiated mouse myotubes are shown. [Figure 4A] Fluorescence microscopy images of low / no immunofluorescence expression of selected receptors on non-myocytes by immunofluorescence are shown. [Figure 4B] Fluorescence microscopy images of low / no immunofluorescence expression of selected receptors on non-myocytes by immunofluorescence are shown. [Figure 5A-5B] Fluorescence microscopy images of receptor-mediated antibody complex internalization by immunofluorescence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Overview The practice of the methods, and the preparation and use of the compositions disclosed herein will employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields, which are within the skill of the art and are fully explained in the literature. For example, Sambrook et al.MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989 and Third edition,2001;Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons,New York,1987 and periodic updates;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS IN ENZYMOLOGY,Vol.304,“Chromatin”(PMWassarman and APWolffe,eds.),Academic Press,San Diego,1999;andMETHODS IN MOLECULAR BIOLOGY,Vol.119,“Chromatin See, for example, "P.B. Becker, ed.," Humana Press, Totowa, 1999.

[0028] definition The term "herein" means the entire application.

[0029] Unless otherwise defined herein, scientific and technical terms used shall have the meanings commonly understood by one of ordinary skill in the art to which this specification pertains. In general, the nomenclature used in connection with the compounds, compositions and methods described herein is that which is well known and commonly used in the art.

[0030] Any of the embodiments described herein, including those described under various aspects of the disclosure and various portions of the specification (including those described in the examples only), may be combined with one or more other embodiments described herein, unless expressly prohibited or inappropriate, and the combination of embodiments is not limited to the specific combinations claimed through multiple dependent claims.

[0031] All of the above, and any other publications, patents and published patent applications mentioned in this application are specifically incorporated herein by reference. In the event of a conflict, the present specification, including its specific definitions, will control.

[0032] Throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" is understood to mean the inclusion of a stated integer (or component) or group of integers (or components) but not the exclusion of any other integer (or component) or group of integers (or components).

[0033] Throughout this specification, when a composition is described as having, including, or comprising certain components (or variations thereof), it is contemplated that the composition may consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process may consist essentially of or consist of the recited processing steps. Furthermore, it should be understood that the order or sequence of steps for performing certain operations is not critical so long as the compositions and methods described herein are operable. Moreover, two or more steps or operations may be performed simultaneously.

[0034] The term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0035] As used herein, "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean plus or minus 10%, as per the practice in the art. Alternatively, "about" may mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, or within 2-fold of a value. When particular values ​​are described in the present application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range for the particular value. Also, when ranges and / or subranges of values ​​are provided, the ranges and / or subranges may include the endpoints of the ranges and / or subranges.

[0036] The term "substantially" as used herein can refer to a value approaching 100% of a given value. In some cases, the term can refer to an amount that can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the total amount. In some cases, the term can refer to a value that can be about 100% of the total amount.

[0037] The use of "a" and "an" and "the" and similar demonstratives in the context of describing elements should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0038] As used herein, the term "or" should be understood to mean "and / or" unless the context clearly dictates otherwise.

[0039] Statistical significance is used to determine whether the null hypothesis should be rejected or retained. The null hypothesis is the default assumption that nothing happened or changed. That is, a difference or deviation, e.g., a significantly lower protein expression in one sample compared to another sample, is significant if the null hypothesis is rejected. To reject the null hypothesis, it is necessary that the observed results are statistically significant, i.e., the observed p-value is less than a pre-specified significance level α. To determine whether a result is statistically significant, a person skilled in the art calculates a p-value, which is the probability of observing an effect of the same magnitude or more extreme, given that the null hypothesis is true. The null hypothesis is rejected if the p-value is less than (or equal to) a pre-specified level α. α is also called the significance level, and is the probability of rejecting the null hypothesis assuming that it is true (type I error). α may be set to 5%. α may be set to 1%. α may be set to 0.1%. α may be set to 0.01%.

[0040] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand notation for individually referring to each separate value falling within the range, unless otherwise indicated, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better clarify the embodiments, and do not pose limitations on the scope of the claims, unless otherwise stated. No language in this specification should be construed as indicating that any non-claimed element is essential.

[0041] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in linear or cyclic conformation and in either single- or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. The term can include known analogs of natural nucleotides as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (e.g., phosphorothioate backbones, 2'-deoxy-, 2'-O-methyl-, 2'-deoxy-2'-fluoro modified nucleotides, and terminal cap molecules at the 3', 5', or both the 3' and 5' termini). In general, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of A base pairs with T. The term also includes polymers that include one or more chemically modified nucleotides. Non-limiting examples of polynucleotides include small interfering RNA (siRNA), microRNA (miRNA), miRNA mimics, short hairpin RNA (shRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), heterogeneous nuclear RNA (hnRNA), antisense oligonucleotides (ASOs, including exon skipping ASOs), messenger RNA (mRNA), complementary DNA (cDNA), plasmids and vectors, and guide RNA.

[0042] The oligonucleotide may comprise a sugar modification. The oligonucleotide may comprise multiple sugar modifications. The sugar modification may comprise glucose or a derivative thereof. The sugar modification may comprise ribose or deoxyribose. The sugar modification may comprise a monosaccharide, a disaccharide, a trisaccharide, or any combination thereof.

[0043] The terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0044] The term "residue" as used herein refers to a position in a protein and its associated amino acid identity.

[0045] The term "homology" can refer to the % identity of a sequence to a reference sequence. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond to a particular nucleic acid sequence described herein), such a particular polypeptide sequence can be routinely determined using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference sequence, parameters can be set so that the percentage of identity is calculated over the full length of the reference sequence, and a maximum of 5% homology gap across the reference sequence is allowed.

[0046] For example, in certain embodiments, the identity between a reference sequence (query sequence, i.e., a sequence described herein) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some cases, identity is interpreted narrowly, and parameters for certain embodiments used in the FASTDB amino acid alignment can include the following: scoring scheme=PAM (mutation tolerance) 0, k-tuple=2, mismatch penalty=1, linkage penalty=20, randomization group length=0, cutoff value=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the subject sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, but not due to internal deletions, the results need to be manually corrected to take into account the fact that the FASTDB program does not consider the N- and C-terminal truncations of the subject sequence when calculating the overall percent identity. For subject sequences that are N- and C-terminally truncated relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal to the subject sequence that do not match / align with the corresponding subject sequence as a percentage of the total bases of the query sequence. The determination of whether a residue matches / aligns can be determined by the results of the FASTDB sequence alignment. This percentage can then be subtracted from the percent identity calculated by the FASTDB program using the specified parameters to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues at the N- and C-termini of the subject sequence, which are not matched / aligned with the query sequence, may be considered for the purposes of manual adjustment of the percent identity score, i.e., only query residue positions other than the farthest N- and C-terminal residues of the subject sequence are considered for this manual correction.For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. These 10 unmatched residues represent 10% of the sequence (number of residues at the N-terminus and C-terminus that do not match / total number of residues in the query sequence), and therefore 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90-residue subject sequence is compared with a 100-residue query sequence. Since this deletion is an internal deletion, there are no residues at the N-terminus or C-terminus of the subject sequence that do not match / align with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions other than the N- and C-termini of the subject sequence, as displayed in the FASTDB alignment, that are not matched / aligned with the query sequence are manually corrected.

[0047] The term "fragment," as used herein, can be a portion of a sequence, i.e., a subset that is less than the full-length sequence. A fragment can be a portion of a gene. A fragment can be a portion of a peptide or protein. A fragment can be a portion of an amino acid sequence. A fragment can be a portion of an oligonucleotide sequence. A fragment can be less than about 20, 30, 40, 50 amino acids in length. A fragment can be less than about 2, 5, 10, 20, 30, 40, 50 oligonucleotides in length.

[0048] As used herein, the terms "Fc", "Fc region" or "Fc domain" are used interchangeably herein and refer to a polypeptide comprising the constant region of an antibody, optionally excluding the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, optionally excluding a portion of the hinge. Thus, Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In the case of IgA and IgM, Fc can include the J chain. In the case of IgG, the Fc domain includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, Fc refers to the truncated CH1 domain of an immunoglobulin, as well as CH2 and CH3. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues E216 or C226 or P230 to the carboxyl terminus, with numbering according to EU numbering. In some embodiments, the Fc domain is derived from a human IgG1 heavy chain Fc domain. In some embodiments, the Fc domain is derived from a human IgG2 heavy chain Fc domain. "EU format as defined by Edelman" or "EU numbering" or "EU index" refers to the residue numbering of a human Fc domain as described in Edelman GM et al. (Proc. Natl. Acad. USA (1969), 63, 78-85, incorporated herein by reference in its entirety).

[0049] As used herein, the term "Fc fusion protein" generally refers to a protein that includes an Fc region linked (optionally via a linker moiety) to a different protein.

[0050] As used herein, the term "antibody" or "Ab" refers to an immunoglobulin molecule (e.g., a complete antibody, an antibody fragment, or a modified antibody) that can recognize and bind to a specific target or antigen, such as, for example, a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc., through at least one antigen recognition site located within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody, including, but not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, engineered antibodies (including humanized antibodies, fully human antibodies, chimeric antibodies, single chain antibodies, artificially selected antibodies, CDR-bearing antibodies, etc.), that specifically binds to a given antigen. In some embodiments, "antibody" and / or "immunoglobulin" (Ig) refers to a polypeptide comprising at least two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa), optionally interconnected by disulfide bonds. There are two types of light chains: λ and κ. In humans, the lambda and kappa light chains are similar, but there is only one type of each antibody. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), which is incorporated by reference in its entirety. In some embodiments, the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no binding ability to Fc receptors. For example, the antibody can be an isotype or subtype, antibody fragment, or mutant that does not support binding to Fc receptors, e.g., with a mutation or deletion of the Fc receptor binding region.

[0051] An "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity), or in other words, an antibody that does not contain the full-length antibody polypeptide, but that contains at least a portion of the full-length antibody polypeptide that is still capable of binding to an antigen. Antibody fragments can include truncated portions of a full-length antibody polypeptide, but the term is not limited to such truncated fragments. Examples of antigen-binding fragments include (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragment (Ward et al., (1989) Nature 341:544-546), consisting of the VH domain; and (vi) isolated complementarity determining regions (CDRs), disulfide-linked Fv (dsFv), and anti-idiotypic (anti-Id) antibodies and intrabodies. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv)), see, e.g., Bird et al. Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Other forms of single-chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123). Useful antibody fragments include, for example, Fab fragments, F(ab')2 fragments, scFv (single-chain Fv) fragments, linear antibodies, monospecific or multispecific antibody fragments, such as bispecific, trispecific, tetraspecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies), monovalent or multivalent antibody fragments, such as bivalent, trivalent, tetravalent, and multivalent antibodies, minibodies, chelating recombinant antibodies, tribodies or bibodies, intrabodies, NANOBODY® molecules, small modular immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins, camelized antibodies, and VHH-containing antibodies. Additional examples of antigen-binding antibody fragments are known in the art.

[0052] The term "immunoglobulin single variable domain" (ISV) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which an antigen-binding site is present on, and formed by, a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V LBoth complementarity determining regions (CDRs) of the ribozyme contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0053] In view of the above definition, fragments of conventional four-chain antibodies (e.g. IgG, IgM, IgA, IgD or IgE molecules known in the art), or Fab fragments, F(ab')2 fragments, Fv fragments such as disulfide-linked Fv or scFv fragments, or bispecific antibodies derived from such conventional four-chain antibodies (all known in the art) are not usually considered as immunoglobulin single variable domains, because in these cases binding to the respective epitope of an antigen is usually not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains such as light and heavy chain variable domains, i.e. the V of immunoglobulin domains which jointly bind to the respective epitope of the antigen. H -V L Because it happens in pairs.

[0054] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with other immunoglobulin variable domains. The binding site of an immunoglobulin single variable domain consists of a single VH, a single V HH or single V L It is formed by domains.

[0055] Thus, a single variable domain may be any combination of a light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that consists essentially of a single variable domain and that does not require interaction with other variable domains to form a functional antigen-binding unit). L -sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., H -sequence or V HH sequence) or a suitable fragment thereof.

[0056] Immunoglobulin single variable domains (ISVs) include, for example, heavy chain ISVs, such as camelized ISVs. Hor humanized V HH Contains V H ,V HH In one embodiment, this can be a camelized V H or humanized V HH Contains V HH The heavy chain ISV can be derived from a traditional four chain antibody or from a heavy chain antibody.

[0057] For example, an immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), a NANOBODY® ISV (as defined herein, V HH (including but not limited to), other single variable domains, or any suitable fragment of any one of these.

[0058] In particular, the immunoglobulin single variable domain is a NANOBODY® ISV (e.g., a humanized V HH Or camelization V H Contains V HH ) or a suitable fragment thereof. [Note: NANOBODY® and NANOBODY® are registered trademarks of Ablynx NV.]

[0059] "V HH Domain" is V HH s, V HH Antigen fragments, and V HH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al. Nature 363:446-448 (1993)). HH The term "variable domain" refers to the heavy chain variable domains present in a conventional four-chain antibody (heavy chain variable domains, herein referred to as "V H domains”) present in conventional four-chain antibodies (herein referred to as “V LThe domain was chosen to distinguish it from the domains that are called "domains." HH For further discussion, see the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0060] The generation of immunoglobulin sequences such as VHHs has been widely described in various published literature, including WO 94 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods camelids are immunized with a target antigen to elicit an immune response against the target antigen. The repertoire of VHHs resulting from the immunization is further screened for VHHs that bind to the target antigen.

[0061] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such sources.

[0062] Immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human, and camelized immunoglobulin sequences, can be sequenced by the methods described herein. Also, fully human, humanized, or chimeric sequences can be sequenced by the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs described by Ward et al. (see, e.g., WO 94 / 04678 and also Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996), can be sequenced by the methods described herein. Additionally, ISVs can be fused to form multivalent and / or multispecific constructs (one or more V HH For multivalent and multispecific polypeptides containing domains and their formulations, see Conrath et al., J. Biol. Chem., Vol. 276.10.7346-7350, 2001, as well as, for example, WO 96 / 34103 and WO 99 / 23221).

[0063] "Humanized V HH " is a naturally occurring V HH domain, but "humanized," i.e., the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the sequence are replaced with a V H The humanized V domain may comprise an amino acid sequence substituted with one or more of the amino acid residues (e.g., as described above) occurring at the corresponding positions in the V domain. This can be carried out in a manner well known per se, as will be clear to the skilled artisan, for example based on the prior art (e.g., WO 2008 / 020079). Again, such a humanized V domain may be substituted with one or more of the amino acid residues (e.g., as described above) occurring at the corresponding positions in the V domain. HH It should be noted that the VHH domain-containing polypeptides can be obtained by any conventional method known per se and are therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material.

[0064] "Camelization V H " is a naturally occurring V H The amino acid sequence of the V domain corresponds to that of the naturally occurring V from a conventional four-chain antibody, but has been "camelized", i.e. H One or more amino acid residues in the amino acid sequence of the domain are replaced by the V HH The term "camelization" includes an amino acid sequence in which one or more of the amino acid residues occurring in the corresponding positions in the V domain are substituted. This can be carried out in a manner known per se, as will be clear to the skilled artisan, for example as described in the prior art (e.g. Davies and Riechman (1994 and 1996) supra). Such "camelization" substitutions, as defined herein, can be carried out by the addition of one or more of the amino acid residues occurring in the corresponding positions in the V domain. H -V L They are inserted at the positions of amino acids that form and / or are present at interfaces and / or so-called camelid hallmark residues (see, for example, WO 94 / 04678 and Davies and Riechmann, supra (1994 and 1996)). H V, which is used as a starting material or starting point for generating or designing H The sequence is V H Sequences, e.g., V H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained by any suitable method known per se, and therefore can be obtained without using the naturally occurring V as starting material. H It should be noted that the polypeptide obtained using the polypeptide containing the domain is not strictly limited.

[0065] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively, interrupted by three "complementarity determining regions" ("CDRs"), which are referred to in the art and herein as "complementarity determining region 1" ("CDR1"), "complementarity determining region 2" ("CDR2"), and "complementarity determining region 3" ("CDR3"), respectively.

[0066] In such immunoglobulin sequences, the framework regions may be any suitable framework region sequence, and examples of suitable framework sequences will be clear to the skilled person, e.g. from standard handbooks and on the basis of the further disclosure and prior art set out herein.

[0067] The framework sequences are immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization) (suitable combinations of such). For example, the framework sequences may be those of a light chain variable domain (e.g., V L -sequence) and / or heavy chain variable domain (e.g., V H -sequence or V HH In one particular embodiment, the framework sequence is derived from the V HH -framework sequences derived from conventional V sequences (wherein said framework sequences are optionally partially or fully humanized) or camelized (as defined herein) H It can be one of the arrays.

[0068] In particular, the framework sequences present in the ISV sequences used in the methods described herein may include one or more Hallmark residues (as defined herein), and the ISV sequence may be, for example, a humanized VSV.HH or Camelization V H Contains V HH NANOBODY® ISVs. Non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the disclosure herein below.

[0069] V H Domain and V HH The total number of amino acid residues in a domain will usually be in the range of 110 to 120, often 112 to 115. It should be noted, however, that shorter and longer sequences may also be suitable for the purposes described herein.

[0070] However, it should be noted that the ISVs contained in the multivalent ISV polypeptides sequenced by the methods of the present application are not limited by the origin of the ISV sequence (or of the nucleotide sequence used to represent it), nor by the manner in which the ISV sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISV sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific, but non-limiting embodiments, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including humanized (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized VHV sequences). HH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized V H ISVs include ISVs derived by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, joining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar immunoglobulin sequence engineering techniques well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0071] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example a sequence isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by mutagenesis (using any suitable technique known per se, such as mismatch PCR) into a naturally occurring nucleotide sequence, a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0072] Generally, NANOBODY® ISVs (especially (partially) humanized VSVs) are HH Sequence and camelized V H V containing arrays HH A NANOBODY® ISV (a sequence) may be characterized by the presence of one or more "hallmark residues" (also as further described herein) in one or more framework sequences (as described herein). Thus, in general, a NANOBODY® ISV may be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1-FR4 refer to framework regions 1-4, respectively, CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein. Specifically, a NANOBODY® ISV can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and framework sequences are as further defined herein. More specifically, a NANOBODY® ISV can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, 108 according to the Kabat numbering are selected from the hallmark residues set out in Table 4 below.

[0073] [Table 1]

[0074] The terms "microRNA", "miRNA" and "miR" are used interchangeably herein to refer to single-stranded non-coding RNAs that function in NRA silencing and post-transcriptional regulation of gene expression. miRNAs function by complementary base pairing with mRNA molecules and, in particular, silence mRNAs by one or more of the following: (a) cleavage of the mRNA strand into two parts, (b) destabilization of the mRNA through shortening of the poly(A) tail, and (c) inefficient translation of the mRNA into protein by ribosomes.

[0075] The terms "subject", "patient" and "individual" are used interchangeably and refer to mammals, including, but not limited to, human patients and non-human primates, as well as rabbits, dogs, cats, rats, mice, and other animals. Thus, the term "subject" or "patient" as used herein means any mammalian patient or subject to which the compositions of the present disclosure can be administered. Subjects include those with a disorder. In one embodiment, the subject is a human.

[0076] The term "genetic disease" as used herein refers to a disease or disorder that can be treated with polynucleotide therapeutics. Examples of genetic disease include, but are not limited to, any disease or disorder caused by gene mutation, cancer, and viral infection, disease or disorder caused by mutation that can be corrected using gene editing (e.g., CRISPR / Cas9, or zinc finger nuclease), disease or disorder caused by overexpression of genes, and disease or disorder caused by reduced or absent expression of genes.

[0077] As used herein, the term "targeting molecule" refers to a molecule that binds to or localizes at a particular target or location. The molecule may be, for example, an antibody or an antigen-binding fragment thereof, or a binding protein. A targeting molecule may be, for example, an immunoglobulin single variable domain (ISV), such as a NANOBODY® molecule.

[0078] The pharmaceutical composition may comprise a first active ingredient. The pharmaceutical composition may be formulated in a unit dose form. The pharmaceutical composition may comprise a pharma- ceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition may comprise a second, third, or fourth active ingredient.

[0079] The compositions described herein may include an excipient. The excipient may include a pH agent (to minimize oxidation or deterioration of the components of the composition), a stabilizer (to prevent modification or deterioration of the components of the composition), a buffer (to increase temperature stability), a solubilizer (to increase protein solubility), or any combination thereof. The excipient may include a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof. The excipients may include sodium carbonate, acetate, citrate, phosphate, polyethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HCl, disodium edetate, lecithin, glycerin, xanthan gum, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. The excipients may be those described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).

[0080] The term "pharmaceutical effective amount", "therapeutically effective amount" or "therapeutically effective dose" refers to an amount effective to treat a disease in a patient, for example, to effect a beneficial and / or desirable change in the overall health, physiological response or condition of a patient suffering from the disease (e.g., a genetic disease as described herein), such as treating, curing, inhibiting or improving. A complete therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for a subject will depend, for example, on the subject's size, health and age, the nature and extent of the disease, the therapeutic agent or combination of therapeutic agents selected for administration, and the method of administration. A skilled practitioner can easily determine the effective amount for a given situation by routine experimentation. A skilled practitioner will recognize that, for example, treating cancer includes, but is not limited to, killing cancer cells, preventing the growth of new cancer cells, causing tumor regression (a reduction in tumor size), causing a reduction in metastasis, improving a patient's vital functions, improving a patient's health, reducing pain, improving appetite, improving a patient's weight, and any combination thereof. The terms "pharmacologically effective amount," "therapeutically effective amount," or "therapeutically effective dose" also refer to the amount necessary to improve the clinical symptoms of a patient. The methods of treatment or methods of treating described herein should not be construed as or otherwise limited to "curing" a disease.

[0081] As used herein, the term "treating" or "treatment" includes reversing, alleviating or arresting symptoms, clinical signs, and underlying pathology of a condition in a manner that improves or stabilizes the condition of a subject. As used herein and as is well understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviating, ameliorating, or slowing the progression of one or more symptoms or conditions associated with a condition, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, delaying or slowing disease progression, improving or palliating, and remission (either partial or total), of the disease state, either detectable or undetectable. "Treatment" can also mean extending survival compared to expected survival in the absence of treatment. Exemplary beneficial clinical results are described herein. The terms "treating" and "treatment" can also relate to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying disease, preventing the onset of symptoms and / or underlying disease, and / or improving or repairing damage.

[0082] "Administering" a composition as disclosed herein to a subject or "administration" of a composition can be performed using one of a variety of methods known to those skilled in the art. Administering can also be performed, for example, once, multiple times, and / or over one or more extended periods. In some aspects, administering includes both direct administration, including self-administration, and joint administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or who instructs another physician to administer a drug, and / or who provides a patient with a prescription for a drug, is administering the drug to the patient. If the method is part of a treatment regimen involving two or more pharmaceutical compositions or therapies, the present disclosure contemplates that the pharmaceutical compositions can be administered at the same or different times and via the same or different routes of administration.

[0083] Administration or application of the compositions disclosed herein provides at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 consecutive or nonconsecutive days of treatment. In some cases, the treatment period is from about 1 to about 30 days, from about 2 to about 30 days, from about 3 to about 30 days, from about 4 to about 30 days, from about 5 to about 30 days, from about 6 to about 30 days, from about 7 to about 30 days, from about 8 to about 30 days, from about 9 to about 30 days, from about 10 to about 30 days, from about 11 to about 30 days, from about 12 to about 30 days, from about 13 to about 30 days, from about 14 to about 30 days, from about 15 to about The incubation period may be about 30 days, about 16 to about 30 days, about 17 to about 30 days, about 18 to about 30 days, about 19 to about 30 days, about 20 to about 30 days, about 21 to about 30 days, about 22 to about 30 days, about 23 to about 30 days, about 24 to about 30 days, about 25 to about 30 days, about 26 to about 30 days, about 27 to about 30 days, about 28 to about 30 days, or about 29 to about 30 days.

[0084] The administration or application of the compositions disclosed herein may be performed for a treatment period of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. Administration may be performed repeatedly over the life of the subject, such as once a month or once a year, for the life of the subject. Administration may be performed repeatedly over a substantial portion of the life of the subject, such as once a month or once a year, for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more.

[0085] Administration or application of the compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day. In some cases, administration or application of the compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some cases, the administration or application of the compositions disclosed herein is for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 104, 105, 106, 107, 108, 109, 1 In one embodiment, the method may be performed 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times.

[0086] In some cases, the composition can be administered / applied as a single dose or as a divided dose. In some cases, the composition described herein can be administered at a first time point and a second time point. In some cases, the composition can be administered such that the first administration precedes the other administration by 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more.

[0087] As used herein, the second therapy may include immunotherapy, hormone therapy, cryotherapy, surgical procedures, or any combination thereof. The second therapy may include administration of a pharmaceutical composition, such as a small molecule. The second therapy may include administration of a pharmaceutical composition, such as one or more antibiotics. The second therapy may include a muscle relaxant, an antidepressant, a steroid, an opioid, a cannabis-based therapeutic, acetaminophen, a nonsteroidal anti-inflammatory drug, a neuropathic agent, cannabis, a progestin, a progesterone, or any combination thereof. The neuropathic agent may include gabapentin. The nonsteroidal anti-inflammatory drug may include naproxen, ibuprofen, a COX-2 inhibitor, or any combination thereof. The second therapy may include administration of a biological agent, a cell therapy, a regenerative medicine therapy, a tissue engineering approach, a stem cell transplant, or any combination thereof. The second therapy may include a medical procedure. The medical treatment may include epidural injections (such as steroid injections), acupuncture, exercise, physical therapy, ultrasound, surgical therapy, chiropractic procedures, orthopedic procedures, chemonucleolysis, or any combination thereof. The second therapy may include the use of a respiratory support device or a ventilator. The second therapy may include administration of a regenerative or immunotherapy therapy, such as a protein, stem cells, cord blood cells, umbilical cord tissue, tissue, or any combination thereof. The second therapy may include a biosimilar.

[0088] Diagnostic tests may include imaging procedures, blood count analysis, histopathology analysis, biomarker analysis, biopsy, magnetic resonance imaging, physical examination, urinalysis, ultrasound, genetic testing, liver function tests, positron emission tomography procedures, x-rays, serology tests, angiography, electrocardiography, endoscopy, diagnostic polymerase chain reaction test (PCR), PAP smear, hematocrit test, skin allergy test, urinalysis, colonoscopy, enzyme-linked immunosorbent assay (ELISA), microscopy, bone marrow test, rapid diagnostic test, pregnancy test, organ function test, toxicity test, infectious disease test, body fluid test, or any combination thereof.

[0089] The term "tissue" as used herein may be any tissue sample. The tissue may be tissue suspected or confirmed to have a disease or condition. The tissue may be substantially healthy, substantially benign, or otherwise substantially free of a disease or condition. The tissue may be tissue removed from a subject, such as a tissue biopsy, tissue resection, aspirate (such as a fine needle aspirate), tissue washing, cytology specimen, bodily fluid, or any combination thereof. The tissue may include cancerous cells, tumor cells, non-cancerous cells, or a combination thereof. The tissue may include a blood sample (e.g., a cell-free DNA sample). The tissue may be a sample that may be genetically modified.

[0090] In some cases, the disease or condition may involve a neuromuscular disorder.

[0091] In some embodiments, the polynucleotide is conjugated to a (therapeutic) targeting molecule / drug. In some embodiments, the targeting moiety includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all these classes of substances. Additional examples of targeting moieties include steroids such as cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, carbohydrates (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some embodiments, the targeting moiety is an antibody or a binding fragment thereof. Targeting moiety is used interchangeably herein with targeting agent or targeting molecule.

[0092] The targeting molecule may be an antibody or an antigen-binding fragment thereof, or a binding protein. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof (e.g., a polynucleotide-antibody complex). In some embodiments, the antibody or a binding fragment thereof is a human antibody or an antigen-binding fragment thereof, a humanized antibody or an antigen-binding fragment thereof, a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a monoclonal antibody or an antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, an immunoglobulin single variable domain (ISV) such as a NANOBODY® molecule, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a vNAR, a mutein based on tenascin-C (also known as a centrin molecule), a camelid antibody or an antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, bispecific antibodies, immunoglobulin single variable domains (ISVs) such as Fab, Fab-Fc, Fv, single chain Fv (scFv), diabodies, minibodies, and NANOBODY® molecules. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. Non-limiting examples of bispecific antibodies include bispecific T cell engagers (BiTEs) and dual affinity retargeting antibodies (DARTs). In some embodiments, the bispecific antibody is a trifunctional antibody or a bispecific miniantibody. In some embodiments, the bispecific antibody is a trifunctional antibody. In some embodiments, the trifunctional antibody is a full-length monoclonal antibody that contains binding sites for two different antigens. In some embodiments, the bispecific antibody is a bispecific miniantibody.In some embodiments, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody or bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein containing two single chain variable fragments (scFv), where the two scFvs target epitopes of two different antigens.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof is a Fab. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab-Fc. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv. In some embodiments, the antibody or antigen-binding fragment thereof is a single chain Fv (scFv). In some embodiments, when the antibody or antigen-binding portion thereof is an scFv, the polynucleotide does not comprise bridging residues. In some embodiments, when the antibody or antigen-binding portion thereof is an scFv, the polynucleotide does not comprise cysteines. In some embodiments, the antibody or antigen-binding fragment thereof is a diabody. In some embodiments, the antibody or antigen-binding fragment thereof is a minibody. In some embodiments, the antibody or antigen-binding fragment thereof is an immunoglobulin single variable domain (ISV), such as a NANOBODY® molecule. The NANOBODY® may be a NANOBODY® molecule-HSA.

[0094] In some embodiments, the antibody or antigen-binding fragment thereof is or is derived from an IgG molecule. The IgG molecule may be an IgG1 or IgG4 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG1 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG2 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG3 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG4 molecule.

[0095] In some embodiments, the targeting molecule is a binding protein. The binding protein may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor comprises the extracellular domain of the receptor. In some embodiments, the soluble receptor is an Fc fusion protein.

[0096] In some embodiments, the targeting molecule is a plasma protein. In some embodiments, the plasma protein comprises albumin. In some embodiments, the albumin is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some examples, the albumin is conjugated to the polynucleotide by native ligation chemistry. In some examples, the albumin is lysine conjugated to the polynucleotide.

[0097] In some examples, the targeting molecule is a steroid. Non-limiting exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, saturated, unsaturated, substituted, or other hydrocarbons, or combinations thereof. In some embodiments, the steroid is cholesterol or a cholesterol derivative. In some embodiments, the targeting molecule is cholesterol. In some embodiments, the steroid is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some embodiments, the steroid is conjugated to the polynucleotide by native ligation chemistry.

[0098] In some embodiments, the targeting molecule is a polymer, including but not limited to a polynucleotide aptamer, that binds to a specific surface marker on a cell. In some embodiments, the targeting molecule is a polynucleotide that does not hybridize to a target gene or mRNA, but instead can selectively bind to a cell surface marker, similar to an antibody that binds to its specific epitope on the cell surface marker.

[0099] In some embodiments, the targeting molecule comprises or is a polypeptide. In some embodiments, the polypeptide has a size of about 1 to about 3 kDa. In some embodiments, the polypeptide has a size of about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some embodiments, the targeting molecule is a polypeptide. In some embodiments, the polypeptide has a size of 1 to 3 kDa. In some embodiments, the polypeptide has a size of 1.2 to 2.8 kDa, 1.5 to 2.5 kDa, or 1.5 to 2 kDa. In some embodiments, the polypeptide is a bicyclic polypeptide. In some embodiments, the bicyclic polypeptide is a constrained bicyclic polypeptide. In some embodiments, the targeting molecule is a bicyclic polypeptide (e.g., bicyclic compounds from Bicycle Therapeutics).

[0100] In additional embodiments, the targeting molecule comprises or is a small molecule. In some embodiments, the small molecule comprises or is an antibody-recruiting small molecule. In some embodiments, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, wherein the target-binding end is capable of recognizing and interacting with a cell surface receptor.

[0101] In some embodiments, the targeting molecule comprises or is a therapeutically or biologically active molecule.

[0102] In some embodiments, an agent (such as a therapeutic agent or a therapeutic targeting agent described herein) comprises a polynucleotide.

[0103] In some embodiments, the polynucleotide is about 5 to about 100 nucleotides in length. In some embodiments, the polynucleotide is about 5 to about 50 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length. In some embodiments, the polynucleotide is about 50 nucleotides in length. In some embodiments, the polynucleotide is about 49 nucleotides in length. In some embodiments, the polynucleotide is about 48 nucleotides in length. In some embodiments, the polynucleotide is about 47 nucleotides in length. In some embodiments, the polynucleotide is about 46 nucleotides in length. In some embodiments, the polynucleotide is about 45 nucleotides in length. In some embodiments, the polynucleotide is about 44 nucleotides in length. In some embodiments, the polynucleotide is about 43 nucleotides in length. In some embodiments, the polynucleotide is about 42 nucleotides in length. In some embodiments, the polynucleotide is about 41 nucleotides in length. In some embodiments, the polynucleotide is about 40 nucleotides in length. In some embodiments, the polynucleotide is about 39 nucleotides in length. In some embodiments, the polynucleotide is about 38 nucleotides in length. In some embodiments, the polynucleotide is about 37 nucleotides in length. In some embodiments, the polynucleotide is about 36 nucleotides in length. In some embodiments, the polynucleotide is about 35 nucleotides in length. In some embodiments, the polynucleotide is about 34 nucleotides in length. In some embodiments, the polynucleotide is about 33 nucleotides in length. In some embodiments, the polynucleotide is about 32 nucleotides in length. In some embodiments, the polynucleotide is about 31 nucleotides in length. In some embodiments, the polynucleotide is about 30 nucleotides in length.In some embodiments, the polynucleotide is about 29 nucleotides in length. In some embodiments, the polynucleotide is about 28 nucleotides in length. In some embodiments, the polynucleotide is about 27 nucleotides in length. In some embodiments, the polynucleotide is about 26 nucleotides in length. In some embodiments, the polynucleotide is about 25 nucleotides in length. In some embodiments, the polynucleotide is about 24 nucleotides in length. In some embodiments, the polynucleotide is about 23 nucleotides in length. In some embodiments, the polynucleotide is about 22 nucleotides in length. In some embodiments, the polynucleotide is about 21 nucleotides in length. In some embodiments, the polynucleotide is about 20 nucleotides in length. In some embodiments, the polynucleotide is about 19 nucleotides in length. In some embodiments, the polynucleotide is about 18 nucleotides in length. In some embodiments, the polynucleotide is about 17 nucleotides in length. In some embodiments, the polynucleotide is about 16 nucleotides in length. In some embodiments, the polynucleotide is about 15 nucleotides in length. In some embodiments, the polynucleotide is about 14 nucleotides in length. In some embodiments, the polynucleotide is about 13 nucleotides in length. In some embodiments, the polynucleotide is about 12 nucleotides in length. In some embodiments, the polynucleotide is about 11 nucleotides in length. In some embodiments, the polynucleotide is about 10 nucleotides in length. In some embodiments, the polynucleotide is about 9 nucleotides in length. In some embodiments, the polynucleotide is about 8 nucleotides in length. In some embodiments, the polynucleotide is about 7 nucleotides in length. In some embodiments, the polynucleotide is about 6 nucleotides in length. In some embodiments, the polynucleotide is about 5 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 50 nucleotides in length.In some embodiments, the polynucleotide is about 10 to about 45 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 40 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 35 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 30 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 25 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 20 nucleotides in length. In some embodiments, the polynucleotide is about 15 to about 25 nucleotides in length. In some embodiments, the polynucleotide is about 15 to about 30 nucleotides in length. In some embodiments, the polynucleotide is about 12 to about 30 nucleotides in length.

[0104] In some embodiments, the polynucleotide is 5-100 nucleotides in length. In some embodiments, the polynucleotide is 5-50 nucleotides in length. In some embodiments, the polynucleotide is 10-30, 15-30, 18-25, 18-24, 19-23, or 20-22 nucleotides in length. In some embodiments, the polynucleotide is 50 nucleotides in length. In some embodiments, the polynucleotide is 49 nucleotides in length. In some embodiments, the polynucleotide is 48 nucleotides in length. In some embodiments, the polynucleotide is 47 nucleotides in length. In some embodiments, the polynucleotide is 46 nucleotides in length. In some embodiments, the polynucleotide is 45 nucleotides in length. In some embodiments, the polynucleotide is 44 nucleotides in length. In some embodiments, the polynucleotide is 43 nucleotides in length. In some embodiments, the polynucleotide is 42 nucleotides in length. In some embodiments, the polynucleotide is 41 nucleotides in length. In some embodiments, the polynucleotide is 40 nucleotides in length. In some embodiments, the polynucleotide is 39 nucleotides in length. In some embodiments, the polynucleotide is 38 nucleotides in length. In some embodiments, the polynucleotide is 37 nucleotides in length. In some embodiments, the polynucleotide is 36 nucleotides in length. In some embodiments, the polynucleotide is 35 nucleotides in length. In some embodiments, the polynucleotide is 34 nucleotides in length. In some embodiments, the polynucleotide is 33 nucleotides in length. In some embodiments, the polynucleotide is 32 nucleotides in length. In some embodiments, the polynucleotide is 31 nucleotides in length. In some embodiments, the polynucleotide is 30 nucleotides in length. In some embodiments, the polynucleotide is 29 nucleotides in length.In some embodiments, the polynucleotide is 28 nucleotides in length. In some embodiments, the polynucleotide is 27 nucleotides in length. In some embodiments, the polynucleotide is 26 nucleotides in length. In some embodiments, the polynucleotide is 25 nucleotides in length. In some embodiments, the polynucleotide is 24 nucleotides in length. In some embodiments, the polynucleotide is 23 nucleotides in length. In some embodiments, the polynucleotide is 22 nucleotides in length. In some embodiments, the polynucleotide is 21 nucleotides in length. In some embodiments, the polynucleotide is 20 nucleotides in length. In some embodiments, the polynucleotide is 19 nucleotides in length. In some embodiments, the polynucleotide is 18 nucleotides in length. In some embodiments, the polynucleotide is 17 nucleotides in length. In some embodiments, the polynucleotide is 16 nucleotides in length. In some embodiments, the polynucleotide is 15 nucleotides in length. In some embodiments, the polynucleotide is 14 nucleotides in length. In some embodiments, the polynucleotide is 13 nucleotides in length. In some embodiments, the polynucleotide is 12 nucleotides in length. In some embodiments, the polynucleotide is 11 nucleotides in length. In some embodiments, the polynucleotide is 10 nucleotides in length. In some embodiments, the polynucleotide is 9 nucleotides in length. In some embodiments, the polynucleotide is 8 nucleotides in length. In some embodiments, the polynucleotide is 7 nucleotides in length. In some embodiments, the polynucleotide is 6 nucleotides in length. In some embodiments, the polynucleotide is 5 nucleotides in length. In some embodiments, the polynucleotide is 10-50 nucleotides in length. In some embodiments, the polynucleotide is 10-45 nucleotides in length.In some embodiments, the polynucleotide is 10-40 nucleotides in length. In some embodiments, the polynucleotide is 10-35 nucleotides in length. In some embodiments, the polynucleotide is 10-30 nucleotides in length. In some embodiments, the polynucleotide is 10-25 nucleotides in length. In some embodiments, the polynucleotide is 10-20 nucleotides in length. In some embodiments, the polynucleotide is 15-25 nucleotides in length. In some embodiments, the polynucleotide is 15-30 nucleotides in length. In some embodiments, the polynucleotide is 12-30 nucleotides in length.

[0105] In some embodiments, the polynucleotide comprises RNA, DNA, or a combination thereof. In some embodiments, the polynucleotide comprises RNA. In some embodiments, the polynucleotide comprises DNA. In some embodiments, the polynucleotide comprises RNA and DNA. In some embodiments, the polynucleotide comprises a combination of DNA, RNA, and / or artificial nucleotide analogs. In some embodiments, the polynucleotide is a regulatory non-coding RNA (ncRNA). In some embodiments, the ncRNA comprises short non-coding RNA sequences expressed in the genome that regulate the expression or function of other biomolecules in mammalian cells. ncRNAs are generally <200 nucleotides in length, can be single-stranded or double-stranded, and can form non-linear secondary or tertiary structures. The ncRNA may include exogenously derived small interfering RNA (siRNA), microRNA (miRNA), nucleolar RNA (U-RNA), small nuclear RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), transfer RNA-derived small RNA (tsRNA), ribosomal RNA-derived small RNA (rsRNA), long non-coding RNA-derived small RNA (lncsRNA), or messenger RNA-derived small RNA (msRNA). In some embodiments, the polynucleotide is an engineered polynucleotide. The engineered polynucleotide may include DNA or RNA. In some embodiments, the engineered polynucleotide includes a plurality of nucleotides. In some embodiments, the engineered polynucleotide includes an artificial nucleotide analog. In some embodiments, the engineered polynucleotide includes DNA. In some embodiments, the DNA is genomic DNA, cell-free DNA, cDNA, fetal DNA, viral DNA, or maternal DNA. In some embodiments, the engineered polynucleotide includes RNA.In some embodiments, the RNA is an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a miximer, a double-stranded RNA (dsRNA), a single-stranded RNAi (ssRNAi), a DNA-dependent RNA interference (ddRNAi), an RNA-activating oligonucleotide (RNAa), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a heterogeneous nuclear RNA (hnRNA), a promoter-associated RNA, a non-coding RNA element that regulates ribosomal RNA transcription by interacting with TIP5 (NoRC RNA), a ribozyme, an anti-microRNA (antimiR), an aptamer, or an exon-skipping oligonucleotide. In some embodiments, the engineered polynucleotide comprises a fully synthetic miRNA. A fully synthetic miRNA is one that is not induced or based on an ncRNA. Alternatively, a fully synthetic miRNA may be based on an analysis of multiple potential target sequences or may be based on an isolated natural non-coding sequence that is not an ncRNA. In some embodiments, the polynucleotide is selected from the group consisting of siRNA, miRNA, miRNA mimic, antisense oligonucleotide (ASO), mRNA, and guide RNA. The polynucleotide may be an siRNA. In some embodiments, the polynucleotide is an miRNA. In some embodiments, the polynucleotide is an miRNA mimic.

[0106] In some embodiments, the polynucleotide is an ASO. In some embodiments, the ASO is a DMPK ASO. In some embodiments, the ASO is a CAPN3 ASO. The ASO may be a DUX4-targeting ASO. DUX4-targeting ASOs are known in the art. See WO 2021 / 203043 and U.S. Provisional Patent Application No. 63 / 221,568, each of which is incorporated by reference in its entirety. Additional non-limiting examples of DUX4-targeting ASOs are provided in Table 1 below. In some embodiments, the DUX4-targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the DUX4-targeting ASO is ASDX2. In some embodiments, the DUX4-targeting ASO is ASDX4. In some embodiments, the DUX4-targeting ASO is ASDX23. In some embodiments, the DUX4-targeted ASO is ASDX26. In some embodiments, the DUX4-targeted ASO is ASDX32.

[0107] [Table 2]

[0108] [Table 3]

[0109] [Table 4]

[0110] In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting dystrophin, DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide comprises an siRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DUX4. In some embodiments, the polynucleotide comprises an ASO targeting DUX4. In some embodiments, the polynucleotide comprises a guide RNA targeting DUX4. In some embodiments, the polynucleotide comprises an siRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DMPK. In some embodiments, the polynucleotide comprises an ASO targeting DMPK. In some embodiments, the polynucleotide comprises an siRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA mimic targeting CAPN3. In some embodiments, the polynucleotide comprises an ASO that targets CAPN3.

[0111] In some embodiments, the polynucleotide is a coding RNA. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is a non-coding RNA. In some embodiments, the polynucleotide is a long non-coding RNA. In some embodiments, the polynucleotide is a guide RNA.

[0112] In some embodiments, a polynucleotide comprises one or more artificial nucleotide analogues. In some examples, the artificial nucleotide analogue comprises a modification at one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or a combination thereof. In some embodiments, one or more of the artificial nucleotide analogues is resistant to nucleases, e.g., ribonucleases such as RNases, deoxyribonucleases such as DNases, or exonucleases, e.g., 5'-3' exonucleases and 3'-5' exonucleases, when compared to natural polynucleotides. In some embodiments, the 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified amino acids are used. Artificial nucleotide analogs, including those modified with LNA, ENA, PNA, HNA, BNA, morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases, e.g., ribonucleases, e.g., RNases, deoxyribonucleases, e.g., DNases, or exonucleases, e.g., 5'-3' exonucleases and 3'-5' exonucleases. In some embodiments, the 2'-O-methyl modified polynucleotides are nuclease-resistant (e.g., RNase, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some embodiments, 2'O-methoxyethyl (2'-O-MOE) modified polynucleotides are nuclease resistant (eg, RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).In some embodiments, 2'-O-aminopropyl modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-deoxy modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, T-deoxy-2'-fluoro modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, LNA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).In some embodiments, ENA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, HNA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). Morpholinos can be nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, PNA modified polynucleotides are resistant to nucleases (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, methylphosphonate nucleotide modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, thiol phosphonate nucleotide modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, polynucleotides comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, the 5' complexes described herein inhibit 5'-3' exonucleolytic cleavage. In some embodiments, the 3' complexes described herein inhibit 3'-5' exonucleolytic cleavage.

[0113] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity to their mRNA targets compared to the equivalent natural polynucleotide. In some embodiments, the artificial nucleotide analogs include nucleic acids having modifications at the 2' hydroxyl group of the ribose moiety. In some embodiments, the modifications include H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some embodiments, the alkyl moiety further comprises a modification. In some embodiments, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some embodiments, the alkyl moiety further comprises a heterosubstitution. In some embodiments, a carbon of a heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In some embodiments, heterocyclic substitution includes, but is not limited to, morpholino, imidazole, and pyrrolidino. One or more of the artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites, may have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleotides.In some embodiments, 2'-O-methyl modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-aminopropyl modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-deoxy modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, T-deoxy-2'-fluoro modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, LNA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides.In some embodiments, ENA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, PNA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, HNA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, morpholino modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, methyl phosphonate nucleotide modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, thiol phosphonate nucleotide modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, polynucleotides comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, the increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0114] In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0115] In some embodiments, the artificial nucleotide analogues include, but are not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2 propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azotimidazole). uridine, 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one or pyridin-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracil and thymine, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moiety, in some embodiments, is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what are known in the art as universal bases.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0116] In some embodiments, the polynucleotide comprises one or more phosphorothioate internucleotide linkages. In some embodiments, the polynucleotide comprises a 2'-5' internucleotide linkage. In some embodiments, the 2'-5' internucleotide linkage is at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In some embodiments, the 2'-5' internucleotide linkage is present at various other locations within one or both strands of the sequence. In some embodiments, the polynucleotide comprises a terminal cap molecule at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus.

[0117] In some embodiments, the targeting molecule and the polynucleotide are combined to provide a synergistic therapeutic or biological effect.

[0118] In some embodiments, the polynucleotide is directly conjugated to the targeting molecule. The polynucleotide may be conjugated to the targeting molecule via a linker. Linkers suitable for conjugating the polynucleotide to the targeting molecule are known in the art. See, for example, International Publication No. WO 2017 / 173408, which is incorporated herein by reference in its entirety. In some embodiments, the linker is a hydrophobic linker. The linker may be a peptide linker. In some embodiments, the linker is a chemical linker. The chemical linker may be a polymer linker. In some embodiments, the chemical linker is linear. In some embodiments, the chemical linker is cyclic.

[0119] In some embodiments, the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, pyrophosphate, or polysarcosine. In some embodiments, the polymer linker comprises PEG. In some embodiments, the polymer linker comprises a sugar. In some embodiments, the polymer linker comprises a fatty acid. In some embodiments, the polymer linker comprises a phosphate. In some embodiments, the polymer linker comprises pyrophosphate. In some embodiments, the polymer linker comprises polysarcosine. The linker may be a high molecular weight PEG linker. In some embodiments, the high molecular weight PEG linker comprises 1,000 to 5,000 PEG monomers (i.e., PEG1k to PEG5k). In some embodiments, the high molecular weight PEG linker is PEG1k. In some embodiments, the high molecular weight PEG linker is PEG1.5k. In some embodiments, the high molecular weight PEG linker is PEG2k. In some embodiments, the high molecular weight PEG linker is PEG3k. In some embodiments, the high molecular weight PEG linker is PEG4k. In some embodiments, the high molecular weight PEG linker is PEG5k.

[0120] In some embodiments, the linker is a low molecular weight PEG linker. In some embodiments, the low molecular weight PEG linker comprises 4 to 100 PEG monomers (i.e., PEG4 to PEG100). In some embodiments, the low molecular weight PEG linker is PEG12 to PEG48. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG24. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG6 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG4. In some embodiments, the low molecular weight PEG linker is PEG5. In some embodiments, the low molecular weight PEG linker is PEG6. In some embodiments, the low molecular weight PEG linker is PEG7. In some embodiments, the low molecular weight PEG linker is PEG8. In some embodiments, the low molecular weight PEG linker is PEG9. In some embodiments, the low molecular weight PEG linker is PEG10. In some embodiments, the low molecular weight PEG linker is PEG11. In some embodiments, the low molecular weight PEG linker is PEG12. In some embodiments, the low molecular weight PEG linker is PEG13. In some embodiments, the low molecular weight PEG linker is PEG14. In some embodiments, the low molecular weight PEG linker is PEG15. In some embodiments, the low molecular weight PEG linker is PEG16. In some embodiments, the low molecular weight PEG linker is PEG17. In some embodiments, the low molecular weight PEG linker is PEG18. In some embodiments, the low molecular weight PEG linker is PEG19. In some embodiments, the low molecular weight PEG linker is PEG20. In some embodiments, the low molecular weight PEG linker is PEG21. In some embodiments, the low molecular weight PEG linker is PEG22. In some embodiments, the low molecular weight PEG linker is PEG23. In some embodiments, the low molecular weight PEG linker is PEG24. In some embodiments, the low molecular weight PEG linker is PEG25.In some embodiments, the low molecular weight PEG linker is PEG26. In some embodiments, the low molecular weight PEG linker is PEG27. In some embodiments, the low molecular weight PEG linker is PEG28. In some embodiments, the low molecular weight PEG linker is PEG29. In some embodiments, the low molecular weight PEG linker is PEG30. In some embodiments, the low molecular weight PEG linker is PEG31. In some embodiments, the low molecular weight PEG linker is PEG32. In some embodiments, the low molecular weight PEG linker is PEG33. In some embodiments, the low molecular weight PEG linker is PEG34. In some embodiments, the low molecular weight PEG linker is PEG35. In some embodiments, the low molecular weight PEG linker is PEG36. In some embodiments, the low molecular weight PEG linker is PEG37. In some embodiments, the low molecular weight PEG linker is PEG38. In some embodiments, the low molecular weight PEG linker is PEG39. In some embodiments, the low molecular weight PEG linker is PEG40. In some embodiments, the low molecular weight PEG linker is PEG41. In some embodiments, the low molecular weight PEG linker is PEG42. In some embodiments, the low molecular weight PEG linker is PEG43. In some embodiments, the low molecular weight PEG linker is PEG44. In some embodiments, the low molecular weight PEG linker is PEG45. In some embodiments, the low molecular weight PEG linker is PEG46. In some embodiments, the low molecular weight PEG linker is PEG47. In some embodiments, the low molecular weight PEG linker is PEG48. In some embodiments, the low molecular weight PEG linker is PEG49. In some embodiments, the low molecular weight PEG linker is PEG50. In some embodiments, the low molecular weight PEG linker is PEG51. In some embodiments, the low molecular weight PEG linker is PEG52. In some embodiments, the low molecular weight PEG linker is PEG53. In some embodiments, the low molecular weight PEG linker is PEG54.In some embodiments, the low molecular weight PEG linker is PEG55. In some embodiments, the low molecular weight PEG linker is PEG56. In some embodiments, the low molecular weight PEG linker is PEG57. In some embodiments, the low molecular weight PEG linker is PEG58. In some embodiments, the low molecular weight PEG linker is PEG59. In some embodiments, the low molecular weight PEG linker is PEG60. In some embodiments, the low molecular weight PEG linker is PEG61. In some embodiments, the low molecular weight PEG linker is PEG62. In some embodiments, the low molecular weight PEG linker is PEG63. In some embodiments, the low molecular weight PEG linker is PEG64. In some embodiments, the low molecular weight PEG linker is PEG65. In some embodiments, the low molecular weight PEG linker is PEG66. In some embodiments, the low molecular weight PEG linker is PEG67. In some embodiments, the low molecular weight PEG linker is PEG68. In some embodiments, the low molecular weight PEG linker is PEG69. In some embodiments, the low molecular weight PEG linker is PEG70. In some embodiments, the low molecular weight PEG linker is PEG71. In some embodiments, the low molecular weight PEG linker is PEG72. In some embodiments, the low molecular weight PEG linker is PEG73. In some embodiments, the low molecular weight PEG linker is PEG74. In some embodiments, the low molecular weight PEG linker is PEG75. In some embodiments, the low molecular weight PEG linker is PEG76. In some embodiments, the low molecular weight PEG linker is PEG77. In some embodiments, the low molecular weight PEG linker is PEG78. In some embodiments, the low molecular weight PEG linker is PEG79. In some embodiments, the low molecular weight PEG linker is PEG80. In some embodiments, the low molecular weight PEG linker is PEG81. In some embodiments, the low molecular weight PEG linker is PEG82. In some embodiments, the low molecular weight PEG linker is PEG83.In some embodiments, the low molecular weight PEG linker is PEG84. In some embodiments, the low molecular weight PEG linker is PEG85. In some embodiments, the low molecular weight PEG linker is PEG86. In some embodiments, the low molecular weight PEG linker is PEG87. In some embodiments, the low molecular weight PEG linker is PEG88. In some embodiments, the low molecular weight PEG linker is PEG89. In some embodiments, the low molecular weight PEG linker is PEG90. In some embodiments, the low molecular weight PEG linker is PEG91. In some embodiments, the low molecular weight PEG linker is PEG92. In some embodiments, the low molecular weight PEG linker is PEG93. In some embodiments, the low molecular weight PEG linker is PEG94. In some embodiments, the low molecular weight PEG linker is PEG95. In some embodiments, the low molecular weight PEG linker is PEG96. In some embodiments, the low molecular weight PEG linker is PEG97. In some embodiments, the low molecular weight PEG linker is PEG98. In some embodiments, the low molecular weight PEG linker is PEG99. In some embodiments, the low molecular weight PEG linker is PEG100.

[0121] In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. The linker may be cleavable in vivo. In some embodiments, the cleavable linker is selected from the group consisting of a disulfide linker, a self-immolative peptide polymer hybrid, and a sulfatase-promoted aryl sulfate linker. In some embodiments, the cleavable linker is a disulfide linker. The cleavable linker may be a self-immolative peptide polymer hybrid. In some embodiments, the cleavable linker is a sulfatase-promoted aryl sulfate linker. In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or a Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX). In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid. In some embodiments, the self-immolative peptide polymer hybrid comprises para-amino-benzoyloxy (PAB). In some embodiments, the self-immolative peptide polymer hybrid comprises 7-amino-3-hydroxyethyl-coumarin (7-AHC). In some embodiments, the self-immolative peptide polymer hybrid comprises an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX).

[0122] In some embodiments, the cleavable linker is cleaved through reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, or bioorthogonal cleavage. In some embodiments, the cleavable linker is cleaved through reduction. In some embodiments, the cleavable linker is cleaved through hydrolysis. In some embodiments, the cleavable linker is cleaved through proteolysis. In some embodiments, the cleavable linker is cleaved through photocleavage. In some embodiments, the cleavable linker is cleaved through chemical cleavage. Chemical cleavage can be by Fe II mediated β-elimination of TRX. In some embodiments, the cell cleavable linker is cleaved through enzymatic cleavage. Enzymatic cleavage can be by non-proteolytic sulfatase, β-galactosidase / glucuronidase, or pyrophosphatase. In some embodiments, the enzymatic cleavage is by non-proteolytic sulfatase. In some embodiments, the enzymatic cleavage is by β-galactosidase / glucuronidase. In some embodiments, the enzymatic cleavage is by pyrophosphatase. In some embodiments, the cleavable linker is cleaved through bioorthogonal cleavage. The bioorthogonal cleavage can be by CuI-BTTAA or free copper ion mediated cleavage. In some embodiments, the linker is an acid cleavable linker.

[0123] In some embodiments, the linker comprises a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group). In some embodiments, the linker comprises a homobifunctional cross-linker, a heterobifunctional cross-linker, etc. In some embodiments, the linker is a traceless linker (or a zero-length linker). In some embodiments, the linker is a non-polymeric linker. In some embodiments, the linker is a non-peptide linker or a linker that does not contain an amino acid residue.

[0124] In some embodiments, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include the Romant reagents dithiobis(succinimidyl propionate) DSP, 3,3′-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3′-dithiobispropionimidate (DTBP), 1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDP), and the like. B), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[2-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-ρ-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0125] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include amine-reactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6 -[a-Methyl-a-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), Succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-maleimidobutyryloxy)succinimide esters (GMBs), N-(gamma-maleimidobutyryloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB ... succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-l-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), etc.Carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), etc., amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA). , sulfosuccinimidyl-(4-azido-salicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azido-salicylamido)ethyl-l,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6 -(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-l,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl Sulfhydryl-reactive and photoreactive crosslinkers, such as sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-l,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), etc.Examples of crosslinkers include, but are not limited to, l-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive crosslinkers, such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as p-azidophenylglyoxal (APG).

[0126] In some embodiments, the linker comprises a reactive functional group. In some embodiments, the reactive functional group comprises a nucleophilic group that reacts with an electrophilic group present on the binding moiety. Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0127] In some embodiments, the linker comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further includes caproic acid to form maleimidocaproyl (mc). In some embodiments, the linker comprises maleimidocaproyl (mc). In some embodiments, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) discussed above.

[0128] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide and provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby precluding the maleimide from undergoing elimination via a retro-Michael reaction. In some embodiments, the self-stabilizing maleimide is a maleimide group described in Lyon, et al, "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol 32(10):1059-1062 (2014). In some embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.

[0129] In some embodiments, the linker comprises a peptide moiety. In some embodiments, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some embodiments, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically), in some embodiments, the peptide moiety is a non-cleavable peptide moiety. In some embodiments, the peptide moiety comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Τφ-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the linker comprises Val-Cit. In some embodiments, the linker is Val-Cit.

[0130] In some embodiments, the linker comprises a benzoic acid group, or a derivative thereof. In some embodiments, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some embodiments, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0131] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the maleimide group is maleimidocaproyl (mc). In some embodiments, the peptide group is val-cit. In some embodiments, the benzoic acid group is PABA. In some embodiments, the linker comprises a mc-val-cit group. In some embodiments, the linker comprises a val-cit-PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.

[0132] In some embodiments, the linker is a self-immolative or self-eliminating linker. In some embodiments, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some embodiments, the linker includes a linker described in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426, each of which is incorporated by reference in its entirety.

[0133] In some embodiments, the linker is a dendritic linker. In some embodiments, the dendritic linker comprises a branched, multifunctional linker moiety. In some embodiments, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some embodiments, the dendritic linker comprises a PAMAM dendrimer.

[0134] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or a linker group) on the polynucleotide or targeting molecule after cleavage.Exemplary traceless linkers include, but are not limited to, germanium linker, silicon linker, sulfur linker, selenium linker, nitrogen linker, phosphorus linker, boron linker, chromium linker, or phenylhydrazine linker.In some embodiments, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497(2013). In some embodiments, the linker is a traceless linker as described in Blaney, et al., 'Traceless solid-phase organic synthesis,' Chem. Rev. 102:2607-2024 (2002). In some embodiments, the linker is a traceless linker as described in U.S. Pat. No. 6,821,783, the entirety of which is incorporated herein by reference.

[0135] In some embodiments, the linker comprises a functional group that exerts steric hindrance at the site of attachment between the linker and the complexing moiety (e.g., a polynucleotide or targeting molecule disclosed herein). In some embodiments, the steric hindrance is around a disulfide bond. Exemplary linkers that exhibit steric hindrance include heterobifunctional linkers, such as the heterobifunctional linkers described above. In some embodiments, linkers that exhibit steric hindrance include SMCC and SPDB.

[0136] In some embodiments, the linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker comprises a hydrazone bond that is susceptible to hydrolytic cleavage. In some embodiments, the acid-cleavable linker comprises a thiomaleamic acid linker. In some embodiments, the acid-cleavable linker is a thiomaleamic acid linker as described in Castaneda, et al, "Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation," Chem. Commun. 49:8187-8189 (2013).

[0137] In some embodiments, the linker is a cyclic amine or cyclic amine as described in U.S. Pat. Nos. 6,884,869, 7,498,298, 8,288,352, 8,609, 105, or 8,697,688; U.S. Patent Application Publication Nos. 2014 / 0127239, 2013 / 028919, 2014 / 2869 70, 2013 / 0309256, 2015 / 037360, or 2014 / 0294851; or a linker described in PCT Publication Nos. WO 2015057699, WO 2014080251, WO 2014197854, WO 2014145090, or WO 2014177042.

[0138] In some embodiments, the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule. In some embodiments, the linker is conjugated to a lysine residue in the targeting molecule. In some embodiments, the linker is conjugated to a cysteine ​​residue in the targeting molecule. In some embodiments, the linker is conjugated to a histidine residue in the targeting molecule. In some embodiments, the linker is conjugated to a non-natural amino acid residue in the targeting molecule.

[0139] In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation or enzymatic conjugation. In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation. Chemical conjugation may include acylation and click chemistry. In some embodiments, the linker is conjugated to the targeting molecule by enzymatic conjugation. Enzymatic conjugation may be via a sortase or transferase enzyme.

[0140] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a chemical ligation process. In some embodiments, the polynucleotide is conjugated to the targeting molecule by native ligation. In some embodiments, conjugation is as described in Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some embodiments, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleotide is conjugated to the targeting molecule via native ligation chemistry, either site-specifically or non-specifically.

[0141] In some embodiments, polynucleotides are conjugated to targeting molecules by a site-directed method utilizing "traceless" coupling technology (Philochem). In some embodiments, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group on the targeting molecule, which is then conjugated to a polynucleotide containing an aldehyde group. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13):5887-5892 (2012)).

[0142] In some embodiments, polynucleotides are conjugated to targeting molecules by site-directed methods that utilize unnatural amino acids incorporated into the targeting molecule. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe selectively binds to an alkoxy-amine derivatized conjugation moiety to form an oxime bond. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40):16101-16106 (2012)).

[0143] In some embodiments, polynucleotides are conjugated to targeting molecules by a site-directed method that utilizes an enzyme-catalyzed process. In some embodiments, the site-directed method utilizes SMARTag™ technology (RedWood). In some embodiments, SMARTag™ technology involves generating formylglycine (FGly) residues from cysteines by formylglycine generating enzyme (FGE) through an oxidation process in the presence of an aldehyde tag, followed by conjugation of FGly to an alkylhydrazine-functionalized polynucleotide via hydrazino-Pictet-Spengler (HIPS) ligation. (See Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9):3000-3005(2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51(2013)).

[0144] In some embodiments, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some embodiments, polynucleotides are conjugated to targeting molecules utilizing a microbial transglutaminase-catalyzed process. In some embodiments, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in a recognition sequence and a primary amine of a functionalized polynucleotide. In some embodiments, mTG is produced from Streptomyces mobarensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2)161-167 (2013))

[0145] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a method such as that described in PCT Publication No. WO 2014 / 140317 (incorporated herein by reference in its entirety), which utilizes a sequence-specific transpeptidase. In some embodiments, the polynucleotide is conjugated to the targeting molecule by a method such as that described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540, each of which is incorporated herein by reference in its entirety.

[0146] In some embodiments, each targeting molecule is conjugated to between 1 and 8 polynucleotide molecules (i.e., a drug:antibody ratio (DAR) of 1 to 8). In some embodiments, each targeting molecule is conjugated to 1 polynucleotide molecule (DAR of 1). In some embodiments, each targeting molecule is conjugated to 2 polynucleotide molecules (DAR of 2). In some embodiments, each targeting molecule is conjugated to 3 polynucleotide molecules (DAR of 3). In some embodiments, each targeting molecule is conjugated to 4 polynucleotide molecules (DAR of 4). In some embodiments, each targeting molecule is conjugated to 5 polynucleotide molecules (DAR of 5). In some embodiments, each targeting molecule is conjugated to 6 polynucleotide molecules (DAR of 6). In some embodiments, each targeting molecule is conjugated to 7 polynucleotide molecules (DAR of 7). In some embodiments, each targeting molecule is conjugated to 8 polynucleotide molecules (DAR of 8).

[0147] In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 30 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 40 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 50 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 60 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of about 7,500 kDa or less.

[0148] In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 30 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 40 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 50 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 60 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of 7,500 kDa or less.

[0149] Polynucleotide Complex Another aspect of the present disclosure provides a polynucleotide complex. In some embodiments, the polynucleotide complex comprises a polynucleotide complexed to a targeting molecule. In such an aspect, the targeting molecule may also be referred to as a targeting agent component, whereas the polynucleotide may be referred to as an example of an active agent component. That is, a (therapeutic) targeting agent comprising an active agent component and a targeting agent component is disclosed herein.

[0150] In some embodiments, the targeting moiety (also referred to in aspects as a targeting agent component) includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all these classes of substances. Additional examples of targeting moieties also include steroids such as cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, carbohydrates (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some embodiments, the targeting moiety is an antibody or a binding fragment thereof.

[0151] The targeting molecule (also referred to in aspects as a targeting agent moiety) is an antibody or an antigen-binding fragment thereof, or a binding protein. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof (e.g., a polynucleotide-antibody conjugate). In some embodiments, the antibody or binding fragment thereof is a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, an immunoglobulin single variable domain (ISV) such as a NANOBODY® molecule, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a vNAR, a mutein based on tenascin-C (also known as a centirin molecule), a camelid antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, bispecific antibodies, immunoglobulin single variable domains (ISVs) such as Fab, Fab-Fc, Fv, single chain Fv (scFv), diabodies, minibodies, and NANOBODY® molecules. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. Non-limiting examples of bispecific antibodies include bispecific T cell engagers (BiTEs) and dual affinity retargeting antibodies (DARTs). In some embodiments, the bispecific antibody is a trifunctional antibody or a bispecific miniantibody. In some embodiments, the bispecific antibody is a trifunctional antibody. In some embodiments, the trifunctional antibody is a full-length monoclonal antibody that contains binding sites for two different antigens. In some embodiments, the bispecific antibody is a bispecific miniantibody.In some embodiments, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody or bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein containing two single chain variable fragments (scFv), where the two scFvs target epitopes of two different antigens.

[0152] In some embodiments, the antibody or antigen-binding fragment thereof is a Fab. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab-Fc. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv. In some embodiments, the antibody or antigen-binding fragment thereof is a single chain Fv (scFv). In some embodiments, when the antibody or antigen-binding portion thereof is an scFv, the polynucleotide does not comprise bridging residues. In some embodiments, when the antibody or antigen-binding portion thereof is an scFv, the polynucleotide does not comprise cysteines. In some embodiments, the antibody or antigen-binding fragment thereof is a diabody. In some embodiments, the antibody or antigen-binding fragment thereof is a minibody. In some embodiments, the antibody or antigen-binding fragment thereof is an immunoglobulin single variable domain (ISV), such as a NANOBODY® molecule. The NANOBODY® may be a NANOBODY® molecule-HSA.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof is or is derived from an IgG molecule. The IgG molecule may be an IgG1 or IgG4 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG1 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG2 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG3 molecule. The antibody or antigen-binding fragment thereof may be or be derived from an IgG4 molecule.

[0154] In some embodiments, the targeting molecule is a binding protein. The binding protein may be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor comprises the extracellular domain of the receptor. In some embodiments, the soluble receptor is an Fc fusion protein.

[0155] In some embodiments, the targeting molecule is a plasma protein. In some embodiments, the plasma protein comprises albumin. In some embodiments, the albumin is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some examples, the albumin is conjugated to the polynucleotide by native ligation chemistry. In some examples, the albumin is lysine conjugated to the polynucleotide.

[0156] In some examples, the targeting molecule is a steroid. Non-limiting exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, saturated, unsaturated, substituted, or other hydrocarbons, or combinations thereof. In some embodiments, the steroid is cholesterol or a cholesterol derivative. In some embodiments, the targeting molecule is cholesterol. In some embodiments, the steroid is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some embodiments, the steroid is conjugated to the polynucleotide by native ligation chemistry.

[0157] In some embodiments, the targeting molecule is a polymer, including but not limited to a polynucleotide aptamer, that binds to a specific surface marker on a cell. In some embodiments, the targeting molecule is a polynucleotide that does not hybridize to a target gene or mRNA, but instead can selectively bind to a cell surface marker, similar to an antibody that binds to its specific epitope on the cell surface marker.

[0158] In some embodiments, the targeting molecule is a polypeptide. In some embodiments, the polypeptide has a size of about 1 to about 3 kDa. In some embodiments, the polypeptide has a size of about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some embodiments, the polypeptide is a bicyclic polypeptide. In some embodiments, the bicyclic polypeptide is a constrained bicyclic polypeptide. In some embodiments, the targeting molecule is a bicyclic polypeptide (e.g., bicyclic compounds from Bicycle Therapeutics).

[0159] In additional embodiments, the targeting molecule is a small molecule. In some embodiments, the small molecule is an antibody-recruiting small molecule. In some embodiments, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, wherein the target-binding end is capable of recognizing and interacting with a cell surface receptor.

[0160] In some embodiments, the targeting molecule is a therapeutically active molecule or a biologically active molecule.

[0161] In some embodiments, the active agent component is a polynucleotide.

[0162] In some embodiments, the polynucleotide comprises RNA, DNA, or a combination thereof. In some embodiments, the polynucleotide comprises RNA. In some embodiments, the polynucleotide comprises DNA. In some embodiments, the polynucleotide comprises RNA and DNA. In some embodiments, the polynucleotide comprises a combination of DNA, RNA, and / or artificial nucleotide analogs. In some embodiments, the polynucleotide is a regulatory non-coding RNA (ncRNA). In some embodiments, the ncRNA comprises short non-coding RNA sequences expressed in the genome that regulate the expression or function of other biomolecules in mammalian cells. ncRNAs are generally <200 nucleotides in length, can be single-stranded or double-stranded, and can form non-linear secondary or tertiary structures. The ncRNA may include exogenously derived small interfering RNA (siRNA), microRNA (miRNA), nucleolar RNA (U-RNA), small nuclear RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), transfer RNA-derived small RNA (tsRNA), ribosomal RNA-derived small RNA (rsRNA), long non-coding RNA-derived small RNA (lncsRNA), or messenger RNA-derived small RNA (msRNA). In some embodiments, the polynucleotide is an engineered polynucleotide. The engineered polynucleotide may include DNA or RNA. In some embodiments, the engineered polynucleotide includes a plurality of nucleotides. In some embodiments, the engineered polynucleotide includes an artificial nucleotide analog. In some embodiments, the engineered polynucleotide includes DNA. In some embodiments, the DNA is genomic DNA, cell-free DNA, cDNA, fetal DNA, viral DNA, or maternal DNA. In some embodiments, the engineered polynucleotide includes RNA.In some embodiments, the RNA is an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a miximer, a double-stranded RNA (dsRNA), a single-stranded RNAi (ssRNAi), a DNA-dependent RNA interference (ddRNAi), an RNA-activating oligonucleotide (RNAa), an aptamer, or an exon-skipping oligonucleotide. In some embodiments, the engineered polynucleotide comprises a fully synthetic miRNA. A fully synthetic miRNA is one that is not guided or based on an ncRNA. Alternatively, a fully synthetic miRNA may be based on an analysis of multiple potential target sequences or may be based on an isolated naturally occurring non-coding sequence that is not an ncRNA. In some embodiments, the polynucleotide is selected from the group consisting of an siRNA, an miRNA, an miRNA mimic, an antisense oligonucleotide (ASO), an mRNA, and a guide RNA. The polynucleotide may be an siRNA. In some embodiments, the polynucleotide is an miRNA. In some embodiments, the polynucleotide is an miRNA mimic.

[0163] In some embodiments, the active agent component is an ASO. In some embodiments, the ASO can target and inhibit multiple genes associated with the disorder. In some embodiments, the ASO targets an autosomal dominant mutant gene that causes a genetic disorder. In some embodiments, the ASO targets DMPK. In some embodiments, the ASO targets CAPN3. The ASO can target DUX4. DUX4-targeted ASOs are known in the art. See WO 2021 / 203043 and U.S. Provisional Patent Application No. 63 / 221,568, each of which is incorporated herein by reference in its entirety. Additional non-limiting examples of DUX4-targeted ASOs are provided in Table 1 above. In some embodiments, the DUX4-targeted ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the DUX4-targeted ASO is ASDX2. In some embodiments, the DUX4 targeting ASO is ASDX4. In some embodiments, the DUX4 targeting ASO is ASDX23. In some embodiments, the DUX4 targeting ASO is ASDX26. In some embodiments, the DUX4 targeting ASO is ASDX32.

[0164] In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide comprises an siRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DUX4. In some embodiments, the polynucleotide comprises an ASO targeting DUX4. In some embodiments, the polynucleotide comprises a guide RNA targeting DUX4. In some embodiments, the polynucleotide comprises an siRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DMPK. In some embodiments, the polynucleotide comprises an ASO targeting DMPK. In some embodiments, the polynucleotide comprises an siRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA mimic targeting CAPN3. In some embodiments, the polynucleotide comprises an ASO that targets CAPN3.

[0165] In some embodiments, the polynucleotide is a coding RNA. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is a non-coding RNA. In some embodiments, the polynucleotide is a long non-coding RNA. In some embodiments, the polynucleotide is a guide RNA.

[0166] In some embodiments, a polynucleotide comprises one or more artificial nucleotide analogs. In some embodiments, one or more of the artificial nucleotide analogs described herein are resistant to, for example, ribonucleases, such as RNases, deoxyribonucleases, such as DNases, or exonucleases, such as 5'-3' exonucleases and 3'-5' exonucleases, when compared to naturally occurring polynucleotides. In some embodiments, the artificial nucleotide analogs are resistant to, for example, ribonucleases, such as RNases, deoxyribonucleases, such as DNases, or exonucleases, such as 5'-3' exonucleases and 3'-5' exonucleases. In some embodiments, the artificial nucleotide analogs are resistant to, for example, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'- Artificial nucleotide analogs including O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are resistant to ribonucleases such as RNases, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some embodiments, the 2'-O-methyl modified polynucleotides are nuclease-resistant (e.g., RNase, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some embodiments, 2'O-methoxyethyl (2'-O-MOE) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-aminopropyl modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).In some embodiments, 2'-deoxy modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, T-deoxy-2'-fluoro modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, LNA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, ENA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant).In some embodiments, HNA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). Morpholinos can be nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, PNA modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, methylphosphonate nucleotide modified polynucleotides are nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, the thiol phosphonate nucleotide modified polynucleotide is nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, the polynucleotide comprising a 2'-fluoro N3-P5'-phosphoramidite is nuclease resistant (e.g., RNase, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some embodiments, the 5' complexes described herein inhibit 5'-3' exonucleolytic cleavage. In some embodiments, the 3' complexes described herein inhibit 3'-5' exonucleolytic cleavage.

[0167] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity to their mRNA target compared to the equivalent natural polynucleotide. In some embodiments, the artificial nucleotide analog comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some embodiments, the modification comprises H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some embodiments, the alkyl moiety further comprises a modification. In some embodiments, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some embodiments, the alkyl moiety further comprises a heterosubstitution. In some embodiments, a carbon of a heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In some embodiments, heterocyclic substitution includes, but is not limited to, morpholino, imidazole, and pyrrolidino. One or more of the artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites, may have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleotides.In some embodiments, 2'-O-methyl modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-aminopropyl modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-deoxy modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, T-deoxy-2'-fluoro modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, LNA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides.In some embodiments, ENA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, PNA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, HNA modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, morpholino modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, methyl phosphonate nucleotide modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, thiol phosphonate nucleotide modified polynucleotides have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, polynucleotides comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity to their mRNA targets compared to equivalent native polynucleotides. In some embodiments, the increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0168] In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0169] In some embodiments, the artificial nucleotide analogues include, but are not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2 propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azotimidazole). uridine, 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one or pyridin-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracil and thymine, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moiety, in some embodiments, is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what are known in the art as universal bases.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0170] In some embodiments, the polynucleotide comprises one or more phosphorothioate internucleotide linkages. In some embodiments, the polynucleotide comprises a 2'-5' internucleotide linkage. In some embodiments, the 2'-5' internucleotide linkage is at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In some embodiments, the 2'-5' internucleotide linkage is present at various other locations within one or both strands of the sequence. In some embodiments, the polynucleotide comprises a terminal cap molecule at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus.

[0171] In some embodiments, the targeting molecule and the polynucleotide are combined to provide a synergistic therapeutic or biological effect.

[0172] In some embodiments, the polynucleotide is directly conjugated to the targeting molecule (such as a targeting agent moiety described herein). The polynucleotide may be conjugated to the targeting molecule via a linker. Linkers suitable for conjugating the polynucleotide to the targeting molecule are known in the art. See, for example, International Publication No. WO 2017 / 173408, which is incorporated herein by reference in its entirety. In some embodiments, the linker is a hydrophobic linker. The linker may be a peptide linker. In some embodiments, the linker is a chemical linker. The chemical linker may be a polymer linker. In some embodiments, the chemical linker is linear. In some embodiments, the chemical linker is cyclic.

[0173] In some embodiments, the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, pyrophosphate, or polysarcosine. In some embodiments, the polymer linker comprises PEG. In some embodiments, the polymer linker comprises a sugar. In some embodiments, the polymer linker comprises a fatty acid. In some embodiments, the polymer linker comprises a phosphate. In some embodiments, the polymer linker comprises pyrophosphate. In some embodiments, the polymer linker comprises polysarcosine. The linker may be a high molecular weight PEG linker. In some embodiments, the high molecular weight PEG linker comprises 1,000 to 5,000 PEG monomers (i.e., PEG1k to PEG5k). In some embodiments, the high molecular weight PEG linker is PEG1k. In some embodiments, the high molecular weight PEG linker is PEG1.5k. In some embodiments, the high molecular weight PEG linker is PEG2k. In some embodiments, the high molecular weight PEG linker is PEG3k. In some embodiments, the high molecular weight PEG linker is PEG4k. In some embodiments, the high molecular weight PEG linker is PEG5k.

[0174] In some embodiments, the linker is a low molecular weight PEG linker. In some embodiments, the low molecular weight PEG linker comprises 4 to 100 PEG monomers (i.e., PEG4 to PEG100). In some embodiments, the low molecular weight PEG linker is PEG12 to PEG48. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG24. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG6 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG4. In some embodiments, the low molecular weight PEG linker is PEG5. In some embodiments, the low molecular weight PEG linker is PEG6. In some embodiments, the low molecular weight PEG linker is PEG7. In some embodiments, the low molecular weight PEG linker is PEG8. In some embodiments, the low molecular weight PEG linker is PEG9. In some embodiments, the low molecular weight PEG linker is PEG10. In some embodiments, the low molecular weight PEG linker is PEG11. In some embodiments, the low molecular weight PEG linker is PEG12. In some embodiments, the low molecular weight PEG linker is PEG13. In some embodiments, the low molecular weight PEG linker is PEG14. In some embodiments, the low molecular weight PEG linker is PEG15. In some embodiments, the low molecular weight PEG linker is PEG16. In some embodiments, the low molecular weight PEG linker is PEG17. In some embodiments, the low molecular weight PEG linker is PEG18. In some embodiments, the low molecular weight PEG linker is PEG19. In some embodiments, the low molecular weight PEG linker is PEG20. In some embodiments, the low molecular weight PEG linker is PEG21. In some embodiments, the low molecular weight PEG linker is PEG22. In some embodiments, the low molecular weight PEG linker is PEG23. In some embodiments, the low molecular weight PEG linker is PEG24. In some embodiments, the low molecular weight PEG linker is PEG25.In some embodiments, the low molecular weight PEG linker is PEG26. In some embodiments, the low molecular weight PEG linker is PEG27. In some embodiments, the low molecular weight PEG linker is PEG28. In some embodiments, the low molecular weight PEG linker is PEG29. In some embodiments, the low molecular weight PEG linker is PEG30. In some embodiments, the low molecular weight PEG linker is PEG31. In some embodiments, the low molecular weight PEG linker is PEG32. In some embodiments, the low molecular weight PEG linker is PEG33. In some embodiments, the low molecular weight PEG linker is PEG34. In some embodiments, the low molecular weight PEG linker is PEG35. In some embodiments, the low molecular weight PEG linker is PEG36. In some embodiments, the low molecular weight PEG linker is PEG37. In some embodiments, the low molecular weight PEG linker is PEG38. In some embodiments, the low molecular weight PEG linker is PEG39. In some embodiments, the low molecular weight PEG linker is PEG40. In some embodiments, the low molecular weight PEG linker is PEG41. In some embodiments, the low molecular weight PEG linker is PEG42. In some embodiments, the low molecular weight PEG linker is PEG43. In some embodiments, the low molecular weight PEG linker is PEG44. In some embodiments, the low molecular weight PEG linker is PEG45. In some embodiments, the low molecular weight PEG linker is PEG46. In some embodiments, the low molecular weight PEG linker is PEG47. In some embodiments, the low molecular weight PEG linker is PEG48. In some embodiments, the low molecular weight PEG linker is PEG49. In some embodiments, the low molecular weight PEG linker is PEG50. In some embodiments, the low molecular weight PEG linker is PEG51. In some embodiments, the low molecular weight PEG linker is PEG52. In some embodiments, the low molecular weight PEG linker is PEG53. In some embodiments, the low molecular weight PEG linker is PEG54.In some embodiments, the low molecular weight PEG linker is PEG55. In some embodiments, the low molecular weight PEG linker is PEG56. In some embodiments, the low molecular weight PEG linker is PEG57. In some embodiments, the low molecular weight PEG linker is PEG58. In some embodiments, the low molecular weight PEG linker is PEG59. In some embodiments, the low molecular weight PEG linker is PEG60. In some embodiments, the low molecular weight PEG linker is PEG61. In some embodiments, the low molecular weight PEG linker is PEG62. In some embodiments, the low molecular weight PEG linker is PEG63. In some embodiments, the low molecular weight PEG linker is PEG64. In some embodiments, the low molecular weight PEG linker is PEG65. In some embodiments, the low molecular weight PEG linker is PEG66. In some embodiments, the low molecular weight PEG linker is PEG67. In some embodiments, the low molecular weight PEG linker is PEG68. In some embodiments, the low molecular weight PEG linker is PEG69. In some embodiments, the low molecular weight PEG linker is PEG70. In some embodiments, the low molecular weight PEG linker is PEG71. In some embodiments, the low molecular weight PEG linker is PEG72. In some embodiments, the low molecular weight PEG linker is PEG73. In some embodiments, the low molecular weight PEG linker is PEG74. In some embodiments, the low molecular weight PEG linker is PEG75. In some embodiments, the low molecular weight PEG linker is PEG76. In some embodiments, the low molecular weight PEG linker is PEG77. In some embodiments, the low molecular weight PEG linker is PEG78. In some embodiments, the low molecular weight PEG linker is PEG79. In some embodiments, the low molecular weight PEG linker is PEG80. In some embodiments, the low molecular weight PEG linker is PEG81. In some embodiments, the low molecular weight PEG linker is PEG82. In some embodiments, the low molecular weight PEG linker is PEG83.In some embodiments, the low molecular weight PEG linker is PEG84. In some embodiments, the low molecular weight PEG linker is PEG85. In some embodiments, the low molecular weight PEG linker is PEG86. In some embodiments, the low molecular weight PEG linker is PEG87. In some embodiments, the low molecular weight PEG linker is PEG88. In some embodiments, the low molecular weight PEG linker is PEG89. In some embodiments, the low molecular weight PEG linker is PEG90. In some embodiments, the low molecular weight PEG linker is PEG91. In some embodiments, the low molecular weight PEG linker is PEG92. In some embodiments, the low molecular weight PEG linker is PEG93. In some embodiments, the low molecular weight PEG linker is PEG94. In some embodiments, the low molecular weight PEG linker is PEG95. In some embodiments, the low molecular weight PEG linker is PEG96. In some embodiments, the low molecular weight PEG linker is PEG97. In some embodiments, the low molecular weight PEG linker is PEG98. In some embodiments, the low molecular weight PEG linker is PEG99. In some embodiments, the low molecular weight PEG linker is PEG100.

[0175] In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. The linker may be cleavable in vivo. In some embodiments, the cleavable linker is selected from the group consisting of a disulfide linker, a self-immolative peptide polymer hybrid, and a sulfatase-promoted aryl sulfate linker. In some embodiments, the cleavable linker is a disulfide linker. The cleavable linker may be a self-immolative peptide polymer hybrid. In some embodiments, the cleavable linker is a sulfatase-promoted aryl sulfate linker. In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or a Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX). In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid. In some embodiments, the self-immolative peptide polymer hybrid comprises para-amino-benzoyloxy (PAB). In some embodiments, the self-immolative peptide polymer hybrid comprises 7-amino-3-hydroxyethyl-coumarin (7-AHC). In some embodiments, the self-immolative peptide polymer hybrid comprises an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX).

[0176] In some embodiments, the cleavable linker is cleaved through reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, or bioorthogonal cleavage. In some embodiments, the cleavable linker is cleaved through reduction. In some embodiments, the cleavable linker is cleaved through hydrolysis. In some embodiments, the cleavable linker is cleaved through proteolysis. In some embodiments, the cleavable linker is cleaved through photocleavage. In some embodiments, the cleavable linker is cleaved through chemical cleavage. Chemical cleavage can be by Fe II mediated β-elimination of TRX. In some embodiments, the cell cleavable linker is cleaved through enzymatic cleavage. Enzymatic cleavage can be by non-proteolytic sulfatase, β-galactosidase / glucuronidase, or pyrophosphatase. In some embodiments, the enzymatic cleavage is by non-proteolytic sulfatase. In some embodiments, the enzymatic cleavage is by β-galactosidase / glucuronidase. In some embodiments, the enzymatic cleavage is by pyrophosphatase. In some embodiments, the cleavable linker is cleaved through bioorthogonal cleavage. The bioorthogonal cleavage can be by CuI-BTTAA or free copper ion mediated cleavage. In some embodiments, the linker is an acid cleavable linker.

[0177] In some embodiments, the linker comprises a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group). In some embodiments, the linker comprises a homobifunctional cross-linker, a heterobifunctional cross-linker, etc. In some embodiments, the linker is a traceless linker (or a zero-length linker). In some embodiments, the linker is a non-polymeric linker. In some embodiments, the linker is a non-peptide linker or a linker that does not contain an amino acid residue.

[0178] In some embodiments, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include the Romant reagents dithiobis(succinimidyl propionate) DSP, 3,3′-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3′-dithiobispropionimidate (DTBP), 1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDP), and the like. B), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[2-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-ρ-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0179] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include amine-reactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6 -[a-Methyl-a-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), Succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-maleimidobutyryloxy)succinimide esters (GMBs), N-(gamma-maleimidobutyryloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB ... succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-l-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), etc.Carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), etc., amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA). , sulfosuccinimidyl-(4-azido-salicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azido-salicylamido)ethyl-l,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6 -(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-l,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl Sulfhydryl-reactive and photoreactive crosslinkers, such as sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-l,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), etc.Examples of crosslinkers include, but are not limited to, l-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive crosslinkers, such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as p-azidophenylglyoxal (APG).

[0180] In some embodiments, the linker comprises a reactive functional group. In some embodiments, the reactive functional group comprises a nucleophilic group that reacts with an electrophilic group present on the binding moiety. Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0181] In some embodiments, the linker comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further includes caproic acid to form maleimidocaproyl (mc). In some embodiments, the linker comprises maleimidocaproyl (mc). In some embodiments, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) discussed above.

[0182] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide and provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby precluding the maleimide from undergoing elimination via a retro-Michael reaction. In some embodiments, the self-stabilizing maleimide is a maleimide group described in Lyon, et al, "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol 32(10):1059-1062 (2014). In some embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.

[0183] In some embodiments, the linker comprises a peptide moiety. In some embodiments, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some embodiments, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically), in some embodiments, the peptide moiety is a non-cleavable peptide moiety. In some embodiments, the peptide moiety comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Τφ-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the linker comprises Val-Cit. In some embodiments, the linker is Val-Cit.

[0184] In some embodiments, the linker comprises a benzoic acid group, or a derivative thereof. In some embodiments, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some embodiments, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0185] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the maleimide group is maleimidocaproyl (mc). In some embodiments, the peptide group is val-cit. In some embodiments, the benzoic acid group is PABA. In some embodiments, the linker comprises a mc-val-cit group. In some embodiments, the linker comprises a val-cit-PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.

[0186] In some embodiments, the linker is a self-immolative or self-eliminating linker. In some embodiments, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some embodiments, the linker includes a linker described in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426, each of which is incorporated by reference in its entirety.

[0187] In some embodiments, the linker is a dendritic linker. In some embodiments, the dendritic linker comprises a branched, multifunctional linker moiety. In some embodiments, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some embodiments, the dendritic linker comprises a PAMAM dendrimer.

[0188] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or a linker group) on the polynucleotide or targeting molecule after cleavage.Exemplary traceless linkers include, but are not limited to, germanium linker, silicon linker, sulfur linker, selenium linker, nitrogen linker, phosphorus linker, boron linker, chromium linker, or phenylhydrazine linker.In some embodiments, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497(2013). In some embodiments, the linker is a traceless linker as described in Blaney, et al., 'Traceless solid-phase organic synthesis,' Chem. Rev. 102:2607-2024 (2002). In some embodiments, the linker is a traceless linker as described in U.S. Pat. No. 6,821,783, the entirety of which is incorporated herein by reference.

[0189] In some embodiments, the linker comprises a functional group that exerts steric hindrance at the site of attachment between the linker and the complexing moiety (e.g., a polynucleotide or targeting molecule disclosed herein). In some embodiments, the steric hindrance is around a disulfide bond. Exemplary linkers that exhibit steric hindrance include heterobifunctional linkers, such as the heterobifunctional linkers described above. In some embodiments, linkers that exhibit steric hindrance include SMCC and SPDB.

[0190] In some embodiments, the linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker comprises a hydrazone bond that is susceptible to hydrolytic cleavage. In some embodiments, the acid-cleavable linker comprises a thiomaleamic acid linker. In some embodiments, the acid-cleavable linker is a thiomaleamic acid linker as described in Castaneda, et al, "Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation," Chem. Commun. 49:8187-8189 (2013).

[0191] In some embodiments, the linker is a cyclic amine or cyclic amine as described in U.S. Pat. Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688; U.S. Patent Application Publication Nos. 2014 / 0127239, 2013 / 028919, 2014 / 2869 70, 2013 / 0309256, 2015 / 037360, or 2014 / 0294851; or a linker described in PCT Publication No. WO 2015057699, WO 2014080251, WO 2014197854, WO 2014145090, or WO 2014177042.

[0192] In some embodiments, the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule. In some embodiments, the linker is conjugated to a lysine residue in the targeting molecule. In some embodiments, the linker is conjugated to a cysteine ​​residue in the targeting molecule. In some embodiments, the linker is conjugated to a histidine residue in the targeting molecule. In some embodiments, the linker is conjugated to a non-natural amino acid residue in the targeting molecule.

[0193] In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation or enzymatic conjugation. In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation. Chemical conjugation may include acylation and click chemistry. In some embodiments, the linker is conjugated to the targeting molecule by enzymatic conjugation. Enzymatic conjugation may be via a sortase or transferase enzyme.

[0194] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a chemical ligation process. In some embodiments, the polynucleotide is conjugated to the targeting molecule by native ligation. In some embodiments, conjugation is as described in Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some embodiments, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleotide is conjugated to the targeting molecule via native ligation chemistry, either site-specifically or non-specifically.

[0195] In some embodiments, polynucleotides are conjugated to targeting molecules by a site-directed method utilizing "traceless" coupling technology (Philochem). In some embodiments, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group on the targeting molecule, which is then conjugated to a polynucleotide containing an aldehyde group. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13):5887-5892 (2012)).

[0196] In some embodiments, polynucleotides are conjugated to targeting molecules by site-directed methods that utilize unnatural amino acids incorporated into the targeting molecule. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe selectively binds to an alkoxy-amine derivatized conjugation moiety to form an oxime bond. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40):16101-16106 (2012)).

[0197] In some embodiments, polynucleotides are conjugated to targeting molecules by a site-directed method that utilizes an enzyme-catalyzed process. In some embodiments, the site-directed method utilizes SMARTag™ technology (RedWood). In some embodiments, SMARTag™ technology involves generating formylglycine (FGly) residues from cysteines by formylglycine generating enzyme (FGE) through an oxidation process in the presence of an aldehyde tag, followed by conjugation of FGly to an alkylhydrazine-functionalized polynucleotide via hydrazino-Pictet-Spengler (HIPS) ligation. (See Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9):3000-3005(2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51(2013)).

[0198] In some embodiments, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some embodiments, polynucleotides are conjugated to targeting molecules utilizing a microbial transglutaminase-catalyzed process. In some embodiments, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in a recognition sequence and a primary amine of a functionalized polynucleotide. In some embodiments, mTG is produced from Streptomyces mobarensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2)161-167 (2013))

[0199] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a method such as that described in PCT Publication No. WO 2014 / 140317 (incorporated herein by reference in its entirety), which utilizes a sequence-specific transpeptidase. In some embodiments, the polynucleotide is conjugated to the targeting molecule by a method such as that described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540, each of which is incorporated herein by reference in its entirety.

[0200] In some embodiments, each targeting molecule is conjugated to between 1 and 8 polynucleotide molecules (i.e., a drug:antibody ratio (DAR) of 1 to 8). In some embodiments, each targeting molecule is conjugated to 1 polynucleotide molecule (DAR of 1). In some embodiments, each targeting molecule is conjugated to 2 polynucleotide molecules (DAR of 2). In some embodiments, each targeting molecule is conjugated to 3 polynucleotide molecules (DAR of 3). In some embodiments, each targeting molecule is conjugated to 4 polynucleotide molecules (DAR of 4). In some embodiments, each targeting molecule is conjugated to 5 polynucleotide molecules (DAR of 5). In some embodiments, each targeting molecule is conjugated to 6 polynucleotide molecules (DAR of 6). In some embodiments, each targeting molecule is conjugated to 7 polynucleotide molecules (DAR of 7). In some embodiments, each targeting molecule is conjugated to 8 polynucleotide molecules (DAR of 8).

[0201] In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 30 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 40 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 50 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than about 60 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of about 7,500 kDa or less.

[0202] In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 30 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 40 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 50 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of more than 60 kDa. In some embodiments, the polynucleotide-complexed targeting molecule has a molecular weight of 7,500 kDa or less.

[0203] The present disclosure further relates to a method of treating a pathology in an individual, comprising administering to the individual a therapeutic targeting agent that specifically binds to a target protein expressed on the muscle tissue cell surface. "Pathology" and "disease" are used interchangeably herein. The pathology may be a genetic disease. Another aspect of the present disclosure provides a method for treating a genetic disease in a subject in need of such treatment. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any of the compositions for delivering a polynucleotide disclosed herein. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any of the polynucleotide complexes disclosed herein.

[0204] The genetic disease as disclosed herein may be cancer, a neurological disorder, a fibrotic disease, a scarring disease, an autoimmune disease, or a genetic disorder.

[0205] In some embodiments, the genetic disease is a neurological disorder. In some embodiments, the neurological disorder is acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (see motor neuron diseases), anencephaly, Angelman syndrome, hemangiomatosis, anoxia, aphasia, apraxia, arachnoid cysts, arachnoiditis, Arnold-Chiari malformation, cerebral arteriovenous malformation, Asperger's syndrome, ataxia-telangiectasia, attention-deficit hyperactivity disorder, autism, auditory processing disorder, autonomic nervous system disorder, and the like. nerve dysfunction, back pain, Batten disease, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, bilateral frontoparietal gyri, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus injury, brain abscess, brain injury, brain tumor, Brown-Séquard syndrome, Canavan disease, carpal tunnel syndrome (CTS), causalgia, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, head disorder, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral gigantism, cerebral palsy, Charcot-Marie-Tooth disease, Chiari Malformations, chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, chronic regional pain syndrome, Coffin-Lowry syndrome, coma including persistent vegetative state, congenital ophthalmoplegia, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cytomegalic inclusion disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, neurological apraxia , diabetic neuropathy, diffuse sclerosis, autonomic neuropathy, dyscalculia, dysgraphia, dyslexia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, cerebral herniation, trigeminal nerve region hemangiomatosis, encopresis, epilepsy, Erb's palsy, erythromelalgia, essential tremor, Fabry's disease, Fahr's syndrome, syncope, familial spastic paralysis, febrile convulsions, Fisher's syndrome, Friedreich's ataxia, FART syndrome, Gaucher's disease, Gerstmann's syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, ectopic gray matter, Guillain-Barré syndrome,HTLV-1-associated myelopathy, Hallervorden-Spatz syndrome, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary polyneuropathic ataxia, otic varicella, herpes zoster, Hirayama syndrome, holoprosencephaly, Huntington's disease, hydranencephaly, hydrocephalus, hyperadrenocorticism, hypertrophic cardiomyopathy, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile phytanic acid storage disease, infantile Refsum disease, infantile spasms, inflammatory myopathy, intracranial cysts, intracranial hypertension, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbone syndrome, Klippel-Feil syndrome, Chlamydia trachomatis, Labbe disease, Kugelberg-Welander disease, Kuru, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Lateral bulbar (Wallenberg) syndrome, Learning disabilities, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Leukodystrophy, Dementia with Lewy bodies, Lissencephaly, Locked-in syndrome, Lou Gehrig's disease, Lumbar disc disease, Lyme disease - neurological sequelae, Machado-Joseph disease (Spinocerebellar ataxia type 3), Megaencephaly, Maple syrup urine disease, Marfan syndrome, Megaencephaly, Melkersson-Rosenthal syndrome, Meniere syndrome, Yale disease, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher syndrome, mild stroke, mitochondrial myopathy, Moebius syndrome, unilateral crural muscular atrophy, motor neuron disease, motor skills disorder, Moyamoya disease, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelinating diffuse sclerosis, infantile developmental epileptic encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neurofibromatosis, neuroleptics Neuroleptic malignant syndrome, Neurological symptoms of AIDS, Neurological sequelae of lupus, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorder, Niemann-Pick disease, Non-24-hour sleep-wake syndrome, Nonverbal learning disorder, O'Sullivan-MacLeod syndrome, Occipital neuralgia, Late onset of latent spinal dysraphism, Ohtahara syndrome, Olivopontocerebellar atrophy, Opsoclonus-myoclonus syndrome, Optic neuritis, Orthostatic hypotension, Overuse syndrome, Recurrent vision, Paresthesias, Parkinson's disease, Congenital paramyotonia, Paraneoplastic disorders, Seizures, Parry-Romberg syndrome, Romberg syndrome,Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, persistent vegetative state, diffuse neuropathy, photophotic sneeze reflex, phytanic acid storage disease, Pick's disease, nerve compression, pituitary tumors, PMG, polio, polymicrogyria, polymyositis, porencephaly, post-polio syndrome, post-herpetic neuralgia (PHN), post-infectious encephalomyelitis, orthostatic hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion diseases, progressive facial hemiatrophy also known as Romberg syndrome, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy , pseudotumor cerebri, Ramsay Hunt syndrome (types I and II), Rasmussen's encephalitis, reflex sympathetic dystrophy, Refsum's disease, repetitive movement disorder, repetitive stress injury, restless legs syndrome, retroviral-associated myelopathy, Rett syndrome, Reye's syndrome, Romberg's syndrome, rabies, chorea, Sandhoff disease, schizophrenia, Schilder's disease, schizencephaly, sensory integration dysfunction, septo-optic dysplasia, shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, snatching syndrome (Sna tiation), Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal tumor, spinal muscular atrophy, spinal stenosis, Steele-Richardson-Olszewski syndrome (see Progressive Supranuclear Palsy), spinocerebellar degeneration, stiff-person syndrome, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial hemosiderosis, Sydenham chorea, syncope, synesthesia, syringomyelia, tardive dyskinesia, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen's disease, thoracic outlet syndrome, pain In some embodiments, the neurological disorder is selected from the group consisting of: sexual tics, Todd's paralysis, Tourette's syndrome, transient ischemic attacks, transmissible spongiform encephalopathies, transverse myelitis, traumatic brain injury, tremors, trigeminal neuralgia, tropical spastic paralysis, trypanosomiasis, tuberous sclerosis, vasculitis including temporal arteritis, von Hippel-Lindau disease (VHL), Viliuisk encephalomyelitis (VE), Wallenberg syndrome, Werdnig-Hoffmann disease, West syndrome, whiplash, Williams syndrome, Wilson's disease, X-linked spinal and bulbar muscular atrophy, and Zellweger syndrome. In some embodiments, the neurological disorder is a movement disorder, such as multiple system atrophy (MSA).

[0206] In some embodiments, the genetic disease is an autoimmune disease. In some embodiments, the autoimmune disease is acute disseminated encephalomyelitis (ADEM), acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM antibody / anti-TBM antibody nephritis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency disease, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune platelet deficiency, autoimmune thyroiditis ... Ataxia purpura (ATP), Autoimmune thyroid disease, Axonal & neuroneuropathy, Baro's disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman's disease, Celiac sprue (non-tropical), Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie virus myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathy, dermatomyositis, Devic's disease (neuromyelitis optica), discoid rash, Dressler's syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, immunoregulatory lipoproteins Interstitial disorders, inclusion body myositis, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing atrophicus, lignified conjunctivitis, linear immunoglobulin A (LAD) disease, lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucca-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular pemphigoid, optic neuritis, relapsing rheumatism,PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polyglandular autoimmune syndrome types I, II & III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary Fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynaud's phenomenon, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome, Scleritis, Scleroderma, Slogren's syndrome, Sperm and testicular autoimmune disorders, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis / Giant cell arteritis, Thrombocytopenic purpura (TPP), Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD) , uveitis, vasculitis, vesicular bullous dermatosis, vitiligo or Wegener's granulomatosis or chronic active hepatitis, primary biliary cirrhosis, dilated cardiomyopathy, myocarditis, autoimmune polyendocrine syndrome type I (APS-I), cystic fibrosis vasculitis, acquired hypoparathyroidism, coronary artery disease, pemphigus foliaceus, pemphigus vulgaris, Rasmussen's encephalitis, autoimmune gastritis, insulin hypoglycemic syndrome (Hirata's disease), type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE), pernicious anemia, resistant Lyme arthritis, polyneuropathy, demyelinating disease, atopic dermatitis, autoimmune hypothyroidism, leukocytosis ... Plaques, thyroid eye disease, autoimmune celiac disease, ACTH deficiency, dermatomyositis, Sjögren's syndrome, systemic sclerosis, progressive systemic sclerosis, localized scleroderma, primary antiphospholipid syndrome, chronic idiopathic urticaria, connective tissue syndrome, necrotizing crescentic glomerulonephritis (NCGN), systemic vasculitis, Raynaud's syndrome, chronic liver disease, visceral leishmaniasis, autoimmune C1 inhibitor deficiency, membranoproliferative glomerulonephritis (MPGN), prolonged clotting time, immunodeficiency, atherosclerosis, neuropathies, paraneoplastic pemphigus, paraneoplastic stiff-man syndrome, paraneoplastic encephalomyelitis,The patient is selected from the group consisting of subacute dysautonomia, cancer-associated retinopathy, paraneoplastic opsoclonus-myoclonus ataxia, lower motor neuron syndrome, and Lambert-Eaton myasthenic syndrome.

[0207] In some embodiments, the genetic disease may be selected from the group consisting of AIDS, anthrax, botulism, brucellosis, chancroid, chlamydia infection, cholera, coccidioidomycosis, cryptosporidiosis, cyclosporiasis, diphtheria, ehrlichiosis, arboviral encephalitis, enterohemorrhagic Escherichia coli infection, giardiasis, gonorrhea, dengue fever, Haemophilus influenzae infection, Hansen's disease (leprosy), Hantavirus pulmonary syndrome, hemolytic uremic syndrome, hepatitis A, hepatitis B, hepatitis C, human immunodeficiency virus infection, legionnaires' disease, listeriosis, Lyme disease, malaria, measles, meningococcal infection, mumps, pertussis (whooping cough), cough), plague, paralytic polio, psittacosis, Q fever, rabies, Rocky Mountain spotted fever, rubella, congenital rubella syndrome, bacterial dysentery, smallpox, streptococcal infections (invasive group A), fulminant hemolytic streptococcal infections, pneumococcal infections, syphilis, tetanus, toxic shock syndrome, trichinellosis, tuberculosis, tularemia, typhoid fever, vancomycin-resistant Staphylococcus aureus infections, chickenpox, yellow fever, variant Creutzfeldt-Jakob disease (vCJD), Ebola hemorrhagic fever, echinococcosis, Hendra virus infection, human monkeypox, influenza A, influenza B, H5N1, Lassa fever, Marburg hemorrhagic fever, Nipah virus infection, Onyong-nyong fever, Rift Valley fever, herpes, HIV, HCV genotype 1, HCV genotype 2, HCV genotype 3, HCV genotype 4, HCV genotype 5, HCV genotype 6, SARS-CoV-2 (COVID-19), SARS-CoV (SARS), MERS-CoV (MERS), 229E coronavirus infection, NL63 coronavirus infection, OC43 coronavirus infection, CoV-HKU1 (HKU1), alphacoronavirus infection, betacoronavirus infection, Venezuelan equine encephalitis and West Nile virus infection.

[0208] In some embodiments, the genetic disease is a fibrotic disease, a scarring disease, or both. In some embodiments, the fibrotic disease or scarring disease is selected from the group consisting of pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, myocardial fibrosis, bridging fibrosis, liver cirrhosis, gliosis, arterial stiffness, fibrotic joint disease, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive diffuse fibrotic lesions, retroperitoneal fibrosis, scleroderma / systemic sclerosis, and adhesive capsulitis.

[0209] In some embodiments, the genetic disease is a muscle-related cancer, such as a sarcoma.

[0210] In some embodiments, the genetic disease is a genetic disorder caused by an abnormality in a gene or chromosome. Genetic disorders can be divided into two categories: single gene disorders and multifactorial and polygenic (complex) disorders. Single gene disorders can be the result of a single mutant gene. Single gene disorders are inherited in ways that include, but are not limited to, autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, Y-linked, and mitochondrial inheritance. In some embodiments, one mutant copy of a gene is required for a person affected by an autosomal dominant disorder. Examples of autosomal dominant disorders include, but are not limited to, Huntington's disease, neurofibromatosis 1, Marfan syndrome, hereditary nonpolyposis colorectal cancer, or hereditary multiple exostoses. In an autosomal recessive disorder, two mutant copies of a gene are required for a subject affected by an autosomal recessive disorder. Examples of this type of disorder may include, but may not be limited to, cystic fibrosis, sickle cell disease (also partial sickle cell disease), Tay-Sachs disease, Niemann-Pick disease, or spinal muscular atrophy. X-linked dominant disorders are caused by mutations in genes on the X chromosome, such as X-linked hypophosphatemic rickets. Some X-linked dominant conditions, such as Rett syndrome, incontinentia pigmenti type 2, and Aicardi syndrome, may be fatal. X-linked recessive disorders are also caused by mutations in genes on the X chromosome. Examples of this type of disorder may include, but are not limited to, hemophilia A, Duchenne muscular dystrophy, red-green color blindness, muscular dystrophy, and male pattern baldness. Y-linked disorders are caused by mutations on the Y chromosome. Examples include, but are not limited to, male infertility and hypertrichosis pinnae. Genetic disorders of mitochondrial inheritance, also known as maternal inheritance, can apply to genes within mitochondrial DNA, and these include Leber's hereditary optic neuropathy.

[0211] Genetic disorders can also be complex, multifactorial or polygenic. Polygenic genetic disorders can be associated with the influence of multiple genes combined with lifestyle and environmental factors. Complex genetic disorders occur in families, but they do not have a clear inheritance pattern. Multifactorial or polygenic disorders include, but are not limited to, heart disease, diabetes, asthma, autism, autoimmune diseases such as multiple sclerosis, cancer, ciliary diseases, cleft palate, hypertension, inflammatory bowel disease, mental retardation or obesity.

[0212] Other exemplary genetic disorders include 1p36 deletion syndrome, 21-hydroxylase deficiency, 22q11.2 deletion syndrome, aceruloplasminemia, achondroplasia type II, achondroplasia, acute intermittent porphyria, adenylosuccinate lyase deficiency, adrenoleukodystrophy, Alexander disease, alkaptonuria, alpha-1 antitrypsin deficiency, Alström syndrome, Alzheimer's disease (types 1, 2, 3, and 4), amelogenesis imperfecta, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis type 2, amyotrophic lateral sclerosis type 4, amyotrophic lateral sclerosis type 5, amyotrophic lateral sclerosis type 6, amyotrophic lateral sclerosis type 7, amyotrophic lateral sclerosis type 8, amyotrophic lateral sclerosis type 9, amyotrophic lateral sclerosis type 10, amyotrophic lateral sclerosis type 11, amyotrophic lateral sclerosis type 12, amyotrophic lateral sclerosis type 13, amyotrophic lateral sclerosis type 14, amyotrophic lateral sclerosis type 15, amyotrophic lateral sclerosis type 16, amyotrophic lateral sclerosis type 17, amyotrophic lateral sclerosis type 18, amyotrophic lateral sclerosis type 19, amyotrophic lateral sclerosis type 20, amyotrophic lateral sclerosis type 21, amyotrophic lateral sclerosis type 22, amyotrophic lateral sclerosis type 23, amyotrophic lateral sclerosis type 24, amyotrophic lateral sclerosis type 25, amyotrophic lateral sclerosis type 26, amyotrophic lateral sclerosis type 27, amyotrophic lateral drogen insensitivity syndrome, anemia, Angelman syndrome, Apert syndrome, ataxia telangiectasia, Bear-Stevenson gyriform scalp syndrome, Benjamin syndrome, beta thalassemia, biotinidase deficiency, Birt-Hogg-Dubé syndrome, bladder cancer, Bloom syndrome, bone disease, breast cancer, ankle dysplasia, Canavan disease, cancer, celiac disease, chronic granulomatous disease (CGD), Charcot-Marie-Tooth disease, Charcot-Marie-Tooth disease type 1, Charcot-Marie-Tooth disease type 4, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 4 type II and type XI collagen disorders, colorectal cancer, congenital absence of the vas deferens, congenital bilateral absence of the vas deferens, congenital diabetes mellitus, congenital erythropoietic porphyria, congenital erythroblastic porphyria, congenital heart disease, congenital hypothyroidism, connective tissue disease, Cowden syndrome, Cri du Chat syndrome, Crohn's disease, fibrostenosis, Crouzon syndrome, Crouzon-cutaneous skeletal syndrome, cystic fibrosis, de Grouchy syndrome, neurodegenerative diseases, Dent's disease, developmental disorders, DiGeorge syndrome, distal spinal muscular atrophy type V, Down's syndrome, low Stature disorder, Ehlers-Danlos syndrome, Ehlers-Danlos syndrome multiple joint laxity type, Ehlers-Danlos syndrome classic type, Ehlers-Danlos syndrome cutis laxity type, Ehlers-Danlos syndrome kyphoscoliosis type, vascular type, erythroblastic protoporphyria, Fabry disease, facial injuries and disorders, factor V Leiden thrombophilia, familial adenopathy, familial dysautonomia, Fanconi anemia, FG syndrome, fragile X syndrome, Friedreich's ataxia, Friedreich's ataxia, G6PD deficiency, galactosemia, Gaucher disease (types 1, 2, and 3), hereditary brain disorders,Glycine encephalopathy, Hemochromatosis type 2, Hemochromatosis type 4, Harlequin ichthyosis, Head and brain malformations, Hearing impairment and hearing loss, Hearing impairment in children, Hemochromatosis (neonatal, type 2 and type 3), Hemophilia, Hepatic erythroblastic porphyria, Hereditary coproporphyria, Hereditary multiple exostoses, Hereditary neuropathy with liability to pressure palsies, Hereditary nonpolyposis colorectal cancer, Homocystinuria, Huntington's disease, Hutchinson-Gilford progeria syndrome, Primary hyperoxaluria, Hyperphenylalaninemia, Hypochondrogenesis, Hypochondroplasia, IDICULOSIS 15, Incontinentia pigmenti, Infantile Gaucher disease, Infantile ascending spastic paralysis, Infertility, Jackson-Wall syndrome Iss syndrome, Joubert syndrome, juvenile primary lateral sclerosis, Kennedy disease, Klinefelter syndrome, Kniest dysplasia, Krabbe disease, learning disabilities, Lesch-Nyhan syndrome, leukodystrophy, Li-Fraumeni syndrome, familial lipoprotein lipase deficiency, male reproductive disorders, Marfan syndrome, McCune-Albright syndrome, McLeod syndrome, familial Mediterranean fever, Menkes disease, Menkes syndrome, metabolic disorders, beta-globin methemoglobinemia, methemoglobinemia, congenital methemoglobinemia, methylmalonic acidemia, Micro syndrome, microcephaly, movement disorders, Mowat-Wilson syndrome, mucopolysaccharidosis (MPS) I), Muenke syndrome, Duchenne and Becker muscular dystrophy, Duchenne and Becker muscular dystrophy, myotonic dystrophy, myotonic dystrophy type 1 and type 2, limb-girdle muscular dystrophy, Pompe disease, neonatal hemochromatosis, neurofibromatosis, neurofibromatosis type 1, neurofibromatosis type 2, neurofibromatosis type I, neurofibromatosis type II, neurological diseases, neuromuscular disorders, Niemann-Pick disease, nonketotic hyperglycinemia, nonsyndromic hearing loss, autosomal recessive nonsyndromic hearing loss Hearing loss, Noonan syndrome, Osteogenesis imperfecta (types I and III), Otospondylomegaphyseal dysplasia, Pantothenate kinase-associated neurodegeneration, Patau syndrome (trisomy 13), Pendred syndrome, Peutz-Jeghers syndrome, Pfeiffer syndrome, Pfeiffer syndrome, Phenylketonuria, Porphyria, Porphyria cutanea tarda, Prader-Willi syndrome, Primary pulmonary hypertension, Prion disease, Progeria, Propionic acidemia, Protein C deficiency, Protein S deficiency, Pseudo-Gaucher disease,Pseudoxanthoma elasticum, Retinopathy, Retinoblastoma, Retinoblastoma, FA-Friedreich's ataxia, Rett syndrome, Rubinstein-Taybi syndrome, Sandhoff disease, Sensory autonomic neuropathy type III, Sickle cell anemia, Skeletal muscle regeneration, Dyschromatosis, Smith-Lemli-Opitz syndrome, Speech and communication disorders, Spinal muscular atrophy, Spinobulbar muscular atrophy, Spinocerebellar degeneration, Strudwick type spondyloepiphyseal dysplasia, Congenital spondyloepiphyseal dysplasia, Stickler syndrome, Stickler syndrome COL2A1, Tay-Sachs disease, Tetrahydrobioptera These may include, but are not limited to, phosphorus deficiency, lethal skeletal dysplasia, diabetes mellitus and thiamine-responsive megaloblastic anemia with sensorineural hearing loss, thyroid disease, Tourette's syndrome, Treacher-Collins syndrome, triple X syndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, variegate porphyria, von Hippel-Lindau disease, Waardenburg syndrome, Weissenbacher-Zweimüller syndrome, Wilson's disease, Wolf-Hirschhorn syndrome, xeroderma pigmentosum, X-linked severe combined immunodeficiency, X-linked sideroblastic anemia, or X-linked spinal and bulbar muscular atrophy.

[0213] In some embodiments, the genetic disease is a viral infection. The viral infection may be by a virus selected from the group consisting of adenovirus, anellovirus, arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, herpesvirus, orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, pneumoniavirus, polyomavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus. In some embodiments, the virus is adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, pneumonia virus, polyomavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus.herpesvirus), Chandipura virus, Chikungunya virus, Cosavirus A, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhoori virus, Djugbe virus, Dubenhage virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus C / G hepatitis virus, Hantavirus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Delta hepatitis virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, Human enterovirus 70, Human herpesvirus 1, Human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, human papillomavirus 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Lassa virus, Lordsdale virus virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, Poliovirus, Punta Toro phlebovirusphlebovirus, Puumala virus, rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sicilian sandfly fever virus, Sapporo virus, SARS coronavirus 2, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tick-borne Powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, and Zika virus.

[0214] In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA that targets a viral gene. In some embodiments, the polynucleotide comprises an siRNA that targets a viral gene. In some embodiments, the polynucleotide comprises an miRNA that targets a viral gene. In some embodiments, the polynucleotide comprises an miRNA mimic that targets a viral gene. In some embodiments, the polynucleotide comprises an ASO that targets a viral gene. In some embodiments, the polynucleotide comprises a guide RNA that targets a viral gene. The polynucleotide may be complexed to a targeting molecule that specifically binds to a viral protein or a protein on the surface of a host cell for the virus. In some embodiments, the polynucleotide and the targeting molecule produce a synergistic effect in treating a viral infection.

[0215] In some embodiments, the genetic disease is cancer. In some embodiments, the cancer is characterized by overexpression of a cancer gene. In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA that targets the cancer gene. In some embodiments, the polynucleotide comprises an siRNA that targets the cancer gene. In some embodiments, the polynucleotide comprises an miRNA that targets the cancer gene. In some embodiments, the polynucleotide comprises an miRNA mimic that targets the cancer gene. In some embodiments, the polynucleotide comprises an ASO that targets the cancer gene. In some embodiments, the polynucleotide comprises a guide RNA that targets the cancer gene.

[0216] In some embodiments, the cancer is characterized by reduced expression of a tumor suppressor gene. The polynucleotide may comprise an mRNA molecule that encodes the tumor suppressor gene. In some embodiments, the polynucleotide comprises a guide RNA that restores expression of the tumor suppressor gene.

[0217] In some embodiments, the genetic disease is a neuromuscular disorder. The neuromuscular disorder may be a muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide comprises an siRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DUX4. In some embodiments, the polynucleotide comprises an ASO targeting DUX4. In some embodiments, the polynucleotide comprises a guide RNA targeting DUX4. In some embodiments, the ASO targeting DUX is selected from the group consisting of DUX4-targeting ASOs disclosed in Table 1. In some embodiments, the DUX4-targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the DUX4 targeting ASO is ASDX2. In some embodiments, the DUX4 targeting ASO is ASDX4. In some embodiments, the DUX4 targeting ASO is ASDX23. In some embodiments, the DUX4 targeting ASO is ASDX26. In some embodiments, the DUX4 targeting ASO is ASDX32. In some embodiments, the polynucleotide comprises an siRNA that targets DMPK. In some embodiments, the polynucleotide comprises an miRNA that targets DMPK. In some embodiments, the polynucleotide comprises an miRNA mimic that targets DMPK. In some embodiments, the polynucleotide comprises an ASO that targets DMPK. In some embodiments, the polynucleotide comprises an siRNA that targets CAPN3. In some embodiments, the polynucleotide comprises an miRNA that targets CAPN3. In some embodiments, the polynucleotide comprises an miRNA mimic that targets CAPN3.In some embodiments, the polynucleotide comprises an ASO that targets CAPN3.

[0218] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. In some embodiments, the polynucleotide is an mRNA encoding dystrophin or utrophin. In some embodiments, the polynucleotide is a guide RNA that restores expression of dystrophin or utrophin.

[0219] In some embodiments, the polynucleotide is complexed to a targeting molecule that specifically binds to a marker (targeted protein) on the surface of the muscle cells of the subject. The marker is selectively expressed on muscle tissue, internalized / recycled on a time scale that allows for drug efficacy (minutes / hours instead of days), and its expression is not adversely affected by disease progression. The targeting molecule may specifically bind to KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, or ACTA1. In some embodiments, the targeting molecule specifically binds to KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, or ACTA1, and the polynucleotide is a DUX4-targeted ASO.

[0220] In some embodiments, the polynucleotide and the targeting molecule produce a synergistic effect in the treatment of muscular dystrophy.

[0221] In the methods herein, drugs are delivered in a tissue-specific manner utilizing drugs that specifically bind to proteins expressed on the muscle cell surface. As the term is used herein, a drug that "specifically binds" to a target protein is a drug that binds preferentially or selectively to the target protein. Although some degree of non-specific interaction may occur between a specifically binding drug and a target protein, specific binding may nevertheless be distinguished as being mediated through specific recognition of all or part of the target protein. Typically, specific binding results in a much stronger association between the drug and the target protein than between the drug and other proteins, e.g., other muscle proteins. The affinity constant (Ka as opposed to Kd) of the drug for its homologue is at least 10 6 or 10 7 and is usually at least 10 8 , alternatively at least 10 9 , alternatively at least 10 10 , or alternatively at least 10 11 M. It should be noted that "specific" binding can be binding that is sufficiently site-specific to be effectively "specific". For example, if the degree of binding is higher (e.g., 10-fold or more, 20-fold or more, or even 100-fold or more), the binding can be functionally equivalent to binding only to the protein targeted at a specific location. Directed effective binding occurs with minimal or no delivery to other tissues. Thus, the amount that is functionally equivalent to specific binding can be determined by evaluating the target of effective delivery of the agent with minimal or no binding to other tissues.

[0222] The targeted protein is tissue specific in certain embodiments; the targeted protein may only be present in one tissue, resulting in a tissue-specific interaction between an agent that binds to the targeted protein and the targeted protein itself.

[0223] In some embodiments, the target protein is enriched in muscle tissue relative to other tissues. "Enriched in muscle tissue relative to other tissues" refers to a higher surface expression of the target protein than that of other non-muscle tissues. In one particular example, the target protein is not expressed (i.e., in an amount that is not detectable by a person skilled in the art) on the cell surface of cells contained in any non-muscle tissue. Non-muscle tissues may be characterized by undetectable expression of one or more of actin alpha 1, myosin heavy chain IV (MYH4), myosin heavy chain VI (MYH6), myosin IA (Myo1A), or caveolin 3 (CAV3).

[0224] Muscle tissue (or muscular tissue) as used herein refers to the soft tissue that constitutes the various types of muscles in animals and gives them the ability to contract. Muscle tissue is formed during embryonic development in a process known as myogenesis. Muscle tissue contains contractile proteins called actin and myosin, which contract and relax to cause movement. Among the many other muscle proteins present are two regulatory proteins, namely troponin and tropomyosin. Muscle tissue varies according to function and location in the body. In mammals, there are three types: skeletal or striated muscle tissue; smooth (non-striated) muscle; and cardiac muscle. In one embodiment, the muscle tissue is skeletal muscle tissue. Skeletal muscle tissue is made up of elongated muscle cells, also called muscle fibers, and is responsible for body movement. Other tissues of skeletal muscle include tendons and perimysium. Without conscious intervention, smooth and cardiac muscles contract involuntarily. These muscle types can be activated through central nervous system interactions as well as by receiving innervation from peripheral plexuses or endocrine (hormonal) activation. Striated or skeletal muscles only contract voluntarily in response to central nervous system influences.

[0225] In some embodiments, the targeted protein has stable expression or increased expression in diseased tissue compared to normal tissue. "Stable expression" requires that the cell surface expression of the targeted protein does not decrease during each stage of disease (or pathology) progression. Normal tissue refers to (muscle) tissue that is not affected by the disease or pathology. The normal tissue may be from the subject to be treated or any other subject that is not affected by the disease or pathology.

[0226] In some embodiments, the targeted protein is KLHL41. In some embodiments, the targeted protein is LMOD2. In some embodiments, the targeted protein is ENO3. In some embodiments, the targeted protein is FABP3. In some embodiments, the targeted protein is CHRNA1. In some embodiments, the targeted protein is SEMA6C. In some embodiments, the targeted protein is XIRP2. In some embodiments, the targeted protein is XIRP1. In some embodiments, the targeted protein is CAVIN4. In some embodiments, the targeted protein is CFL2. In some embodiments, the targeted protein is SVIL. In some embodiments, the targeted protein is MUSK. In some embodiments, the targeted protein is ART1. In some embodiments, the targeted protein is CACNA1S. In some embodiments, the targeted protein is CDH15. In some embodiments, the targeted protein is CLCN1. In some embodiments, the targeted protein is CLCN1. In some embodiments, the targeted protein is MYMX. In some embodiments, the targeted protein is ACTA1.

[0227] In some embodiments, the methods herein include the steps of detecting, comparing, evaluating, or any combination thereof. In some cases, detecting may include running a computer program on a computer. In some cases, comparing may include running a computer program on a computer. In some cases, evaluating may include running a computer program on a computer. In some embodiments, detecting, comparing, evaluating, or any combination thereof uses a computer processor. In some embodiments, detecting, comparing, evaluating, or any combination thereof uses computer readable memory. In some embodiments, detecting, comparing, evaluating, or any combination thereof uses computer readable instructions on a computer readable memory. In some cases, the computer programs may be the same computer program. In some cases, the computer programs may be different computer programs. In some cases, the computers herein may include a graphical user interface. In some cases, the computers herein may include an electronic display.

[0228] In some cases, a computer system for performing the methods herein includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor"), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system may also include memory or memory locations (random access memory, read-only memory, flash memory), electronic storage (hard disk), communication interfaces (e.g., network adapters) for communicating with one or more other systems, and peripheral devices such as cache, other memory, data storage, and / or electronic display adapters. The memory, storage units, interfaces, and peripheral devices may communicate with the CPU via a communication bus, such as a motherboard. The storage units may be data storage units (or data repositories) for storing data. The computer system may be operatively coupled to a computer network ("network") with the aid of a communication interface. The network may be the Internet, an Internet, and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network may be a telecommunications and / or data network in some cases. The network may include one or more computer servers, enabling distributed computing, such as cloud computing. The network, possibly with the aid of a computer system, can implement a peer-to-peer network, whereby devices coupled to the computer system can act as clients or servers. The CPU can execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as a memory. The instructions may be directed to the CPU, which may then be programmed or otherwise configured to implement the methods of the present disclosure.

[0229] Numbered embodiments Various methods are disclosed herein. Specific exemplary embodiments of these methods are disclosed below. The following embodiments list non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is considered to be dependent on or related to every preceding or following numbered embodiment, regardless of the order in which they are listed.

[0230] Implementation Section 1: Embodiment 1. A method of in vivo delivery of an agent to muscle tissue in a tissue specific manner, comprising contacting a surface of a muscle cell with an agent that specifically binds to a target protein expressed on the cell surface of the muscle tissue, wherein the target protein is enriched in muscle tissue relative to other tissues, the target protein has stable or increased expression in diseased tissue relative to normal tissue, and the target protein is expressed for about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about wherein the compound is internalized and reused within 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0231] Embodiment 2. The method of embodiment 1, wherein the targeted protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0232] Embodiment 3. The method of any one of embodiments 1 or 2, wherein the agent is a specific binding agent for the target protein.

[0233] Embodiment 4. The method of embodiment 3, wherein the specific binding agent is a soluble receptor or a soluble ligand.

[0234] Embodiment 5 The method of embodiment 4, wherein the soluble receptor comprises the extracellular domain of the receptor.

[0235] Embodiment 6 The method of any one of embodiments 4 or 5, wherein the soluble receptor is an Fc fusion protein.

[0236] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the agent is an antibody or an antigen-binding fragment thereof.

[0237] Embodiment 8. The method of embodiment 7, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and a nanobody.

[0238] Embodiment 9. A method of treating a pathology in an individual, comprising administering to the individual a therapeutic targeting agent that specifically binds to a target protein expressed on a muscle tissue cell surface, wherein the target protein is enriched in muscle tissue relative to other tissues, the target protein has stable or increased expression in diseased tissue relative to normal tissue, and the target protein is expressed for about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours. wherein the compound is internalized and reused within about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0239] Embodiment 10. The therapeutic targeting agent is a drug comprising an active agent component and a targeting agent component, wherein the active agent component is a radionuclide, a chemotherapeutic agent, an immunostimulant, an antitumor agent, an anti-inflammatory agent, a pro-inflammatory agent, a pro-apoptotic agent, a pro-coagulant, a toxin, an antibiotic, a hormone, an enzyme, a protein, a carrier protein, a lytic agent, a small molecule, an aptamer, a cell, a vaccine-induced cell or other immune cell, a nanoparticle, transferrin, an immunoglobulin, a multivalent antibody, a lipid, a lipoprotein, a liposome, a modified natural ligand, a gene or a nucleic acid, an oligonucleotide, an RNA, an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), 10. The method of embodiment 9, wherein the targeting agent component is selected from the group consisting of dicer-dependent siRNA (di-siRNA), antisense oligonucleotide (ASO), gapmer, miximer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-dependent RNA interference (ddRNAi), RNA activating oligonucleotide (RNAa), aptamer, exon skipping oligonucleotide, miRNA, miRNA mimic, mRNA, guide RNA, viral or non-viral gene delivery vector, prodrug, and promolecule, and the targeting agent component specifically binds to the targeted protein.

[0240] Embodiment 11 The method of embodiment 10, wherein the targeting agent component comprises a specific binding agent for the targeted protein.

[0241] Embodiment 12 The method of embodiment 11, wherein the specific binding agent is a soluble receptor or a soluble ligand.

[0242] Embodiment 13 The method of embodiment 12, wherein the soluble receptor comprises the extracellular domain of the receptor.

[0243] Embodiment 14 The method of any one of embodiments 12 or 13, wherein the soluble receptor is an Fc fusion protein.

[0244] Embodiment 15 The method of any one of embodiments 10 or 11, wherein the targeting agent component comprises an antibody or an antigen-binding fragment thereof.

[0245] Embodiment 16. The method of embodiment 15, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and a nanobody.

[0246] Embodiment 17. The method of embodiment 10, wherein the active agent component is an oligonucleotide and the targeting agent component is an antibody or an antigen-binding fragment thereof, the active agent component is conjugated to the targeting agent component, and the oligonucleotide targets a disease gene expressed in muscle tissue.

[0247] Embodiment 18. The method of any one of embodiments 9 to 17, wherein the target protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0248] Embodiment 19. A method of delivering an imaging agent to muscle tissue in a tissue-specific manner, comprising contacting a surface of a muscle cell with an imaging agent comprising an imaging agent component and a targeting agent component, wherein the targeting agent component specifically binds to a target protein expressed on the cell surface of the tissue, the target protein being enriched in muscle tissue relative to other tissues, the target protein having stable or increased expression in diseased tissue relative to normal tissue, and the target protein being enriched in muscle tissue relative to other tissues, and the target protein being enriched in muscle tissue relative to normal tissues, and the target protein is enriched in muscle tissue relative to other tissues, and the target protein is ... wherein the compound is internalized and reused within 2 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0249] Embodiment 20. The method of embodiment 19, wherein the targeted protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0250] Embodiment 21 The method of any one of embodiments 19 or 20, wherein the targeting agent component is a specific binding agent for the targeted protein.

[0251] Embodiment 22 The method of embodiment 21, wherein the specific binding agent is a soluble receptor or a soluble ligand.

[0252] Embodiment 23 The method of embodiment 22, wherein the soluble receptor comprises the extracellular domain of the receptor.

[0253] Embodiment 24 The method of any one of embodiments 22 or 23, wherein the soluble receptor is an Fc fusion protein.

[0254] Embodiment 25 The method of any one of embodiments 19 to 21, wherein the targeting agent component is an antibody or an antigen-binding fragment thereof.

[0255] Embodiment 26. The method of embodiment 25, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and a nanobody.

[0256] Embodiment 27. The method of any one of embodiments 19 to 26, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a genetic vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0257] Embodiment 28. A method of delivering an imaging agent to a tissue sample in a tissue-specific manner, comprising contacting the tissue sample with an imaging agent comprising an imaging agent component and a targeting agent component, wherein the targeting agent component specifically binds to a targeted protein expressed on a muscle cell surface of the tissue, the targeted protein being enriched in muscle tissue relative to other tissues, the targeted protein having stable or increased expression in diseased tissue relative to normal tissue, and the targeted protein being enriched in muscle tissue relative to normal tissue within about 2 minutes to about 1 minute. wherein the compound is internalized and reused within 2 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0258] Embodiment 29. The method of embodiment 28, wherein the targeted protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0259] Embodiment 30 The method of any one of embodiments 28 or 29, wherein the targeting agent component is a specific binding agent for the targeted protein.

[0260] Embodiment 31 The method of embodiment 30, wherein the specific binding agent is a soluble receptor or a soluble ligand.

[0261] Embodiment 32 The method of embodiment 31, wherein the soluble receptor comprises the extracellular domain of the receptor.

[0262] Embodiment 33 The method of any one of embodiments 31 or 32, wherein the soluble receptor is an Fc fusion protein.

[0263] Embodiment 34 The method of any one of embodiments 28 to 30, wherein the targeting agent component is an antibody or an antigen-binding fragment thereof.

[0264] Embodiment 35. The method of embodiment 34, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and a nanobody.

[0265] Embodiment 36. The method of any one of embodiments 28 to 35, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a genetic vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0266] Embodiment 37. A method of performing physical imaging of muscle tissue of an individual, comprising administering to the individual an imaging agent comprising a targeting agent component and an imaging agent component, wherein the targeting agent component specifically binds to a target protein expressed on a cell surface of the muscle tissue, the target protein is enriched in the muscle tissue relative to other tissues, the target protein has stable or increased expression in the diseased tissue relative to normal tissue, and the target protein is enriched in the diseased tissue relative to normal tissue for about 2 minutes to about 12 hours. , about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours,

[0267] Embodiment 38. The method of embodiment 37, wherein the targeted protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0268] Embodiment 39. The method of any one of embodiments 37 or 38, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0269] Embodiment 40. A method of evaluating an individual for the presence or absence of muscle tissue pathology comprising: a) administering to the individual an imaging agent comprising an imaging agent component and a targeting agent component, wherein the targeting agent component specifically binds to a targeted protein expressed on the cell surface of muscle tissue; b) evaluating the individual for the presence or absence of a concentration of the imaging agent; and c) the presence or absence of the concentration of the imaging agent indicates the presence of the pathology.

[0270] Embodiment 41 The method of embodiment 40, wherein the targeting agent component is a specific binding agent for the targeted protein.

[0271] Embodiment 42 The method of embodiment 41, wherein the specific binding agent is a soluble receptor or a soluble ligand.

[0272] Embodiment 43 The method of embodiment 42, wherein the soluble receptor comprises the extracellular domain of the receptor.

[0273] Embodiment 44 The method of any one of embodiments 42 or 43, wherein the soluble receptor is an Fc fusion protein.

[0274] Embodiment 45 The method of any one of embodiments 40 or 41, wherein the targeting agent component is an antibody or an antigen-binding fragment thereof.

[0275] Embodiment 46. The method of embodiment 45, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and a nanobody.

[0276] Embodiment 47. The method of any one of embodiments 40 to 46, wherein the target protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0277] Embodiment 48. The method of any one of embodiments 40 to 47, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0278] Embodiment 49. A method of assessing the response of muscle tissue from an individual to treatment with a therapeutic targeting agent, wherein the therapeutic targeting agent specifically binds to a target protein expressed on the cell surface of muscle tissue, comprising: a) assessing the level of the target protein in a sample from the individual prior to treatment with the therapeutic targeting agent; b) assessing the level of the target protein in a sample from the individual during or after treatment with the therapeutic targeting agent; and c) comparing the pre-treatment level with the during- or post-treatment level, wherein a level of the target protein during or after treatment that is significantly lower than the pre-treatment level of the target protein indicates the effectiveness of treatment with the therapeutic targeting agent and is indicative of a response to the treatment of the target protein. 13. A method according to claim 12, wherein the protein is enriched in muscle tissue relative to other tissues, the target protein has stable or increased expression in diseased tissue relative to normal tissue, and the target protein is internalized and reused within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

[0279] Embodiment 50. The method of embodiment 49, wherein the targeted protein is selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0280] Implementation Section 2: Embodiment 1. A method of delivering an agent to muscle tissue in vivo in a tissue specific or tissue selective manner, the method comprising contacting a surface of muscle cells of the muscle tissue with an effective amount of an agent that specifically or selectively binds to a target protein expressed on the cell surface of muscle cells of the muscle tissue, wherein the target protein is enriched in the muscle tissue relative to other tissues, wherein the target protein has stable or increased expression in the diseased tissue relative to an otherwise comparable normal tissue, and optionally wherein the target protein is expressed for about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 9 hours, about 2 minutes to about 12 ... the agent is internalized and reused within about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours, thereby delivering the agent to muscle tissue in vivo in a tissue-specific or tissue-selective manner.

[0281] Embodiment 2. The method of embodiment 1, wherein the targeted protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0282] Embodiment 3. The method of any one of embodiments 1 or 2, wherein the agent is a specific or selective binding agent of the target protein.

[0283] Embodiment 4. The method of embodiment 3, wherein the specific or selective binding agent is a soluble receptor or a soluble ligand.

[0284] Embodiment 5. The method of embodiment 4, wherein the specific or selective binding agent is a soluble receptor, and the soluble receptor comprises the extracellular domain of the receptor.

[0285] Embodiment 6 The method of any one of embodiments 4 or 5, wherein the soluble receptor is an Fc fusion protein.

[0286] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the agent is an antibody or an antigen-binding fragment thereof.

[0287] Embodiment 8. The method of embodiment 7, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV).

[0288] Embodiment 9. A method of treating a pathology in an individual, comprising administering to the individual an effective amount of a therapeutic targeting agent that specifically or selectively binds to a target protein expressed on the cell surface of muscle tissue, wherein the target protein is enriched in muscle tissue relative to other tissues, wherein the target protein has stable or increased expression in diseased tissue relative to an otherwise comparable normal tissue, and optionally wherein the target protein is expressed for between about 2 minutes and about 12 hours, between about 2 minutes and about 10 hours, between about 2 minutes and about 8 hours, between about 2 minutes and about 12 ... the compound is internalized and reused within about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours, thereby treating a pathology in an individual.

[0289] Embodiment 10. The therapeutic targeting agent is a drug comprising an active agent component and a targeting agent component, wherein the active agent component is a radionuclide, a chemotherapeutic agent, an immunostimulant, an antitumor agent, an anti-inflammatory agent, a pro-inflammatory agent, a pro-apoptotic agent, a pro-coagulant, a toxin, an antibiotic, a hormone, an enzyme, a protein, a carrier protein, a lytic agent, a small molecule, an aptamer, a cell, a vaccine-induced cell or other immune cell, a nanoparticle, transferrin, an immunoglobulin, a multivalent antibody, a lipid, a lipoprotein, a liposome, a modified natural ligand, a gene or a nucleic acid, an oligonucleotide, an RNA, an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicRNA, a medicament ... 10. The method of embodiment 9, wherein the targeting agent component is selected from the group consisting of er-dependent siRNA (di-siRNA), antisense oligonucleotide (ASO), gapmer, miximer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-dependent RNA interference (ddRNAi), RNA activating oligonucleotide (RNAa), aptamer, exon skipping oligonucleotide, miRNA, miRNA mimic, mRNA, guide RNA, viral or non-viral gene delivery vector, prodrug, and pro-molecule, and the targeting agent component specifically or selectively binds to the targeted protein.

[0290] Embodiment 11 The method of embodiment 10, wherein the targeting agent component comprises a specific or selective binding agent for the targeted protein.

[0291] Embodiment 12. The method of embodiment 11, wherein the specific or selective binding agent is a soluble receptor or a soluble ligand.

[0292] Embodiment 13 The method of embodiment 12, wherein the specific or selective binding agent is a soluble receptor, and the soluble receptor comprises the extracellular domain of the receptor.

[0293] Embodiment 14 The method of any one of embodiments 12 or 13, wherein the soluble receptor is an Fc fusion protein or a biologically active fragment thereof.

[0294] Embodiment 15 The method of any one of embodiments 10 or 11, wherein the targeting agent component comprises an antibody or an antigen-binding fragment thereof.

[0295] Embodiment 16. The method of embodiment 15, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV).

[0296] Embodiment 17. The method of embodiment 10, wherein the active agent component is an oligonucleotide or polynucleotide and the targeting agent component is an antibody or an antigen-binding fragment thereof, the active agent component is conjugated to the targeting agent component, and the oligonucleotide targets a disease gene expressed in muscle tissue.

[0297] Embodiment 18. The method of any one of embodiments 9 or 10, wherein the target protein is encoded by a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0298] Embodiment 19. A method of delivering an imaging agent to muscle tissue in a tissue-specific or tissue-selective manner, comprising contacting a surface of muscle cells of the muscle tissue with an effective amount of an imaging agent comprising an imaging agent component and a targeting agent component, wherein the targeting agent component specifically or selectively binds to a targeted protein expressed on the cell surface of muscle cells of the muscle tissue, wherein the targeted protein is enriched in the muscle tissue relative to other tissues, wherein the targeted protein has stable or increased expression in the diseased tissue relative to an otherwise comparable normal tissue, and optionally wherein the targeted protein is enriched in the diseased tissue relative to an otherwise comparable normal tissue for about 2 minutes to about 12 hours. 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours, thereby delivering the imaging agent to muscle tissue in a tissue-specific or tissue-selective manner.

[0299] Embodiment 20. The method of embodiment 19, wherein the targeted protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0300] Embodiment 21 The method of any one of embodiments 19 or 20, wherein the targeting agent component is a specific or selective binding agent for the targeted protein.

[0301] Embodiment 22. The method of embodiment 21, wherein the specific or selective binding agent is a soluble receptor or a soluble ligand.

[0302] Embodiment 23 The method of embodiment 22, wherein the specific or selective binding agent is a soluble receptor, and the soluble receptor comprises the extracellular domain of the receptor.

[0303] Embodiment 24 The method of any one of embodiments 22 or 23, wherein the soluble receptor is an Fc fusion protein.

[0304] Embodiment 25 The method of any one of embodiments 19 to 21, wherein the targeting agent component is an antibody or an antigen-binding fragment thereof.

[0305] Embodiment 26. The method of embodiment 25, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV).

[0306] Embodiment 27. The method of any one of embodiments 19 to 26, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a genetic vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0307] Embodiment 28. A method of delivering an imaging agent to a tissue sample in a tissue-specific or selective manner, the method comprising contacting the tissue sample with an effective amount of an imaging agent comprising an imaging agent component and a targeting agent component, the targeting agent component specifically or selectively binding to a targeted protein expressed on a muscle cell surface of the tissue, the targeted protein having stable or increased expression in the diseased tissue compared to an otherwise comparable normal tissue, and optionally, the targeted protein is maintained for about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours, thereby delivering the imaging agent to the tissue sample in a tissue-specific or selective manner.

[0308] Embodiment 29. The method of embodiment 28, wherein the targeted protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0309] Embodiment 30 The method of any one of embodiments 28 or 29, wherein the targeting moiety is a specific or selective binding agent for the targeted protein.

[0310] Embodiment 31 The method of embodiment 30, wherein the specific or selective binding agent is a soluble receptor or a soluble ligand.

[0311] Embodiment 32 The method of embodiment 31, wherein the specific or selective binding agent is a soluble receptor, and the soluble receptor comprises the extracellular domain of the receptor.

[0312] Embodiment 33 The method of any one of embodiments 31 or 32, wherein the soluble receptor is an Fc fusion protein.

[0313] Embodiment 34 The method of any one of embodiments 28 to 30, wherein the targeting agent component is an antibody or an antigen-binding fragment thereof.

[0314] Embodiment 35. The method of embodiment 34, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV).

[0315] Embodiment 36. The method of any one of embodiments 28 to 35, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a genetic vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0316]

[0041] Embodiment 37. A method of performing physical imaging of muscle tissue of an individual, the method comprising administering to the individual an effective amount of an imaging agent comprising a targeting agent component and an imaging agent component, wherein the targeting agent component specifically or selectively binds to a targeted protein expressed on the cell surface of the muscle tissue, wherein the targeted protein is enriched in the muscle tissue relative to other tissues, wherein the targeted protein has stable or increased expression in the diseased tissue relative to an otherwise comparable normal tissue, and optionally wherein the targeted protein is enriched in the diseased tissue relative to an otherwise comparable normal tissue, and wherein the targeted protein is enriched in the diseased tissue relative to an otherwise comparable normal tissue, and optionally ... 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours, thereby performing physical imaging of muscle tissue of the individual.

[0317] Embodiment 38. The method of embodiment 37, wherein the targeted protein is selected from and expressed by a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0318] Embodiment 39. The method of any one of embodiments 37 or 38, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0319] Embodiment 40. A method of assessing an individual for the presence or absence of muscle tissue pathology, the method comprising: a) administering to the individual an imaging agent comprising an effective amount of an imaging agent component and a targeting agent component, wherein the targeting agent component specifically or selectively binds to a targeted protein expressed on the cell surface of muscle tissue; b) detecting in the individual the presence or absence of a concentration of the imaging agent by using MRI or other medical imaging, an in vitro diagnostic, or any combination thereof; and c) the presence or absence of the concentration of the imaging agent indicates the presence of pathology, thereby assessing the individual for the presence or absence of muscle tissue pathology.

[0320] Embodiment 41 The method of embodiment 40, wherein the targeting agent component is a specific or selective binding agent for the targeted protein.

[0321] Embodiment 42. The method of embodiment 41, wherein the specific or selective binding agent is a soluble receptor or a soluble ligand.

[0322] Embodiment 43 The method of embodiment 42, wherein the specific or selective binding agent is a soluble receptor comprising the extracellular domain of the receptor.

[0323] Embodiment 44 The method of any one of embodiments 42 or 43, wherein the soluble receptor is an Fc fusion protein.

[0324] Embodiment 45 The method of any one of embodiments 40 or 41, wherein the targeting agent component is an antibody or an antigen-binding fragment thereof.

[0325] Embodiment 46. The method of embodiment 45, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV).

[0326] Embodiment 47. The method of any one of embodiments 40 to 46, wherein the target protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0327] Embodiment 48. The method of any one of embodiments 40 to 47, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

[0328] Embodiment 49. A method of assessing the response of muscle tissue from an individual to treatment with a therapeutic targeting agent, wherein the therapeutic targeting agent specifically binds to a target protein expressed on the cell surface of muscle tissue, the method comprising: a) determining the level of the target protein in a sample obtained from the individual before treatment with the therapeutic targeting agent; b) detecting the level of the target protein in a sample obtained from the individual during or after treatment with the therapeutic targeting agent by using MRI or other medical imaging, in vitro diagnostics, or any combination thereof; c) optionally, using a computer, comparing the pre-treatment level with the mid-treatment or post-treatment level, wherein a lower level of the target protein during or after treatment than the pre-treatment level indicates the effectiveness of treatment with the therapeutic targeting agent; the target protein is enriched in muscle tissue relative to other tissues, the target protein has stable or increased expression in diseased tissue relative to an otherwise comparable normal tissue, and optionally the target protein is expressed for about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 2 hours, and being internalized and reused within about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours, thereby assessing the response of muscle tissue from the individual to treatment with the therapeutic targeting agent.

[0329] Embodiment 50. The method of embodiment 49, wherein the targeted protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

[0330] Embodiment 51. The method of embodiment 9, wherein the pathology comprises a neuromuscular disorder, including a muscular dystrophy or myopathy.

[0331] Embodiment 52. The method of embodiment 51, wherein the pathology is Duchenne muscular dystrophy (DMD), myotonic dystrophy (MD), facioscapulohumeral muscular dystrophy (FSHD), limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, or distal muscular dystrophy.

[0332] Embodiment 53 The method of any one of the preceding embodiments, wherein the individual is a human.

[0333] Embodiment 54. The method of any one of the preceding embodiments, wherein administering comprises orally, by inhalation, intranasally, by injection, subcutaneously, intramuscularly, administering directly to a tissue or cell, administering indirectly to a tissue or cell, intravenously, rectally, intrathecally, intraocularly, auricularly, intraperitoneally, or topically.

[0334] Embodiment 55. The method of any one of the preceding embodiments, wherein the contacting is performed in an individual and the contacting results from administering an agent to the individual.

[0335] Embodiment 56 The method of any one of the preceding embodiments, wherein the evaluating comprises a biopsy.

[0336] Embodiment 57. The method of any one of the preceding embodiments, wherein the agent is administered in the form of a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient, diluent, carrier, or any combination thereof.

[0337] Embodiment 58. The method of any one of the preceding embodiments, wherein the contacting or administering is once daily, twice daily, three times daily, four times daily, weekly, twice weekly, three times weekly, four times weekly, monthly, twice monthly, quarterly, quarterly, twice yearly, annually, on demand, or lifelong.

[0338] Embodiment 59. The method of embodiment 57, wherein the pharmaceutical composition is in a unit dose form.

[0339] Embodiment 60. The method of any one of the preceding embodiments, wherein the agent is administered in an amount ranging from about 1 ng to about 25,000 mg, about 10 ng, about 100 ng, about 1 microgram, about 10 micrograms, about 100 micrograms, about 1 mg, about 10 mg, about 100 mg, about 1000 mg, about 10000 mg, or about 25000 mg.

[0340] Embodiment 61. The method of any one of the preceding embodiments, wherein the agent is optionally in the form of a pharma- ceutically acceptable salt. EXAMPLES

[0341] Example 1: Identification of targets for muscle-specific delivery To identify ideal targets to enable muscle-specific delivery of ADCs, transcript gene expression from human diseased and healthy muscle tissues from 28 data sets from 21 different muscle-related life spans was performed, which are listed in Table 2 below.

[0342] [Table 5]

[0343] An overview of this analysis is shown in Figure 1. First, genes with stable or increased expression in diseased muscle tissue compared to control muscle tissue were identified using statistical methods from the dataset and each study. This data was overlaid to identify approximately 17,000 genes that remained stably expressed across all muscle disorders analyzed. Next, RNA expression data from the GTEx (https: / / gtexportal.org / home / ) and FANTOM5 (PMID:24670764) databases were utilized to identify genes significantly enriched in muscle tissue (skeletal, smooth, cardiac and tongue muscles) versus non-muscle tissue. The list was filtered to only stably expressed disease genes, resulting in the identification of approximately 600 genes. To identify genes suitable for antibody targeting, the Gene Ontology database (PMID:10802651) was used to identify genes listed as extracellular and associated with the outer cell membrane, resulting in a list of approximately 120 genes.

[0344] Finally, the RNA expression data was validated at the protein expression level using the Human Protein Atlas (PMID: 25613900), whereby both protein and RNA expression data showed extracellular expression and muscle specificity. This data was filtered for genes that were highly expressed at the protein level, i.e., genes expressed at expression levels 2 or 3 (medium to high, if available), according to the immunohistochemistry scoring data available in the Human Protein Atlas. This resulted in a list of 19 genes, as shown in Table 3 below.

[0345] [Table 6]

[0346] As a validation of the robustness of this approach, we note that previous studies have suggested that SLC2A4 (GLUT4) is a good target for muscle-specific delivery. From this list, we selected ART-1, CDH15, MUSK, CACNA1S, and SLC2A4 for further validation.

[0347] Example 2: Validation of muscle expression and antibody accessibility. Figure 2 shows fluorescence microscopy validation of selected receptor expression in differentiated myotubes. Differentiated human myotubes were fixed with 4% PFA and blocked with PBS+5% serum+1% BSA for 1 hour at room temperature. Cells were stained with primary antibodies against A-ALK2, B-GLUT4, C-CDH15, D-MuSK, E-ART1, F-CHRND, GN / A (secondary antibody only and no primary antibody as negative control) and H-MYHC4 at 5ug / mL in blocking solution for 1 hour at room temperature or overnight at 4°C, then washed 3 times with PBS+0.1% tween20. Cells were then co-stained with Hoechst 33342 and AF647-conjugated secondary antibodies for 1 hour at room temperature. After washing 3 times with PBS+0.1% tween20, cells were imaged to detect cells in DAPI and Cy5 channels (shown).

[0348] Figure 3 shows fluorescence microscopy validation of receptor expression in differentiated mouse myotubes. Differentiated C2C12 myotubes were fixed with 4% PFA and blocked with PBS+5% serum+1% BSA for 1 hour at room temperature. Cells were stained with primary antibodies against A-GLUT4, B-CDH15, C-MuSK, D-ART1, E-CHRND, F-CACNA1S, and GN / A (secondary antibody only and no primary antibody as negative control) at 5ug / mL in blocking solution for 1 hour at room temperature or overnight at 4°C, then washed 3 times with PBS+0.1% tween20. Next, cells were co-stained with Hoechst 33342 and AF647-conjugated secondary antibodies for 1 hour at room temperature. After washing 3 times with PBS+0.1% tween20, cells were imaged to detect cells in DAPI and Cy5 channels (shown). Panel H is a representative image of the same treatment as panel G, but in the DAPI channel to show stained nuclei.

[0349] Figures 4A-4B show fluorescence microscopy of low / no expression of receptors in non-muscle cells. Figure 4A shows HuVEC cells fixed with 4% PFA and blocked with PBS+5% serum+1% BSA for 1 hour at room temperature. Cells were stained with primary antibodies against A-ALK2, B-CHRND, C-GLUT4, D-CDH15, E-MuSK, F-ART1, and GN / A at 2ug / mL in blocking solution for 1 hour at room temperature or overnight at 4°C, then washed 3 times with PBS+0.1% tween20. Cells were then co-stained with Hoechst 33342 and AF647-conjugated secondary antibodies for 1 hour at room temperature. After washing 3 times with PBS+0.1% tween20, cells were imaged for detection on DAPI and Cy5 (shown) channels. Panel H represents the same treatment as panel G, but in the DAPI channel to show stained nuclei.

[0350] FIG. 4B shows HepG2 cells fixed with 4% PFA and blocked with PBS+5% serum+1% BSA for 1 hour at room temperature. Cells were stained with primary antibodies against A-ALK2, B-CHRND, C-GLUT4, D-CDH15, E-MuSK, F-ART1, GN / A at 2ug / mL in blocking solution for 1 hour at room temperature or overnight at 4°C, then washed 3 times with PBS+0.1% tween 20. Cells were then co-stained with Hoechst 33342 and AF647-conjugated secondary antibodies for 1 hour at room temperature. After washing 3 times with PBS+0.1% tween 20, cells were imaged for detection on DAPI and Cy5 (shown) channels. Panel H represents the same treatment as panel G, but in the DAPI channel to show stained nuclei.

[0351] Example 3: Internalization and delivery of conjugated antibodies to muscle cells. Antibody internalization can be observed by labeling the antibody with a pH-sensitive fluorophore such as pHrodo. pHrodo is non-fluorescent at neutral pH when present in the cell culture medium or when bound to an antigen on the cell surface. After cellular internalization into endosomal or lysosomal compartments where a lower pH is present, pHrodo fluoresces in the red spectrum (RFP channel). Thus, cells are treated with labeled antibodies and the fluorescence can be observed and quantified over time by fluorescence microscopy or by measuring fluorescence intensity. This data can be used to determine the time required for receptor internalization after antibody binding. To measure receptor recycling after treatment with fluorescent antibodies, free antibody is removed, cells are washed, and a reagent to inhibit protein synthesis and degradation is added. Fluorescence intensity over time can then be measured to calculate receptor recycling. Figures 5A-5B show examples of fluorescence microscopy of receptor-mediated antibody internalization for muscle-specific receptors. Differentiated human myotubes were treated with antibodies against muscle-expressed receptors labeled with pHrodo dye. Cells were incubated with pHrodo antibody (5ug / mL) on ice for 1 hour and then washed with PBS to remove unbound antibody. Cells were then incubated at 37°C to allow internalization and imaged after another hour. Internalized antibody was detected in the RFP channel (illustrated in 5A) and scored by visual inspection of several wells for +low internalization, ++moderate internalization, and +++high internalization. (A-ALK2+++, B-GLUT4++, C-CDH15+++, D-MuSK+) Transferrin-pHrodo+++ (panel E) and secondary Ab-pHrodo (panel F) were included as positive and negative controls, respectively. Figure 5B shows cell nuclei from the same wells as in Figure 5A (stained with Hoechst 33342) detected in the DAPI channel (blue).

[0352] Extensive mining of RNA and protein expression profiles from healthy and diseased tissues has revealed a number of muscle-selective surface antigens whose expression remains stable with disease progression. The surface receptors described herein were selected based on the following criteria: ·Cell membrane localization; · Accessibility via the extracellular space; Expression in muscle tissue; Selective / enriched expression in muscle compared to other tissue types; and · Time required for internalization and recycling to the cell surface.

[0353] Surface antigens described in this disclosure include ALK2, KLHL41, SLC2A4, FABP3, LMOD2, CDH15, MUSK, ART1, CACNA1S, CLCN1, ENO3, KLHL41, ACTA1, MYMX, CHARNA1, SEMA6C, XIRP1, XIRP2, CFL2, SVIL, and CAVIN4.

[0354] Previous attempts to use neutralizing monoclonal antibodies to treat DMD did not result in an approved therapy (domagrozumab / anti-myostatin / Pfizer). Structural and bioinformatics analysis of the described receptors allowed the identification of sequences useful for the development of antibodies. The developed antibodies were selected based on their ability to bind to surface antigens and deliver therapeutic agents to skeletal muscle without adversely affecting endogenous receptor function.

[0355] While exemplary embodiments have been shown and described herein, such embodiments are merely exemplary. Numerous variations, changes, and substitutions may be made to the exemplary embodiments. It is to be understood that various alternatives to the embodiments described herein may be used.

Claims

1. 1. An agent that specifically or selectively binds to a target protein expressed on the cell surface of muscle cells in muscle tissue for use in delivering an agent to said muscle tissue in a tissue-specific or tissue-selective manner, comprising: the surface of the muscle cell is contacted with an effective amount of the agent; the target protein is enriched in muscle tissue relative to other tissues; the target protein has stable or increased expression in diseased tissue compared to otherwise identical normal tissue; Optionally, the target protein is internalized and recycled within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

2. the target protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1; or the drug is a specific or selective binding agent for the target protein; Preferably, the specific or selective binding agent is a soluble receptor or a soluble ligand; More preferably, said specific or selective binding agent is said soluble receptor, said soluble receptor comprising the extracellular domain of the receptor; or The method of claim 1, wherein the soluble receptor is an Fc fusion protein.

3. the agent is an antibody or an antigen-binding fragment thereof; Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody, 2. The agent for use according to claim 1, wherein the agent is selected from the group consisting of a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV).

4. 1. A therapeutic targeting agent that specifically or selectively binds to a target protein expressed on the surface of muscle tissue cells for use in treating a pathology in an individual, comprising: a therapeutically effective amount of the agent is administered to the individual; the target protein is enriched in muscle tissue relative to other tissues; the target protein has stable or increased expression in diseased tissue compared to otherwise identical normal tissue; Optionally, the therapeutic targeting agent wherein the targeted protein is internalized and recycled within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

5. the target protein is encoded by a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1; or the therapeutic targeting agent is a drug comprising an active agent component and a targeting agent component; the active agent component is selected from the group consisting of a radionuclide, a chemotherapeutic agent, an immunostimulant, an anti-tumor agent, an anti-inflammatory agent, a pro-inflammatory agent, a pro-apoptotic agent, a pro-coagulant, a toxin, an antibiotic, a hormone, an enzyme, a protein, a carrier protein, a lytic agent, a small molecule, an aptamer, a cell, a vaccine-induced cell or other immune cell, a nanoparticle, transferrin, an immunoglobulin, a polyvalent antibody, a lipid, a lipoprotein, a liposome, a modified natural ligand, a gene or nucleic acid, an oligonucleotide, RNA, siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a miximer, a double-stranded RNA (dsRNA), a single-stranded RNAi (ssRNAi), a DNA-dependent RNA interference (ddRNAi), an RNA activating oligonucleotide (RNAa), an aptamer, an exon-skipping oligonucleotide, a miRNA, a miRNA mimic, an mRNA, a guide RNA, a viral or non-viral gene delivery vector, a prodrug, and a promolecule; the targeting agent component specifically or selectively binds to the target protein; Preferably, the targeting agent component comprises a specific or selective binding agent for the target protein; More preferably, the specific or selective binding agent is a soluble receptor or a soluble ligand; Even more preferably, the specific or selective binding agent is a soluble receptor, wherein the soluble receptor comprises the extracellular domain of the receptor; or the soluble receptor is an Fc fusion protein or a biologically active fragment thereof; or the targeting agent component comprises an antibody or antigen-binding fragment thereof; Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a diabody, a Fab, a Fab-Fc, a Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV), or the active agent component is an oligonucleotide or polynucleotide; the targeting agent component is an antibody or an antigen-binding fragment thereof; the active agent component is conjugated to the targeting agent component; the oligonucleotide targets a disease gene expressed in muscle tissue; or the pathology comprises a neuromuscular disorder, including a muscular dystrophy or myopathy, 5. The therapeutic targeting agent of claim 4, wherein the pathology is Duchenne muscular dystrophy (DMD), myotonic dystrophy (MD), facioscapulohumeral muscular dystrophy (FSHD), limb gland muscular dystrophy (LGMD), Becker muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, or distal muscular dystrophy.

6. 6. The therapeutic targeting agent of claim 5, wherein the active agent component is an oligonucleotide or polynucleotide that targets DUX4, and optionally the polynucleotide is an antisense oligonucleotide (ASO), and / or the neuromuscular disorder is facioscapulohumeral muscular dystrophy (FSHD).

7. 1. An imaging agent comprising an imaging agent component and a targeting agent component for use in delivering the imaging agent component to muscle tissue or a tissue sample in a tissue-specific or tissue-selective manner, comprising: the surface of muscle cells in said tissue or tissue sample is contacted with an effective amount of said imaging agent; the targeting agent component specifically or selectively binds to a target protein expressed on the cell surface of muscle cells in the tissue; the target protein is enriched in muscle tissue relative to other tissues; the target protein has stable or increased expression in diseased tissue compared to otherwise identical normal tissue; Optionally, the target protein is internalized and recycled within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

8. the target protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1; or the targeting agent component is a specific or selective binding agent for the target protein; Preferably, the specific or selective binding agent is a soluble receptor or a soluble ligand; More preferably, the specific or selective binding agent is a soluble receptor, wherein the soluble receptor comprises the extracellular domain of the receptor; or the soluble receptor is an Fc fusion protein; or the targeting agent component is an antibody or an antigen-binding fragment thereof; Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a diabody, a Fab, a Fab-Fc, a Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV), or The imaging agent component may be a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or a virus that induces a detection agent.

8. The imaging agent for use according to claim 7, wherein the imaging agent is selected from the group consisting of a dye, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

9. 1. An imaging agent for use in performing physical imaging of muscle tissue in an individual, comprising: the imaging agent comprises a targeting agent moiety and an imaging agent moiety; the targeting agent component specifically or selectively binds to a target protein expressed on the cell surface of the muscle tissue; the imaging agent moiety is administered to the individual in an effective amount; the target protein is enriched in muscle tissue relative to other tissues; the target protein has stable or increased expression in diseased tissue compared to otherwise identical normal tissue; Optionally, the target protein is internalized and recycled within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

10. the target protein is selected from and expressed by a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1; and / or 10. The imaging agent for use according to claim 9, wherein the imaging agent moiety is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

11. 1. An imaging agent for use in assessing an individual for the presence or absence of muscle tissue pathology, comprising: a) administering to the individual an effective amount of an imaging agent comprising an imaging agent component and a targeting agent component, wherein the targeting agent component specifically or selectively binds to a target protein expressed on the cell surface of the muscle tissue; b) the presence or absence of a concentration of said imaging agent is detected in said individual using MRI or other medical imaging, in vitro diagnostics, or any combination thereof; An imaging agent, the presence or absence of a concentration of said imaging agent being indicative of the presence of said pathology.

12. the targeting agent component is a specific or selective binding agent for the target protein; Preferably, the specific or selective binding agent is a soluble receptor or a soluble ligand; More preferably, said specific or selective binding agent is said soluble receptor comprising the extracellular domain of the receptor, or the soluble receptor is an Fc fusion protein; or the targeting agent component is an antibody or an antigen-binding fragment thereof; Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, a VNAR, and an immunoglobulin single variable domain (ISV). selected and / or the target protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1; and / or 12. The imaging agent for use according to claim 11, wherein the imaging agent is selected from the group consisting of a radiopharmaceutical, a radioisotope or radiopharmaceutical, a contrast agent, a magnetic or paramagnetic agent, a liposome, an ultrasound agent, a gene vector or virus directing a detection agent, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material.

13. 1. A therapeutic targeting agent for use in assessing the response of muscle tissue from an individual to treatment with the therapeutic targeting agent, comprising: the therapeutic targeting agent specifically binds to a target protein expressed on the cell surface of the muscle tissue; a) the level of the target protein is determined in a sample obtained from the individual prior to treatment with the therapeutic targeting agent; b) detecting the level of the target protein in a sample obtained from the individual during or after treatment with the therapeutic targeting agent by using MRI or other medical imaging, in vitro diagnostics, or any combination thereof; c) optionally, said levels before treatment and said levels during or after treatment are compared using a computer; a level of the target protein during or after treatment that is lower than the level of the target protein before treatment indicates efficacy of treatment with the therapeutic targeting agent; the target protein is enriched in muscle tissue relative to other tissues; the target protein has stable or increased expression in diseased tissue compared to otherwise identical normal tissue; Optionally, the therapeutic targeting agent wherein the targeted protein is internalized and recycled within about 2 minutes to about 12 hours, about 2 minutes to about 10 hours, about 2 minutes to about 8 hours, about 2 minutes to about 6 hours, about 2 minutes to about 4 hours, about 2 minutes to about 2 hours, about 2 minutes to about 60 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 12 hours, about 20 minutes to about 12 hours, about 40 minutes to about 12 hours, about 60 minutes to about 12 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, about 10 hours to about 12 hours, or about 11 hours to about 12 hours.

14. 14. The therapeutic targeting agent for use according to claim 13, wherein the targeting protein is expressed from a gene selected from the group consisting of KLHL41, LMOD2, ENO3, FABP3, CHRNA1, SEMA6C, XIRP2, XIRP1, CAVIN4, CFL2, SVIL, MUSK, ART1, CACNA1S, CDH15, CLCN1, MYMX, and ACTA1.

15. the individual is a human, and / or the drug, imaging agent or therapeutic targeting agent is administered orally, by inhalation, intranasally, by injection, subcutaneously, intramuscularly, directly to a tissue or cell, indirectly to a tissue or cell, intravenously, intrarectally, intrathecally, intraocularly, otically, intraperitoneally or topically; and / or said contacting is performed on an individual, said contacting resulting from administering an agent to said individual; and / or The agent, imaging agent or therapeutic targeting agent for use according to any one of claims 1 to 14, wherein said assessing comprises a biopsy.

16. The agent may be administered in a pharmaceutically acceptable excipient, diluent, carrier, or any combination thereof. and / or the contacting or administering is once daily, twice daily, three times daily, four times daily, weekly, twice weekly, three times weekly, four times weekly, monthly, twice monthly, quarterly, quarterly, twice yearly, annually, on demand, or lifelong; and / or the pharmaceutical composition is in unit dose form, and / or the agent is administered in an amount ranging from about 1 ng to about 25,000 mg, about 10 ng, about 100 ng, about 1 microgram, about 10 micrograms, about 100 micrograms, about 1 mg, about 10 mg, about 100 mg, about 1000 mg, about 10000 mg, or about 25000 mg; and / or 16. The agent, imaging agent or therapeutic targeting agent for use according to any one of claims 1 to 15, wherein the agent is optionally in the form of a pharmaceutically acceptable salt.

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