Polymyxins for delivering an agent to the kidney

Abstract

Disclosed herein are conjugate agents comprising a targeting moiety (in certain embodiments, a polymyxin or related compound) conjugated directly or indirectly to a payload moiety, compositions comprising the conjugate agents, and methods of making and using the conjugate agents.

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 585,806, filed September 27, 2023, and U.S. Provisional Patent Application No. 63 / 655,321, filed June 3, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure generally relates to the molecular targeting of renal cells by compositions and related methods. Background Technology

[0003] Regulatory nucleic acid therapy has shown significant clinical and commercial success in treating liver diseases, at least in part because the fenestrations in hepatic sinusoidal epithelial cells make these cells particularly vulnerable to the utilization of injected nucleic acids, especially those carrying N-acetylgalactosamine (GalNAc) modifications. Non-hepatic tissues (including the kidneys) are often less readily utilized by regulatory nucleic acid therapies. However, recent findings describe the successful use of portions targeting renal cell surface factors to facilitate the efficient delivery of partially linked regulatory nucleic acids to renal cells (see, for example, PCT / US23 / 16319).

[0004] There is a need for extended-range renal cell-targeting agents capable of delivering payload portions (e.g., regulatory nucleic acids) to renal cells. Summary of the Invention

[0005] This disclosure provides, in particular, techniques for the targeted delivery of therapeutic agents and / or nucleic acid agents, especially to renal cells, via compounds comprising polymyxin compounds and other compounds believed to interact with megalin cell surface receptors. In some embodiments, the provided compositions and techniques achieve delivery by targeting cell surface factors (e.g., cell surface receptors) that are internalized upon binding to the targeted portion (e.g., the megalin targeting portion). In some embodiments, the targeted delivery according to this disclosure (e.g., megalin-targeted delivery) may be directed against renal cells. In some embodiments, this disclosure provides techniques particularly suitable for delivery to, for example, proximal tubular epithelial cells, podocytes, and / or renal cysts (e.g., in polycystic kidney disease).

[0006] This disclosure particularly recognizes that some of the challenges typically associated with targeted delivery (e.g., macroprotein targeted delivery) are inefficient and / or insufficiently specific delivery; unwanted off-target effects; or effects in cells or tissues that do not represent the intended site of action, which can be particularly problematic.

[0007] This disclosure specifically provides conjugated pharmaceutical agents comprising a polymyxin (e.g., polymyxin B, polymyxin E (colistin), etc.) or a polymyxin-related compound as a targeting moiety (e.g., a macroprotein targeting moiety); directly or indirectly conjugated to a payload moiety comprising nucleic acids. It is not intended to be theoretically feasible for the targeting moiety, as described herein, to specifically bind to factors present on the surface of target cells of interest (e.g., kidney-related cells). In some embodiments, the provided techniques enable targeted delivery of the nucleic acid payload moiety to target cells, tissues, organs, or organisms of interest, e.g., with minimal off-target effects. In some embodiments, the targeting moiety (e.g., a macroprotein targeting moiety) as described herein specifically binds to factors preferentially present on the surface of target cells or tissues of interest (e.g., relative to one or more non-target cells or tissues). In some embodiments, the targeting moiety (e.g., a polymyxin or a polymyxin-related compound as the targeting moiety) as described herein specifically binds to factors specific to target cells or tissues of interest.

[0008] This disclosure provides, in particular, the insight that targeting macroproteins with polymyxin (or polymyxin-related compounds) presenting conjugates represents a particularly useful strategy for delivering certain nucleic acid agents into cells. This disclosure provides a particular insight that targeting macroproteins represents a particularly useful strategy for delivering certain agents, and especially nucleic acid agents, into kidney-related cells (e.g., renal cells).

[0009] Furthermore, this specification specifically teaches that conjugated pharmaceutical agents, as described herein, comprising a macroprotein-binding moiety conjugated (optionally via a linker) to a nucleic acid agent, are particularly useful for delivering such nucleic acid agents to cells expressing macroproteins. This specification specifically identifies the use of such conjugated pharmaceutical agents in delivering nucleic acid agents to kidney cells.

[0010] In some embodiments, the conjugated agents disclosed herein are characterized, for example, when they are provided to an associated system (e.g., comprising one or more cells, tissues, organs, or organisms), they affect the expression and / or activity of one or more targets or their forms significantly more than when the system is in contact with an unconjugated nucleic acid payload under other comparable conditions.

[0011] In one aspect, this disclosure provides a pharmaceutical agent comprising a nucleic acid conjugated to a polymyxin moiety or analogue, wherein the nucleic acid is conjugated to the polymyxin moiety or analogue via an adapter.

[0012] In some implementations, the connector includes C 2-22 An alkylene or branched alkylene chain, wherein the carbon atoms of the alkylene chain are optionally interrupted by one or more -O-.

[0013] In some embodiments, the connector comprises one or more -(OCH2CH2)- units. Optionally, the connector comprises at least two -(OCH2CH2)- units.

[0014] In some embodiments, the agent is characterized by enhanced delivery of nucleic acids to cells, tissues, or subjects, compared to a comparative agent, when delivered to them. Optionally, the comparative agent is a similar cell, tissue, or subject delivering unconjugated nucleic acids. In one related embodiment, the enhanced delivery of nucleic acids is mediated by a polymyxin moiety or analogue.

[0015] In one embodiment, the agent is characterized in that, upon delivery to cells, tissues, or subjects, the level of the target gene is reduced in said cells, tissues, or subjects compared to a comparative agent. In a related embodiment, the comparative agent is a similar cell, tissue, or subject delivered with unconjugated nucleic acid. In some embodiments, the reduction in target gene levels is mediated by nucleic acid.

[0016] On the other hand, this disclosure provides a conjugate agent for regulating the expression of a target gene, comprising (i) a payload containing nucleic acid conjugated to (ii) a structure of formula I: Wherein R1 is an optionally substituted C3-C7 alkyl or optionally substituted C3-C7 aryl. L is Or a branch link header; n is an integer from 1 to 16; and AA1, AA2, and AA3 are each independently chemical bonds or selected from the following amino acids: , Where X is: Where L is an optional connector, and Where M is: , Where Y = O, S or NSO2Me, and Z = nucleic acid.

[0017] In one implementation scheme, AA2 is or .

[0018] In another implementation, n is an integer from 5 to 12.

[0019] In some implementations, R1 is or .

[0020] In some implementations, the structure of Formula I is selected from the following: , , , ,as well as .

[0021] In one embodiment, the conjugate comprises a plurality of Formula I structures. Optionally, the nucleic acid conjugate agent comprises at least two, at least three, at least four, or at least five Formula I structures. In one embodiment, the plurality of Formula I structures are conjugated to the payload portion via a branch linker (L). Optionally, the branch linker L has a structure selected from: as well as , Where Comp1, Comp2, and / or Comp3 independently contain compounds of formula I': .

[0022] In one implementation, the nucleic acid conjugate comprises two structures of formula I'.

[0023] In another embodiment, the nucleic acid conjugate comprises three I' structures.

[0024] In some implementations, the nucleic acid conjugate consists of two, three, four, or five structures of Formula I' conjugated to the nucleic acid payload via one or more adapters (optionally branched).

[0025] In one implementation, the nucleic acid is conjugated with a ligand selected from the following: , , , ,as well as .

[0026] In some implementations, the nucleic acid is or contains an antisense sequence element. Optionally, the antisense sequence element is complementary to at least a portion of one or more of the following in the target sequence: exons, introns, untranslated regions, splice sites, promoter regions, enhancer regions, or non-coding regions.

[0027] In some implementations, the nucleic acid is or contains a sense sequence element. Optionally, the sense sequence element is substantially similar (e.g., at least 80% identical) to at least a portion of one or more of the following in the target sequence: exons, introns, untranslated regions, splice sites, promoter regions, enhancer regions, or non-coding regions.

[0028] In one implementation, the nucleic acid contains sequence elements that are at least 80% complementary to the target sequence in the sense strand.

[0029] In another implementation, the nucleic acid contains sequence elements that are at least 80% complementary to the target sequence in the antisense strand.

[0030] In some embodiments, the nucleic acid comprises at least one sequence element having at least three consecutive nucleotides that are at least 80% complementary to a portion of the target sequence.

[0031] In one implementation, the nucleic acid is single-stranded.

[0032] In another implementation, the nucleic acid is double-stranded.

[0033] In some implementations, nucleic acids are or contain RNA.

[0034] In some implementations, the nucleic acid is an inhibitory RNA agent (RNAi). Optionally, the RNAi is or contains short interfering RNA (siRNA).

[0035] In one implementation, the nucleic acid comprises an oligonucleotide chain of approximately 15-25 nucleotides in length.

[0036] In one implementation, the nucleic acid comprises one or more modified nucleotides.

[0037] In another embodiment, the nucleic acid is or comprises DNA. Optionally, the DNA is or comprises a DNA analog. Optionally, the DNA analog comprises one or more morpholino subunits linked together by phosphorus-containing bonds. In some embodiments, the DNA analog is or comprises phosphoryldiamine morpholino nucleic acid (PMO). In some embodiments, the PMO comprises about 12-40 nucleotides.

[0038] In some implementations, the nucleic acid is or contains antisense oligonucleotides (ASO).

[0039] In another implementation, the nucleic acid is or contains peptide nucleic acid (PNA).

[0040] In some implementations, the nucleic acid includes one or more of the following modifications: a modified backbone, a modified nucleobase, a modified ribose, a modified deoxyribose, or a combination thereof.

[0041] In some implementations, the nucleic acid includes one or more modifications to the 5' end of the nucleic acid.

[0042] In one embodiment, the effective load portion is attached to the remainder of the compound of formula I at either the 5' end or the 3' end of the effective load portion.

[0043] In another embodiment, the nucleic acid of the payload portion includes one or more extended nucleic acid (“exNA”) modifications. Optionally, the one or more exNA modifications are located at or near the 3' end of the nucleic acid of the payload portion.

[0044] In some implementations, the nucleic acid of the payload portion comprises one or more phosphoryl guanidine backbones (“PN backbones”) and / or methanesulfonyl aminophosphate modifications.

[0045] Another aspect of this disclosure provides a pharmaceutical composition comprising the conjugate pharmaceutical agent of this disclosure and a pharmaceutically acceptable carrier.

[0046] Another aspect of this disclosure provides a cell comprising a conjugate pharmaceutical agent of this disclosure bound thereto.

[0047] In one embodiment, the cell is a kidney cell. Optionally, the cell is a subject's kidney cell. Optionally, the cell is an in vivo kidney cell. In some embodiments, the cell may be a mammalian cell, such as a human cell. In some embodiments, the cell expresses a macroprotein. In other embodiments, the cell expresses cubilin. In still other embodiments, the cell expresses both macroprotein and cubilin. In a particular embodiment, the cell is a human cell expressing macroprotein and / or cubilin.

[0048] Another aspect of this disclosure provides a method for delivering a conjugated pharmaceutical agent to cells, tissues, or a subject, the method involving administering the conjugated pharmaceutical agent, pharmaceutical composition, and / or cells of the present disclosure to cells, tissues, or a subject, thereby delivering the conjugated pharmaceutical agent.

[0049] Another aspect of this disclosure provides a method for regulating the expression of target genes in cells, the method involving providing cells with conjugated pharmaceutical agents, pharmaceutical compositions and / or cells of this disclosure.

[0050] Another aspect of this disclosure provides a method for treating a subject suffering from or susceptible to a disease or condition, the method involving administering to the subject a conjugate pharmaceutical agent, pharmaceutical composition, and / or cells of this disclosure.

[0051] In one implementation, the disease is a disease associated with the expression of cell surface receptors. Optionally, the disease is a disease comprising cells in which both cell surface receptors and targets recognized by the payload portion are present.

[0052] Another aspect of this disclosure provides a method for improving the delivery of a pharmaceutical agent to cells, the method involving contacting a system or subject having at least one cell with the conjugated pharmaceutical agent, pharmaceutical composition and / or cells of this disclosure.

[0053] In one embodiment, the cells are selected from: kidney cells, thyroid cells, parathyroid cells, inner ear cells, or nervous system cells, or combinations thereof. Optionally, the kidney cells are selected from proximal tubular epithelial cells and / or podocytes.

[0054] In some embodiments, this disclosure provides for administering the conjugated pharmaceutical agent of this disclosure to cells, tissues, or subjects to deliver a payload portion to at least 5% more target cells than: (a) other similar cells, tissues, or subjects to which an unconjugated payload portion is delivered; (b) non-target cells; or (c) both (a) and (b).

[0055] In some implementations, the target cells are or include kidney cells.

[0056] In another embodiment, the target cell is or contains a cell expressing a renal cell surface factor. Optionally, the renal cell surface factor is a macroprotein or a cuboprotein.

[0057] In one embodiment, the nucleic acid payload is an antisense compound having an antisense strand as the first strand. In one embodiment, the antisense compound comprises a second strand of 15 to 60 nucleotides in length and complementary to the first strand.

[0058] In some embodiments, the compound is an antisense oligonucleotide (ASO).

[0059] In one embodiment, the payload regulatory nucleic acid compound of this disclosure (e.g., antisense compound, dsRNA, etc.) has at least one modified nucleoside internucleotide bond, sugar moiety, or nucleobase. Optionally, the modified nucleoside internucleotide bond is or contains a phosphate thioester bond.

[0060] In some embodiments, at least one modified nucleoside interbond, sugar moiety, or nucleobase comprises one or more of the following modifications: deoxynucleoside, 3'-terminal deoxythymidine (dT) nucleoside, 2'-O-methyl (2'-OMe) modified nucleoside, 2'-fluoro (2'-F) modified nucleoside, 2'-deoxy-modified nucleoside, locked nucleotide (LNA), unlocked nucleoside, conformation-restricted nucleoside, restricted ethyl nucleoside, debaseted nucleoside, 2'-O,4'-C-ethylidene bridged nucleic acid (ENA), 2'-amino modified nucleoside, 2'-O-allyl modified nucleoside, 2'-C-alkyl Modified nucleosides, 2'-hydroxy modified nucleosides, 2'-methoxyethyl (2'-MOE) modified nucleosides, 2'-O-alkyl modified nucleosides, morpholino nucleosides, aminophosphates, non-natural bases containing nucleosides, tetrahydropyran modified nucleosides, 1,5-dehydrohexanol modified nucleosides, cyclohexenyl modified nucleosides, nucleotides containing 5'-thiophosphate groups, nucleotides containing 5'-methylphosphonate groups, nucleotides containing 5'-phosphates or 5'-phosphate mimics, nucleotides containing vinylphosphonates, nucleosides containing adenosine diol nucleic acids (GNA), nucleosides containing thymidine diol nucleic acids (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleosides linked to cholesterol derivatives and dodecanoic acid bisdecamide groups; and combinations thereof.

[0061] In some embodiments, the modified nucleobase is or contains 5-methylcytosine.

[0062] In some embodiments, the double-stranded nucleic acid payload of this disclosure has a double-stranded region of 19-30 nucleotide pairs in length; a double-stranded region of 19-25 nucleotide pairs in length; a double-stranded region of 19-23 nucleotide pairs in length; each strand independently having a length not exceeding 30 nucleotides; the sense strand having a length of 21 nucleotides and the antisense strand having a length of 23 nucleotides; and / or the complementary region having a length of at least 17 nucleotides. Optionally, the complementary region has a length between 19 and 23 nucleotides. Optionally, the complementary region has a length of 19 nucleotides; and / or at least one strand contains a 3' overhang of at least 2 nucleotides.

[0063] In one embodiment, the payload nucleic acid of this disclosure is or contains exon-skipping antisense oligonucleotides.

[0064] In one embodiment, the nucleic acid payload of this disclosure (e.g., antisense compound, RNAi compound, exon-skipping antisense oligonucleotide, etc.) has at least one modified nucleoside internucleotide, sugar moiety, or nucleobase. Optionally, the modified nucleoside internucleotide comprises a phosphate thioester bond. In some embodiments, at least one modified nucleoside internucleotide, sugar moiety, or nucleobase comprises a modification selected from: deoxynucleosides, 3'-terminal deoxythymidine (dT) nucleosides, 2'-O-methyl (2'-OMe) modified nucleosides, 2'-fluoro (2'-F) modified nucleosides, 2'-deoxy-modified nucleosides, locked nucleotides (LNA), unlocked nucleosides, conformationally restricted nucleosides, restricted ethyl nucleosides, debaseted nucleosides, 2'-O,4'-C-ethylidene bridged nucleic acids (ENA), 2'-amino modified nucleosides, 2'-O-allyl modified nucleosides, 2'-C-alkyl modified nucleosides, etc. Nucleosides containing 2'-hydroxyl, 2'-methoxyethyl (2'-MOE), 2'-O-alkyl, morpholino, aminophosphate, non-natural bases containing nucleosides, tetrahydropyran-modified nucleosides, 1,5-dehydrohexanol-modified nucleosides, cyclohexenyl-modified nucleosides, nucleotides containing 5'-thiophosphate groups, nucleotides containing 5'-methylphosphonate groups, nucleotides containing 5'-phosphates or 5'-phosphate mimics, nucleotides containing vinylphosphonates, nucleosides containing adenosine diol nucleic acid (GNA), nucleosides containing the S-isomer of thymidine diol nucleic acid (GNA), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleosides linked to cholesterol derivatives and dodecanoic acid bisdecamide groups; and combinations thereof. Optionally, the nucleic acid payload (e.g., an antisense compound, a dsRNA agent, an exon-jumping antisense oligonucleotide, etc.) comprises one or more 2'-methoxyethyl (2'-MOE) modified nucleosides. In some embodiments, all nucleosides in the nucleic acid payload are 2'-methoxyethyl (2'-MOE) modified nucleosides.

[0065] In some embodiments, the nucleic acid payload comprises at least one modified nucleotide. Optionally, substantially all nucleotides of the sense strand of the nucleic acid payload; or substantially all nucleotides of the antisense strand of the antisense compound of the nucleic acid payload contain a modification; or substantially all nucleotides of both the sense and antisense strands of the nucleic acid payload contain a modification.

[0066] In some embodiments, all nucleotides of the sense strand of the dsRNA agent nucleic acid payload contain modifications; all nucleotides of the antisense strand of the antisense compound of this disclosure or other nucleic acid payload contain modifications; or all nucleotides of both the sense strand and the antisense strand of the nucleic acid payload contain modifications.

[0067] In one embodiment, at least one modified nucleotide is a deoxynucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluorine modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-restricted nucleotide, a restricted ethyl nucleotide, a debased nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, or a morpholino-modified nucleotide. Nucleotides (e.g., phosphoryldiamine morpholinonucleotide (PMO)), aminophosphates, non-natural bases containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-dehydrohexanol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing a phosphoryl guanidine-based backbone, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, heat-destabilized nucleotides, diol-modified nucleotides (GNA) and / or 2-O-(N-methylacetamide)-modified nucleotides; and / or combinations thereof.

[0068] In some embodiments, the modification on the nucleotide is selected from the following: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, 2'-hydroxy, and ethylene glycol; and combinations thereof; C7-modified denitro-adenine, C7-modified denitro-guanosine, C5-modified cytosine, C5-modified uridine, N1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, 5-methoxy Cytidine or combinations thereof; phosphate thioester (PS) modification, phosphoryl guanidine (PN) modification, borophosphate modification, alkyl phosphonate nucleic acid (phNA), peptide nucleic acid (PNA) or combinations thereof; deoxyribonucleic acid (DNA), optionally wherein the DNA is or contains a DNA analog, optionally wherein the DNA analog contains one or more morpholino subunits linked together by phosphorus-containing bonds, optionally wherein the DNA analog is or contains phosphorylated dimorpholino nucleic acid (PMO), optionally wherein the PMO contains about 12-40 nucleotides; peptide nucleic acid (PNA) modification; and / or one or more modifications at the 5' end of an antisense compound or dsRNA agent, optionally wherein the 5' end modification is a 5' amino modification.

[0069] In some embodiments, at least one modified nucleotide is selected from the following: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, diol modified nucleotides (GNA) and / or vinylphosphonate nucleotides; and / or combinations thereof.

[0070] In some embodiments, at least one modified nucleotide is selected from the following: deoxynucleotides, 2'-O-methyl modified nucleotides, and / or 2'-fluorine modified nucleotides; and / or combinations thereof.

[0071] In one embodiment, the nucleic acid payload of this disclosure comprises one or more deoxynucleotides. Optionally, the nucleic acid payload is a component of heterodistranded oligonucleotides (HDOs) and / or heterodistranded oligonucleotides.

[0072] In one embodiment, the adapter of this disclosure is a cleavable adapter. Optionally, the adapter is cleaved upon exposure to the intracellular environment.

[0073] Optionally, the pharmaceutical compositions disclosed herein are formulated for intravenous, subcutaneous, intramuscular, parenteral, or oral delivery.

[0074] Another aspect of this disclosure provides a kit for carrying out the methods disclosed herein, the kit comprising a conjugated pharmaceutical agent and instructions for use thereof, the conjugated pharmaceutical agent comprising the regulatory loaded nucleic acid disclosed herein. Optionally, the kit further comprises means for administering the conjugated pharmaceutical agent to a subject.

[0075] definition To facilitate understanding of this disclosure, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​is referenced, the intermediate values ​​and ranges of those values ​​are also part of this disclosure.

[0076] The articles “a” and “a kind” are used in this text to refer to one or more of the grammatical objects of the article (i.e., at least one). For example, “a component” means one or more components, such as multiple components.

[0077] The term “including” is used in this document to mean the phrase “including but not limited to”, and is used interchangeably with it.

[0078] Unless the context clearly indicates otherwise, the term “or” is used herein to mean the term “and / or” and is used interchangeably with the term “and / or”.

[0079] The term “about” is used herein to mean a typical tolerance range in this field. For example, “about” can be understood as approximately 2 standard deviations from the mean. In some embodiments, “about” means ±10%. In some embodiments, “about” means ±5%. When “about” appears before a series of numbers or ranges, it should be understood that “about” may modify each number in that series or range.

[0080] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that are logically possible to include, as is clearly apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range.

[0081] As used herein, “no more than” or “less than” is understood to be a value adjacent to a phrase and a logically low value or an integer that is logically zero in context. For example, a double-stranded strand with “no more than 2 nucleotides” overhangs has 2, 1, or 0 nucleotide overhangs. When “no more than” appears before a series of numbers or ranges, it should be understood that “no more than” can modify each number in that series or range. As used herein, ranges include upper and lower limits.

[0082] As used herein, detection methods may include determining that the amount of analyte present is below the detection level of the method.

[0083] In the event of a conflict between the specified target site and the nucleotide sequence of the sense or antisense strand, the specified sequence takes precedence.

[0084] In the event of a conflict between chemical structure and chemical name, the chemical structure shall prevail.

[0085] As used herein, the term "joint" refers to the organic part that connects two parts of a compound (e.g., covalently connects two parts of a compound). Joints typically contain direct bonds or atoms, such as oxygen or sulfur, units, such as NH, C(O), C(O)NH, SO, SO2, SO2NH, or atomic chains, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalenyl, arylalkynyl, heteroarylalkyl, heteroarylalenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkenyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalenyl, alkenylarylalkynyl, alkenylarylalkyl, alkenylarylalenyl, alkenylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalenyl Alkyl, alkyl-heteroaryl-alkynyl, alkenyl-heteroaryl-alkyl, alkenyl-heteroaryl-alkenyl, alkenyl-heteroaryl-alkynyl, alkenyl-heteroaryl-alkyl, alkenyl-heteroaryl-alkenyl, alkenyl-heteroaryl-alkynyl, alkyl-heterocyclic alkyl, alkyl-heterocyclic alkenyl, alkyl-heterocyclic alkyl-alkenyl, alkenyl-heterocyclic alkynyl, alkenyl-heterocyclic alkyl-alkenyl, alkenyl-heterocyclic alkynyl, alkyl-aryl, alkenyl-heterocyclic alkynyl, alkenyl-heterocyclic alkynyl, alkenyl-aryl, alkenyl-heterocyclic alkynyl, alkyl-aryl, alkenyl-hetero ...

[0086] In some embodiments, the linker or bond can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, wherein the linker can be a straight chain, branched chain, cyclic chain or a single atom. In some cases, the linker refers to a branched linker connecting three or more groups. In some cases, one, two, three, four or five or more carbon atoms in the linker backbone may optionally be substituted with sulfur, nitrogen or oxygen heteroatoms. In some cases, the linker backbone includes linking functional groups such as ethers, thioethers, amino groups, amides, sulfonamides, carbamates, thiocarbamates, ureas, thioureas, esters, thioesters or imines. The bonds between backbone atoms can be saturated or unsaturated, and in some cases, there are no more than one, two or three unsaturated bonds in the linker backbone. The linker may include one or more substituent groups, such as alkyl, aryl or alkenyl groups. The linker may include, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, and alkyl groups, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein two or more atoms (e.g., 2, 3, or 4 atoms) of the cyclic group are contained within the backbone. The linker may be cleavable or non-cleavable. In some cases, the linker is a branched linker, such as a branched linker as described herein (e.g., a linker that branches to allow a multivalent targeting portion, a multivalent loading portion, or both within a single conjugate).

[0087] Certain compositions disclosed herein provide compounds having aliphatic hydrocarbons. Aliphatic chains include the alkyl, alkenyl, and alkynyl categories as defined below.

[0088] Straight-chain aliphatic chains are limited to the unbranched carbon chain portion. As used herein, the term "aliphatic group" refers to a straight-chain, branched, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as alkyl groups, alkenyl groups, or alkynyl groups.

[0089] "Alkyl" refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having a specified number of carbon atoms, or, if not specified, a maximum of 30 carbon atoms. For example, alkyl groups with 1-8 carbon atoms refer to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, as well as positional isomers of these moieties. Alkyl groups with 10 to 30 carbon atoms include decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecyl, tridecyl, and tetradecyl. In some embodiments, straight-chain or branched alkyl groups have 30 or fewer carbon atoms in their backbone (e.g., for straight-chain C1-C1 alkyl groups). 30 For C3-C branches 30 ), and optionally, 20 or fewer carbon atoms. The alkyl group may be substituted or unsubstituted.

[0090] As used herein, the term “heteroalkyl” refers to an alkyl moiety as defined above, which contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of a carbon atom.

[0091] As used herein, the term "alkylene" refers to an alkyl group having a specific number of carbon atoms (e.g., 2 to 12 carbon atoms) and containing two connection points to the remainder of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include methylene-(CH2)-, ethylene-(CH2CH2)-, n-propylene-(CH2CH2CH2)-, isopropylene-(CH2CH(CH3))-, etc. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain portions, and may optionally be substituted with one or more substituents.

[0092] "Cycloalkyl" refers to a monocyclic, bicyclic, bridged, spirocyclic, or polycyclic saturated carbon ring, each having 3 to 12 carbon atoms. Some cycloalkyl groups have 3-10 carbon atoms in their ring structure, and optionally 3-6 carbon atoms in the ring structure. Cycloalkyl groups can be substituted or unsubstituted.

[0093] Unless otherwise specified, “lower alkyl” as used herein means an alkyl group as defined above, but having one to ten carbon atoms in its skeletal structure, optionally one to six carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Similarly, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the disclosure, certain alkyl groups are lower alkyl groups. In some embodiments, substituents referred to herein as alkyl are lower alkyl groups.

[0094] "Alkenyl" refers to any cyclic or acyclic, branched or unbranched unsaturated carbon chain moiety having a specified number of carbon atoms, or, if no limit is specified, a maximum of 26 carbon atoms; and having one or more double bonds in the moiety. Examples of alkenyl chains with 6-26 carbon atoms are hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosene, dodecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosene, dodecenyl, dodecenyl, tridecenyl, and dodecenyl, wherein the unsaturated bonds can be located at any position in the moiety and can have a (Z) or (E) configuration of double bonds.

[0095] "Alkyne" refers to a hydrocarbon group within the alkenyl group range, but with one or more triple bonds in that group.

[0096] As used herein, the term "aryl" includes 3 to 12-membered substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is a carbon (i.e., carbocyclic aryl), or one or more of the atoms are heteroatoms (i.e., heteroaryl). Optionally, aryl groups include 5 to 12-membered rings, and optionally 6 to 10-membered rings. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are common to two adjacent rings, wherein at least one of the rings is aromatic; for example, other cyclic rings may be cycloalkyl, cycloalkenyl, cycloynyl, aryl, heteroaryl, and / or heterocyclic. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc. Heteroaryl groups include substituted or unsubstituted aromatic 3 to 12-membered ring structures, optionally 5 to 12-membered rings, optionally 5 to 10-membered rings, whose ring structures include one to four heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Aryl and heteroaryl groups can be monocyclic, bicyclic, or polycyclic.

[0097] The term "heterocyclic group" or "heterocyclic group" refers to a 3- to 12-membered ring structure, optionally a 5- to 12-membered ring, optionally a 5- to 10-membered ring, whose ring structure includes one to four heteroatoms. Heterocyclic rings can be monocyclic, bicyclic, spirocyclic, or polycyclic. Heterocyclic groups include, for example, thiophene, thiam, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthidine, quinoxaline, quinazolin, cinnoline, pteridine, carbazole, and carboline. The heterocycle can be substituted with one or more of the following substituents: phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxopentane, thiocyclopentane, oxazole, piperidine, piperazine, morpholine, lactone, lactam (such as azacyclobutanone and pyrrolidone), sulfonamide, sulfonyl lactone, etc. The heterocycle can be substituted with the above substituents at one or more positions, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, mercapto, imino, amide, phosphate ester, phosphonate, hypophosphonate, carbonyl, carboxyl, silyl, aminosulfonyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3, -CN, etc.

[0098] The term "substituted" refers to the portion of the skeleton in which a hydrogen atom on one or more carbons is replaced by a substituent. It will be understood that "substitution" or "replacement with..." includes the implicit condition that such substitution conforms to the permissible valence of the substituted atom and the substituent, and that the substitution yields a stable compound, for example, one that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is considered to include all permissible substituents in organic compounds. In a broader sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents in organic compounds.

[0099] For suitable organic compounds, permissible substituents can be one or more and can be the same or different. For the purposes of this disclosure, heteroatoms (such as nitrogen) can have hydrogen substituents as described herein and / or any permissible substituents of the organic compound, said substituents satisfying the valence of the heteroatom. Substituents can include any substituents as described herein, such as halogens, hydroxyl groups, carbonyl groups (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate esters, phosphonates, hypophosphonates, amino groups, amide groups, amidine groups, imine groups, cyano groups, nitro groups, azide groups, mercapto groups, alkylthio groups, sulfate esters, sulfonates, aminosulfonyl groups, sulfonamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties. In some embodiments, the substituents on the substituted alkyl group are selected from C10. 1-6 Alkyl, C 3-6 Cycloalkyl, halogen, carbonyl, cyano, or hydroxyl groups. In some embodiments, the substituent on the substituted alkyl group is selected from fluorine, carbonyl, cyano, or hydroxyl groups. Those skilled in the art will understand that the substituent itself can be substituted if appropriate. Unless specifically stated as “unsubstituted,” references to the chemical groups herein should be understood to include substituted variants. For example, references to an “aryl” group or in part implicitly include both substituted and unsubstituted variants.

[0100] As used herein, the term "pharmaceutical" can refer to a physical entity or phenomenon. In some embodiments, a pharmaceutical can be characterized by specific characteristics and / or effects. In some embodiments, a pharmaceutical can be a compound, molecule, or entity of any chemical class, including, for example, small molecules, peptides, nucleic acids, sugars, lipids, metals, or combinations or complexes thereof. In some embodiments, the term "pharmaceutical" can refer to a compound, molecule, or entity comprising a polymer. In some embodiments, the term can refer to a compound or entity containing one or more polymeric moieties. In some embodiments, the term "pharmaceutical" can refer to a compound, molecule, or entity substantially free of a particular polymer or polymeric moieties. In some embodiments, the term can refer to a compound, molecule, or entity lacking or substantially free of any polymer or polymeric moieties.

[0101] As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, the amino acid has the general structure H₂N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. In some embodiments, the amino acids (including carboxyl and / or amino-terminal amino acids) in the polypeptide may contain structural modifications compared to the general structure described above. For example, in some embodiments, the amino acid may be modified by methylation, amidation, acetylation, polyethylene glycolation, glycosylation, phosphorylation, and / or substitution (e.g., amino group, carboxylic acid group, one or more protons and / or hydroxyl groups) compared to the general structure. In some embodiments, such modification can, for example, alter the cycling half-life of a peptide containing the modified amino acid compared to a peptide containing otherwise identical, unmodified amino acids. In some embodiments, such modification does not significantly alter the relevant activity of a peptide containing the modified amino acid compared to a peptide containing otherwise identical, unmodified amino acids. As will be clear from the context, in some embodiments, the term "amino acid" can be used to refer to a free amino acid; in some embodiments, it can be used to refer to the amino acid residues of a peptide.

[0102] In some embodiments, antisense compounds, including antisense oligonucleotides and other antisense compounds, are disclosed for regulating the expression and / or activity of target nucleic acid molecules, including, for example, regulating the expression of target mRNA molecules. Not wishing to be bound by theory, in some embodiments, the regulation of target mRNA expression can be achieved by providing antisense compounds that hybridize with one or more target nucleic acid molecules (including with one or more target mRNAs).

[0103] In some embodiments, double-stranded RNA (dsRNA) agents (e.g., siRNA) for regulating the expression of nucleic acid molecules encoding target mRNA are disclosed. Not wishing to be bound by theory, in some embodiments, the expression regulation of target genes is achieved by providing dsRNA agents that participate in RNA interference mechanisms (particularly RNA-induced silencing complexes (RISC)) to guide the sequence-specific cleavage and degradation of one or more target nucleic acid molecules. In some other embodiments, the dsRNA compositions disclosed herein (e.g., heteroduplex oligonucleotides (HDOs), known in the art, and comprising antisense oligonucleotide chains double-stranded with complementary RNA oligonucleotide chains (typically "gapmer" antisense oligonucleotide structures)) can achieve the expression regulation of target nucleic acids via antisense oligonucleotide (ASO) mechanisms that do not involve RISC.

[0104] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably herein and refer to an agent containing RNA as defined herein, which mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of target genes in cells (e.g., cells within a subject, such as a mammalian subject).

[0105] In one embodiment, the RNAi agent of this disclosure comprises a single-stranded RNA that interacts with a target RNA sequence to guide the cleavage of the target RNA. Not wishing to be bound by theory, it is believed that long double-stranded RNA introduced into the cell is cleaved into siRNA by a type III nuclease called Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer (a ribonuclease-III-like enzyme) processes dsRNA into short interfering RNA of 19–23 base pairs with a characteristic two-base 3' overhang (Bernstein et al. (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to guide target recognition (Nykanen et al. (2001) Cell 107:309). After binding to a suitable target mRNA, one or more nucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188). Therefore, in one aspect, this disclosure relates to intracellularly generated single-stranded RNA (siRNA) that promotes the formation of RISC complexes to achieve silencing of target genes. Thus, the term "siRNA" is also used herein to refer to iRNA as described above.

[0106] In some implementations, the regulatory nucleic acid agent may be a single-stranded antisense oligonucleotide (ASO) or a single-stranded siRNA (ssRNAi), either of which is introduced into a cell or organism to repress the target mRNA. Not wishing to be bound by theory, the single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides long and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al. (2012) Cell 150:883-894, the entire contents of which are hereby incorporated by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs as described herein, or chemically modified using the methods described in Lima et al., (2012) Cell 150:883-894.

[0107] In some embodiments, the “iRNA” used in the compositions, uses, and methods of this disclosure is a double-stranded RNA and is referred to herein as a “double-stranded RNA agent,” a “double-stranded RNA (dsRNA) molecule,” a “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules (optionally including modified nucleotides, as defined herein, substituted at one, more, or all of such ribonucleic acids on one or both strands of such a dsRNA agent) having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, described as having “sense” and “antisense” orientations relative to the target RNA. In some embodiments of this disclosure, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA through a post-transcriptional gene silencing mechanism, referred herein as RNA interference or RNAi.

[0108] As used herein, the term “antisense oligonucleotide (ASO)” refers to an oligonucleotide that can interact with and / or hybridize with pre-mRNA or mRNA having a complementary nucleotide sequence, thereby altering gene expression.

[0109] As used herein, the term "exon skipping" refers to the modification of premRNA splicing by targeting splice donors and / or receptors and branching sites within the premRNA with one or more complementary antisense oligonucleotides (ASOs). By blocking the spliceosome's access to one or more splice donors, receptors, or branching sites, ASOs can inhibit the splicing response, resulting in the exclusion of one or more exons from the fully processed mRNA. Exon skipping is achieved in the cell nucleus during the maturation process of premRNA. It involves masking key sequences involved in the splicing of target exons by using antisense oligonucleotides (ASOs) complementary to the splice donor / receptor, branching site sequences and / or by overlapping ESEs (within exons) / ISEs (within introns) within the premRNA.

[0110] Typically, most nucleotides on each strand of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, as used herein, “iRNA” or ASO may include chemically modified ribonucleotides; iRNA or ASO may include substantial modifications at multiple nucleotide sites.

[0111] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term modified nucleotide includes, for example, substitution, addition, or removal of functional groups or atoms in the internucleotide bond, sugar moiety, or nucleobase. Modifications applicable to pharmaceuticals disclosed herein include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modification, if used in siRNA-type molecules, is covered by "iRNA" or "RNAi agent," or if used in ASO-type molecules, is covered by "antisense oligonucleotide" or "ASO."

[0112] In some embodiments of this disclosure, if the deoxynucleotide is present in the RNAi agent, or in ASO or other regulatory nucleic acid agents, then the presence of the deoxynucleotide can be considered as constituting a modified nucleotide.

[0113] As used herein, the term "targeting portion" refers to a portion that specifically binds to such target cells when in contact with a system containing one or more target cells of interest (e.g., in a culture, in a tissue, and / or in an organism). In many embodiments, the targeting portion binds to cell surface factors (e.g., binds to factors preferentially or specifically found on the surface of such target cells of interest). In some embodiments, the binding of the targeting portion to the cell surface factor results in the internalization of the targeting portion. Typically, when included in a conjugate pharmaceutical agent as described herein, the targeting portion useful according to this disclosure retains its specific binding characteristics; in some embodiments, the binding of such a conjugate pharmaceutical agent to the associated cell surface factor results in the internalization of the conjugate pharmaceutical agent. In some embodiments, the targeting portion specifically binds to factors on the surface of kidney cells. In some embodiments, the targeting portion specifically binds to cuboproteins. In some embodiments, the targeting portion specifically binds to macroproteins. In some embodiments, the targeting portion of this disclosure comprises compounds of formula I or I', or as otherwise disclosed herein.

[0114] As used herein, the term "cell surface factor" refers to a factor (e.g., a peptide) present on the surface of a cell of interest (e.g., a target cell as described herein, which in many embodiments may be a kidney cell). In some embodiments, cell surface factors are preferentially present on the surface of target cells (e.g., kidney cells) compared to cells of one or more other tissues. In some embodiments, cell surface factors are also present on certain non-target cells in addition to target cells. In some embodiments, cell surface factors are not preferentially or specifically present on the associated target cells of interest. In some embodiments, cell surface factors are or contain receptors. In some embodiments, cell surface factors are internalized by binding to one or more specific ligands (e.g., with a target motif as described herein). In some embodiments, cell surface factors may interact (e.g., bind to, form complexes with, etc.) with one or more other components of the cell found on its surface. In some embodiments, cell surface factors and / or specific forms or variants thereof and / or any of the aforementioned cell surface factors may be associated with a specific cell state or condition (e.g., developmental stage, disease state, etc.).

[0115] In some embodiments, the “conjugated agent” has a structure represented by the formula: (Xn1-Yn2-Zn3), where X is a target moiety (e.g., polymyxin or other target moiety structures disclosed herein), and n1 is an integer (i.e., 1 or greater, typically less than 5); Y is a linker (optionally, a branch linker, e.g., to allow a conjugated structure to include multiple target moieties associated with one or more payload moieties in a single structure), and n2 is 0 or an integer (i.e., 1 or greater, typically less than 5); and Z is a regulatory nucleic acid of this disclosure, and n3 is an integer (i.e., 1 or greater, typically less than 5); in many embodiments, n2 = n1 and / or n2 = n3. In many embodiments, n1 and / or n3 is 1. In many embodiments, the conjugated agent has a structure represented by the formula (XYZ). In some embodiments, the conjugated agent has a structure represented by the formula: (XY)nZ, where n is an integer greater than 1, and the conjugated agent contains more than one target moiety. In some implementations, the conjugate agent has a structure represented by the following formula: X-(YZ)n, where n is an integer greater than 1, and the conjugate agent contains more than one regulatory nucleic acid.

[0116] As used in this article, the term "macroprotein" refers to the receptor of a member of the low-density lipoprotein receptor (LDLR) family. Macroproteins are composed of... LRP2Gene encoding. The amino acid sequences of the full-length macroprotein and / or the nucleic acid encoding it can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human macroprotein (SEQ ID NO: 1, where residues 27-4411 represent extracellular domains containing LDL receptor class A domains, LDL receptor class B domains, and EGF-like domains; residues 4589-4602 represent DAB2 interaction domains; and residues 4453-4622 represent cytoplasmic domains containing NPXY motifs, SH2 binding domains, SH3 binding domains, and proline-rich domains) can be found as UniProt / Swiss-Prot accession number P98164, and the nucleic acid sequence encoding human macroprotein can be found as accession number NM_004525.3. For example, macroproteins are also referred to as low-density lipoprotein receptor-associated protein 2 (LRP2), glycoprotein 330 (Gp330), calcium sensor protein, or homologs of Heyman's nephritis antigen. Those skilled in the art will understand that the sequence of SEQ ID NO: 1 is exemplary, and certain variants (including, for example, conserved substitutions in SEQ ID NO: 1, and codon-optimized variants of the associated nucleic acid sequence, etc.) are understood to also encode or encode human macroproteins. Furthermore, those skilled in the art will understand that homologs and orthologs of human macroproteins are known and / or are known by practice or common techniques, for example, based on the degree of sequence identity, the presence of one or more characteristic sequence elements, and / or one or more shared activities. In some embodiments, the macroprotein comprises a full-length macroprotein or a variant or fragment thereof. In some embodiments, the macroprotein targeted according to this disclosure is a macroprotein expressed by a specific target cell and / or tissue of interest (e.g., in an organism of interest). In some embodiments, the macroprotein targeted according to this disclosure is an engineered macroprotein. In many embodiments, the macroproteins targeted according to this disclosure are present on the surface of target cells of interest (e.g., in target tissues of interest, such as the kidneys) and are internalized by such cells upon binding to the macroprotein-binding portions as described herein. Macroproteins have been reported to be expressed in one or more of the following tissues and / or cells: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils, and / or platelets); nervous system cells (e.g., brain tissue, cortex, cerebellum, retinal cells, spinal cord cells, nerve cells, neurons, and / or supporting cells); endothelial cells; muscles (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle); small intestine; colon; adipocytes; kidneys; liver; lungs; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; pituitary gland; breast; skin; ovaries; uterus; placenta; prostate; and testes.Macroprotein expression has been reported to be particularly enriched (e.g., higher than one or more other tissues) in the following tissues and / or cells: kidney tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissues. Macroprotein expression (e.g., at relatively high levels) on the surface of kidney cells (such as proximal tubular epithelial cells and podocytes) has been specifically reported. See Nielsen R. et al. (2016). Kidney Int . 89(1): 58-67.

[0117] As used herein, the term "macroprotein-binding moiety" refers to a portion that binds to a macroprotein upon contact with it. Typically, macroprotein-binding moieties useful according to this disclosure specifically bind to macroproteins upon contact. In some embodiments, the macroprotein-binding moiety is or comprises: the polymyxin or related structures disclosed herein, peptides, aminoglycosides, endogenous substances, xenobiotics, antibodies, fragments thereof, or combinations thereof. In some embodiments, the macroprotein-binding moiety is internalized upon binding to a macroprotein on the cell surface.

[0118] As used in this article, the term "cubic protein" refers to proteins composed of... CUBNGenetically encoded receptors. The amino acid sequences of the full-length cuboprotein and / or the nucleic acid encoding it can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of the human cuboprotein (SEQ ID NO: 2, where residues 1-23 represent the signal peptide; residues 24-35 represent the propeptide that can be removed in the mature form; and residues 36-3623 represent the mature cuboprotein polypeptide) can be found as UniProt / SwissProt accession number O60494, and the nucleic acid sequence encoding the human cuboprotein can be found as accession number NM_001081.3. For example, the cuboprotein is also known as IFCR, Gp280, intrinsic factor-vitamin B12 receptor, MGA1, or IGS1. Those skilled in the art will understand that the cuboprotein sequence of SEQ ID NO: 2 is exemplary, and certain variants (including, for example, conserved substitutions in SEQ ID NO: 2, codon-optimized variants of the nucleic acid sequence encoding the cuboprotein, etc.) are understood to also encode or encode the human cuboprotein. Furthermore, those skilled in the art will understand that homologs and orthologs of human cuboproteins are known and / or are identifiable by practice or common techniques, for example, based on the degree of sequence identity, the presence of one or more characteristic sequence elements, and / or one or more shared activities. In some embodiments, the cuboprotein comprises a full-length cuboprotein or a variant or fragment thereof. In some embodiments, the cuboprotein targeted according to this disclosure is a cuboprotein expressed by a specific target cell and / or tissue of interest (e.g., in an organism of interest). In some embodiments, the cuboprotein targeted according to this disclosure is an engineered cuboprotein. In many embodiments, the cuboprotein targeted according to this disclosure is present on the surface of target cells of interest (e.g., in a target tissue of interest) and is internalized by such cells upon binding to the cuboprotein binding portion as described herein. Cubic proteins have been reported to be expressed in one or more of the following tissues and / or cells: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils, and / or platelets); the nervous system (e.g., brain tissue, cortex, cerebellum, retinal cells, spinal cord cells, nerve cells, neurons, and / or supporting cells; endothelial cells); muscle (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle); small intestine; colon; adipocytes; kidney; liver; lung; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; pituitary gland; breast; skin; ovary; uterus; placenta; prostate; and testis). Cubic protein expression has been reported to be particularly enriched (e.g., higher than one or more other tissues) in the following tissues and / or cells: kidney tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissues.Cubic proteins have been specifically reported to be expressed (e.g., at relatively high levels) on the surface of kidney cells, such as proximal tubular epithelial cells and podocytes. See Nielsen R. et al. (2016). Kidney Int 89(1):58-67.

[0119] As used herein, the term "cubic protein binding portion" refers to a portion that binds to a cubic protein upon contact. Typically, cubic protein binding portions useful according to this disclosure specifically bind to cubic proteins upon contact. In some embodiments, the cubic protein binding portion is internalized upon binding to a cubic protein on the cell surface.

[0120] As used herein, the phrase "kidney-associated cells" refers to cells present in or potentially present in the kidney (e.g., during development, during tissue homeostasis, or during disease or condition). In some embodiments, kidney-associated cells are also referred to herein as "kidney cells." In some embodiments, kidney-associated cells include any or all of the following cell types: proximal tubular epithelial cells, podocytes, renal cyst cells (e.g., in polycystic kidney disease), parietal epithelial cells, mesangial cells, renal stem cells, epithelial progenitor cells, fibroblasts, myofibroblasts, pericytes, ascending loops of Henlecells, descending loops of Henlecells, distal tubular cells, connecting tubular cells, intercalated cells, and chief cells. Exemplary kidney cell populations are provided by Schumacher et al., (2021). npj Regen Med 6, 45 The entire contents of this document are incorporated herein by reference. In some embodiments, kidney cells are or comprise cells derived from the kidney, such as kidney tumor cells and / or metastatic kidney tumor cells.

[0121] As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., the first regimen of all "doses" is administered before any dose of the second regimen); in some embodiments, such agents are administered with overlapping dosing regimens. In some embodiments, "administration" of combination therapy may involve administering one or more agents or forms to a subject who is receiving other agents or forms in combination. For clarity, combination therapy does not require the administration of a single agent together as a single composition (or even necessarily simultaneous administration), although in some embodiments, two or more agents or their active portions may be administered together as a combination composition, or even as a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0122] Compositions or methods described herein as “comprising” one or more named elements or steps are open-ended, meaning that the named elements or steps are necessary, but other elements or steps may be added within the scope of the composition or method. To avoid verbosity, it should also be understood that any composition or method described as “comprising” (or “comprises”) one or more named elements or steps also describes a corresponding, more limited composition or method “consisting essentially” (or “consists essentially of”) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not significantly affect the essential and novel characteristics of the composition or method. It should also be understood that any composition or method described herein as “comprising” or “essentially” one or more named elements or steps also describes a corresponding, more limited and closed-ended composition or method “consisting of” (or “consists of”) named elements or steps to exclude any other unnamed elements or steps. In any composition or method disclosed herein, any known or disclosed equivalent of any named basic element or step may be substituted for that element or step.

[0123] As used in this article, the term “peptide” refers to polypeptides that are typically relatively short, such as those less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids in length.

[0124] As used herein, a "peptide" refers to a polymeric chain of amino acids. In some embodiments, the peptide has a naturally occurring amino acid sequence. In some embodiments, the peptide has an amino acid sequence that is not naturally occurring. In some embodiments, the peptide has an engineered amino acid sequence because it is designed and / or generated by human intervention. In some embodiments, the peptide may comprise or consist of natural amino acids, non-natural amino acids, or both.

[0125] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent (e.g., a regulatory nucleic acid agent disclosed herein) is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, shows a statistically significant probability of achieving the intended therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in a particular form (e.g., in solid or liquid form), and / or may be particularly suitable for, for example: oral administration (e.g., as an oral drenching [aqueous or non-aqueous solution or suspension], tablet, capsule, pill, powder, granule, paste, etc., which may be specifically formulated for, for example, buccal, sublingual, or systemic absorption); parenteral administration (e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, a sterile solution or suspension, or a sustained-release formulation, etc.); topical application (e.g., as a cream, ointment, patch, or spray applied to, for example, the skin, lungs, or mouth); intravaginal or rectal administration (e.g., as a vaginal suppository, suppository, cream, or foam); ocular application; nasal or pulmonary application, etc.

[0126] As used herein, the term "subject" refers to an organism, such as a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, the subject has a disease, condition, or disorder, such as a disease, condition, or disorder that can be treated as provided herein. In some embodiments, the subject is susceptible to a disease, condition, or disorder; in some embodiments, a susceptible subject is susceptible to and / or exhibits an increased risk of developing a disease, condition, or disorder (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, condition, or disorder. In some embodiments, the subject does not exhibit a specific symptom (e.g., clinical manifestations of a disease) or characteristic of a disease, condition, or disorder. In some embodiments, the subject does not exhibit any symptom or characteristic of a disease, condition, or disorder. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is receiving and / or has received a diagnostic and / or therapeutic treatment. In one implementation, the subject is a person who, such as a person treating or assessing a disease or condition that would benefit from reduced target mRNA expression; a person at risk of benefiting from a disease or condition that would benefit from reduced target mRNA expression; a person suffering from a disease or condition that would benefit from reduced target mRNA expression; or a person being treated for a disease or condition that would benefit from reduced target mRNA expression as described herein. In some implementations, the subject is female. In other implementations, the subject is male. In one implementation, the subject is an adult subject. In another implementation, the subject is a pediatric subject.

[0127] As used herein, “reference” describes a standard or control for comparison. For example, in some embodiments, a drug, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control drug, animal, individual, population, sample, sequence, or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally contained in a tangible medium. Generally, as those skilled in the art will understand, a reference or control is determined or characterized under conditions or settings comparable to those of the subject being evaluated. Those skilled in the art will understand that when sufficient similarity exists, there is reason to rely on and / or compare a particular possible reference or control.

[0128] As used herein, the term "specific binding" refers to the ability to identify potential binding partners within the context in which binding occurs. When other potential targets are present, a binding agent that interacts with a specific target is referred to as a "specific binding agent." Specific binding"To the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binder and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the binder-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of the binder to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination within a concentration range."

[0129] The term "specificity," as used herein to refer to an active agent, is understood by those skilled in the art to mean that the agent distinguishes a potential target entity or state. For example, in some embodiments, an agent is said to "specifically" bind to its target if, in the presence of one or more competing alternative targets, the agent preferentially binds to the target. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitopes, fissures, binding sites). It should be understood that specificity need not be absolute. In some embodiments, specificity can be evaluated relative to the specificity of the binder to one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binder. In some embodiments, specificity is evaluated relative to the specificity of a reference non-specific binder. In some embodiments, the agent or entity binds undetectably to a competing alternative target when bound to its target entity. In some embodiments, the binder binds to its target entity with a higher association rate, a lower dissociation rate, increased affinity, reduced dissociation, and / or increased stability compared to a competing alternative target.

[0130] As is known in the art, "specificity" is a measure of a particular ligand's ability to distinguish its binding mate from other potential binding mates.

[0131] As used herein, "complementary" refers to a nucleic acid molecule that can form hydrogen bonds with another nucleic acid molecule through conventional Watson-Crick base pairing or other non-conventional types of pairing (e.g., Hoogsteen or reverse Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. Regarding the oligonucleotides of this disclosure, the binding free energy of the antisense oligonucleotide / antisense strand to its complementary sequence is sufficient to allow the relevant function of the oligonucleotide agent to continue, and there is a sufficient degree of complementarity to prevent non-specific binding of the antisense oligonucleotide / antisense strand to non-target sequences under conditions requiring specific binding (i.e., physiological conditions under in vivo therapeutic treatment). Measuring the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., CSH Symp. Quant. Biol. 1 / 7:123-133 (1987); Frier et al., Proc. Nat. Acad. Sci. USA 83:9373-77 (1986); and Turner et al., J. Am. Chem. Soc. 109:3783-3785 (1987)). Therefore, “complementary” (or “specifically hybridizable”) refers to a sufficient degree of complementarity or precise pairing that allows for stable and specific binding between the antisense oligonucleotide / antisense strand and the pre-mRNA or mRNA target. It is understood in the art that a nucleic acid molecule does not need to be 100% complementary to the target nucleic acid sequence for specific hybridization. That is, two or more nucleic acid molecules may not be perfectly complementary.

[0132] Complementarity is defined by the minimum percentage of consecutive residues in one nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if both the first and second nucleic acid molecules have 10 nucleotides, then base pairings of 5, 6, 7, 8, 9, or 10 nucleotides between the two molecules represent 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. “Perfect” or “completely” complementary nucleic acid molecules mean that all consecutive residues in the first nucleic acid molecule will form hydrogen bonds with the same number of consecutive residues in the second nucleic acid molecule, wherein the nucleic acid molecules either both have the same number of nucleotides (i.e., the same length) or the two molecules have different lengths.

[0133] As used herein, the term "variant" refers to a molecule or entity that exhibits significant structural identity with a reference molecule or entity but differs structurally from the reference molecule or entity (e.g., is or comprises a nucleic acid, protein, or small molecule), for example, differing from the reference molecule or entity in the presence or absence or level of one or more chemical motifs. In some embodiments, a "variant" may be referred to as a "derivative." In some embodiments, a variant is functionally different from its reference molecule or entity. In many embodiments, whether a particular molecule or entity is properly considered a "variant" of the reference is based on the degree of its structural identity with the reference molecule. As understood by those skilled in the art, biological or chemical reference molecules are typically characterized by certain characteristic structural elements. By definition, a variant is a unique molecule or entity that shares one or more such characteristic structural elements but differs from the reference molecule or entity in at least one respect. To cite just a few examples, a polypeptide may have a characteristic sequence element consisting of multiple amino acids at designated positions relative to each other in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element consisting of multiple nucleotide residues at designated positions relative to each other in linear or three-dimensional space. In some embodiments, variant peptides or nucleic acids may differ from reference peptides or nucleic acids due to one or more differences in the amino acid or nucleotide sequence and / or one or more differences in the chemical portions (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the peptide or nucleic acid (e.g., linked to the peptide or nucleic acid backbone). In some embodiments, variant peptides or nucleic acids exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% of the overall sequence identity with the reference peptide or nucleic acid. In some embodiments, variant peptides or nucleic acids do not share at least one characteristic sequence element with the reference peptide or nucleic acid. In some embodiments, the reference peptide or nucleic acid has one or more biological activities. In some embodiments, variant peptides or nucleic acids share one or more of the biological activities of the reference peptide or nucleic acid. In some embodiments, variant peptides or nucleic acids lack one or more of the biological activities of the reference peptide or nucleic acid. In some embodiments, variant peptides or nucleic acids exhibit a reduced level of one or more biological activities compared to the reference peptide or nucleic acid. In some implementations, a polypeptide or nucleic acid of interest is considered a “variant” of the reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference polypeptide or nucleic acid, but with minor sequence changes at specific positions. Typically, compared to the reference, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of residues in the variant are substituted, inserted, or deleted.In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference. Typically, the variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) substituted, inserted, or deleted functional residues (i.e., residues involved in a specific biological activity) compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 added or deleted residues compared to the reference, and in some embodiments, no added or deleted residues are included. In some embodiments, the variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, or about 6, and typically fewer than about 5, about 4, about 3, or about 2 additions or deletions, compared to a reference. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0134] In some implementations, regulatory RNA compounds (e.g., repressive RNA compounds, such as siRNA, antisense compounds, including repressive antisense oligonucleotides and those designed to regulate splicing, etc.) are used to prevent, improve, and / or treat diseases or disorders for which such prevention, improvement, and / or treatment of the disease or disorder can be provided by delivering nucleic acid molecules capable of selectively and specifically regulating the expression of target mRNAs to the kidney cells or other organ cells of the subject.

[0135] As used herein, the term "treating" or "treatment" refers to a beneficial or desired outcome, such as a reduction in at least one sign or symptom of a target RNA-related disease in a subject. Treatment also includes reducing one or more signs or symptoms associated with the expression of unwanted target RNAs and / or target peptides; reducing the degree of activation or stabilization of unwanted target RNAs and / or target peptides; and improving or alleviating the activation or stabilization of unwanted target RNAs and / or target peptides. "Treatment" can also refer to an extension of survival compared to expected survival without treatment. The term "lower" in the context of the level of target RNAs and / or target peptides in a subject or in a disease marker or symptom refers to a statistically significant reduction in such levels. This reduction can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the reduction is at least 20%. In some embodiments, the reduction is at least 50% in disease markers (e.g., protein or gene expression levels). In the context of target RNA and / or target peptide levels in subjects, "lower" is preferably lower than levels acceptable within the normal range for individuals without such a condition. In some embodiments, "lower" refers to a reduced difference between the levels of markers or symptoms in subjects with a disease and levels acceptable within the normal range for an individual, such as the level of weight loss between obese individuals and individuals with acceptable weight within the normal range.

[0136] As used herein, “prevention” or “preventing” when referring to a disease, condition, or disorder can be treated or improved by reducing the expression of target genes. This means reducing the likelihood that a subject will develop symptoms associated with such a disease, condition, or disorder, such as symptoms of unwanted or excessive expression of target RNAs and / or target peptides, such as metabolic disorders and / or kidney diseases or disorders, such as glomerular diseases, tubular diseases, other kidney diseases, congenital metabolic defects, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, viral infections, etc. For example, the likelihood of developing, for instance, a kidney disease or disorder is reduced when an individual with one or more risk factors for kidney disease or disorder fails to develop one or more severe kidney diseases or disorders, or when, relative to a group with the same risk factors who did not receive the treatments described herein, they develop one or more severe kidney diseases or disorders. Effective prevention is defined as the failure to develop into a disease, condition, or disorder, or a reduction in the development of symptoms associated with such a disease, condition, or disorder (e.g., a reduction of at least about 10% at a clinically acceptable level), or a delay in the onset of symptoms (e.g., a delay of days, weeks, months, or years).

[0137] As used herein, the term "target RNA-related disease" refers to a disease, condition, or disorder caused by or associated with unwanted or excessive levels and / or expression of target RNAs. The term "target RNA-related disease" includes diseases, conditions, or disorders that can be treated or improved by reducing target RNA expression. The term "target RNA-related disease" includes metabolic disorders, including kidney diseases and various other kidney diseases or disorders such as glomerular disorders, tubular disorders, other kidney diseases, congenital metabolic defects, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, viral infections, phenylketonuria (PKU), and related amino acid disorders.

[0138] "Effective quantity" refers to a quantity that is sufficient to achieve a beneficial or desired result.

[0139] As used herein, “therapeutic effective dose” is intended to include an amount of regulatory nucleic acid agent that, when administered to a subject with a target RNA-related disease, is sufficient to treat the disease (e.g., by reducing, improving, or maintaining the existing disease or one or more symptoms of the disease). “Therapeutic effective dose” can vary depending on the regulatory nucleic acid agent, the method of administration, the disease and its severity and history, age, weight, family history, genetic makeup, the type of prior or concomitant treatment (if any), and other individual characteristics of the subject being treated.

[0140] As used herein, “preventive effective dose” is intended to include an amount of regulatory nucleic acid agent that, when administered to a subject with a target RNA-related disease, is sufficient to prevent or improve the disease or one or more symptoms of the disease. Improving the disease includes slowing its progression or reducing the severity of later-stage disease. The “preventive effective dose” can vary depending on the regulatory nucleic acid agent, the method of administration, the risk level of the disease, medical history, age, weight, family history, genetic makeup, the type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0141] "Therapeutic effective amount" or "preventive effective amount" also includes the amount of regulatory nucleic acid agent that produces some desired effect with a reasonable benefit / risk ratio applicable to any treatment. The conjugated regulatory nucleic acid (e.g., conjugated iRNA) used in the methods of this disclosure can be administered in sufficient amounts to produce a reasonable benefit / risk ratio applicable to such treatment.

[0142] The phrase “pharmaceutically acceptable” as used in this article refers to compounds, materials, compositions, or dosage forms that, within reasonable medical judgment, are suitable for tissue contact with human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a reasonable benefit / risk ratio.

[0143] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate, or zinc, or stearic acid), or solvent encapsulation material, relating to the delivery or transfer of a subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and harmless to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers intended for administration by injection.

[0144] As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, eye discharge, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized areas. For example, a sample may be derived from a specific organ, a portion of an organ, or fluids or cells within those organs. In some embodiments, a sample may be derived from the kidney (e.g., the entire kidney or certain segments of the kidney or certain types of cells in the kidney, such as, for example, proximal tubular epithelial cells, podocytes, etc.). In some embodiments, "sample derived from a subject" refers to urine obtained from a subject. "Sample derived from a subject" may refer to blood from a subject or blood-derived serum or plasma.

[0145] As used herein, the terms “target nucleic acid,” “nucleic acid molecule encoding target RNA,” “nucleic acid molecule encoding target polypeptide,” and “nucleic acid molecule encoding non-coding RNA” conveniently encompass RNA (including pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding target RNA and / or target polypeptide, as well as cDNA derived from such RNA, and non-coding RNA and / or regulatory RNA species. In some embodiments, the target nucleic acid is mRNA encoding a human target polypeptide.

[0146] Regulation of target nucleic acid expression can be achieved by altering the function of any number of nucleic acids (DNA or RNA). "Regulation" or "regulation of expression" refers to interference with function, such as an increase (stimulation or induction) or decrease (inhibition or reduction) in the expression of target mRNA. As another example, regulation of expression can include interference with splicing site selection before mRNA processing. "Expression" encompasses all functions of translating gene-encoded information into structures present and functioning in the cell. These structures include the products of transcription and translation. The function of the RNA to be regulated can include translocation functions, including but not limited to RNA translocation to protein translation sites, RNA translocation to intracellular sites distant from RNA synthesis sites, and translation of proteins from RNA. Regulated RNA processing functions include, but are not limited to, RNA splicing (to produce one or more RNA species), RNA capping, RNA 3' maturation, and catalytic activity or complex formation involving RNA (which may be involved in or promoted by RNA). Regulation of expression can result in an increase or decrease in the levels of one or more nucleic acid species, whether transient or at net homeostatic levels. One result of such interference with target nucleic acid function is the regulation of the expression of target RNA (e.g., target mRNA) and / or target polypeptides. Therefore, in one embodiment, regulation of expression may refer to an increase or decrease in the level of the target RNA or protein. In another embodiment, regulation of expression may refer to an increase or decrease in one or more RNA splicing products, or a change in the ratio of two or more splicing products.

[0147] The effects of regulatory RNA compounds on target nucleic acid expression can be tested in any of a variety of cell types, provided the target nucleic acid is present at measurable levels and can be routinely determined using methods such as PCR or Northern blotting. Cell lines are derived from normal tissues and cell types, as well as cells associated with various conditions (e.g., hyperproliferative disorders). Cell lines derived from a variety of tissues and species can be obtained from the American Type Culture Collection (ATCC, Manassas, Va) and other public sources. Primary cells, or those isolated from animals and not subjected to continuous culture, can be prepared according to methods known in the art or obtained from various commercial suppliers. Furthermore, primary cells include cells obtained from donor subjects (i.e., blood donors, surgical patients) in a clinical setting. These techniques are well known to those skilled in the art.

[0148] As used herein, the definition of each expression (e.g., alkyl, m, η, etc.) is intended to be independent of its definition elsewhere in the same structure when it appears more than once in any structure.

[0149] This disclosure is further illustrated by the following detailed description. Detailed Implementation

[0150] This article especially Disclosed are conjugated pharmaceutical agents comprising a targeting moiety (e.g., a polymyxin compound, its variants or derivatives, or other targeting compounds disclosed herein) directly or indirectly conjugated to a payload moiety. In some embodiments, the targeting moiety specifically binds to a surface factor on a target cell of interest (e.g., a kidney cell). In some embodiments, the payload moiety is or comprises a nucleic acid agent. In some embodiments, the payload moiety is or comprises a therapeutic agent (e.g., a therapeutic oligonucleotide).

[0151] This disclosure provides, in particular, the insight that conjugated agents as described herein may be especially useful or effective for delivering nucleic acid agents to kidney cells and / or other cells that express or contain surface factors (e.g., macroproteins) specifically bound by the target moieties as described herein.

[0152] Polymyxins and polymyxin-related target components In some respects, the conjugated pharmaceutical agents disclosed herein comprise a polybasic structure (e.g., polymyxin, polymyxin-type, or other related compounds) as a targeting moiety. The targeting moiety as disclosed herein may bind to (e.g., selectively bind to) surface factors (e.g., portions thereof, and / or specific forms thereof, such as disease-related forms) present on the surface of target cells of interest (e.g., kidney cells) disclosed herein.

[0153] Polymyxin B (PMB) has the following structure: Polymyxin E (colistin) has the following structure: In this field, both PMB and colistin have been identified as binding to macroproteins with high affinity, leading to the internalization of such compounds in the kidneys (Moestrup et al.). J Clin Invest (96: 1404-1413). Certain aspects of this disclosure relate to the identification and use of a class of polybasic compounds associated with PMB and colistin as targeting moieties for conjugation to a molecular payload (e.g., a nucleic acid agent) via a linker moieties (optionally linked to such linkers comprising one or more amino acid structures).

[0154] Therefore, the target moiety of some currently disclosed conjugate compositions includes compounds of formula I: R1 is an optionally substituted C3-C7 alkyl or optionally substituted C3-C7 aryl; and L is a linker as disclosed herein.

[0155] Not wishing to be bound by theory, this disclosure proposes that the binding of a targeting moiety (polymyxin, polymyxin-type, or other related compound) to a cell surface factor present on the surface of a related (e.g., kidney) cell (e.g., tissue cell) can achieve the internalization of both the cell surface factor and the bound targeting moiety (e.g., which may be a portion of a conjugated pharmaceutical agent as described herein). In some embodiments, such internalization may mean that the related cell surface factor is no longer (at least for a period of time) present on the surface of the cell (e.g., tissue cell) for purposes such as signal transduction and / or binding to a ligand.

[0156] This disclosure provides, in particular, the insight that the internalization of triggering surface factors can usefully enable the delivery of a target moiety (and / or agent, such as the conjugate agent described herein, which includes the target moiety) to, for example, internal compartments (such as vesicles and / or organelles and / or the cytoplasm of the cell). This disclosure further provides the insight that such internalization may be particularly useful for the delivery of conjugate agents as described herein and / or a portion thereof (e.g., its payload portion) into the cell. This disclosure provides a specific insight that such internalization may be particularly useful for the delivery of nucleic acid agents as described herein, specifically including the case of conjugate agents as described herein (e.g., as its payload portion).

[0157] In some embodiments, at least 5% of the cell surface factors (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, or at least 95% of the cell surface factors) are internalized upon binding to the target moiety. In some embodiments, substantially all or all of the cell surface factors are internalized upon binding to the target moiety.

[0158] In some implementations, the binding of the targeting portion to cell surface factors on the surface of cells (e.g., cells of a tissue) does not internalize the cell surface factors.

[0159] In some embodiments, the conjugate agents described herein comprise one or more payload portions and / or one or more target portions.

[0160] In some embodiments, the conjugate agent described herein comprises a payload portion and one or more targeting portions.

[0161] In some embodiments, the conjugate agent described herein comprises one or more payload portions and a targeting portion.

[0162] In some embodiments, cell surface factors are or comprise polypeptides present on the surface of cells (e.g., cells of tissues) (e.g., detectable thereon). In some embodiments, cell surface factors are present on the surface of cells expressing macroproteins (e.g., detectable thereon), for example, as described herein. In some embodiments, cell surface factors comprise receptors.

[0163] In some embodiments, the cell surface factor is or includes renal cell surface factors. In some embodiments, the renal cell surface factor is present on the surface of kidney-related cells (e.g., renal cells or cells that can be found in the kidney, such as during development, tissue homeostasis, or during disease or condition) (e.g., detectable thereon). In some embodiments, the renal cell surface factor is present on proximal tubular epithelial cells, podocytes, and / or renal cyst cells (e.g., detectable thereon).

[0164] In some implementations, renal cell surface factors are present on the surface (e.g., detectable thereon) of kidney-related tissues (e.g., tissues that are part of or can be found in the kidney, such as during development, during tissue homeostasis, and / or during disease or condition).

[0165] In some embodiments, the renal cell surface factor is or comprises receptors present on the surface of cells (such as kidney-related cells as described herein) or kidney-related tissues as described herein (e.g., detectable thereon). In some embodiments, the renal cell surface factor may bind to one or more co-receptors on the surface of cells (e.g., cells of tissues). In some embodiments, the renal cell surface factor may be internalized when a kidney-specific binding portion of the conjugate agent binds to the renal cell surface factor. In some embodiments, as a result of binding to the kidney-specific binding portion of the conjugate agent, the internalization of the renal cell surface factor also internalizes the conjugate agent (e.g., a portion thereof, such as the payload portion) into the cells. In some embodiments, the internalized conjugate agent (e.g., a portion thereof, such as the payload portion) is delivered to vesicles (e.g., lysosomes, endosomes, clathrin-coated pits, or intracellular membrane organelles, or combinations thereof) within the cells. In some embodiments, an internalized conjugate agent (e.g., a portion thereof, such as a payload portion) is delivered to a compartment in the cell, such as the cytoplasm, mitochondria, ribosomes, nucleus, nucleolus, or any other compartment in the cell, or a combination thereof.

[0166] In some embodiments, an internalized conjugate agent (e.g., a portion thereof, such as a payload portion) within a cell (e.g., in a vesicle or compartment within the cell) can reduce the expression and / or activity of the target of the payload portion.

[0167] In some embodiments, internalizing the conjugated agent (e.g., a portion thereof, such as the payload portion) into cells (e.g., vesicles or compartments within the cell) uncouples (e.g., segregates) the target portion from the payload portion. In some embodiments, the target portion is uncoupled (e.g., segregates) from the payload portion by chemical reaction and / or mechanical separation. In some embodiments, the chemical reaction includes an enzymatic reaction to cleave the connector linking the target portion to the payload portion.

[0168] In some embodiments, the conjugate agent (e.g., a portion thereof, such as the payload portion) is internalized into cells (e.g., vesicles or compartments within the cell) to uncouple the targeting portion from the payload portion.

[0169] In some embodiments, the conjugated agents disclosed herein can be filtered by glomerular capillaries into, for example, Bowman's capsule. In some embodiments, the conjugated agents disclosed herein have size, charge, conformation, and / or other properties that allow them to be filtered by glomerular capillaries. In some embodiments, the glomerular filtration threshold is in the range of 30-50 kDa.

[0170] In some implementations, cell surface factors (e.g., renal cell surface factors) are or contain receptors selected from macroproteins, cuboproteins, or both.

[0171] connector Some aspects of this disclosure are characterized by connectors that link a target moiety (e.g., a polybasic moiety as described herein) to a payload moiety (e.g., a nucleic acid agent). In some embodiments, the bioconjugate or conjugate agent comprises a regulatory nucleic acid chemically conjugated or covalently linked to the target moiety.

[0172] In some embodiments, conjugated agents are prepared by conjugating or covalently linking a regulatory nucleic acid agent to a target moiety. In some embodiments, the regulatory nucleic acid can be linked to the target moiety by, for example, a reaction between the regulatory nucleic acid and the target moiety in solution. Conjugated agents can also be prepared in a single synthesis, for example, by solid-phase preparation of GalNAc-conjugated nucleic acids using known synthetic methods. (e.g., U.S. Patent Nos. 9,422,562, WO2009073809, 8,106,022, 8,828,956, 9,133,461, and 10,131,907, each of which is incorporated herein by reference in its entirety).

[0173] Regardless of how it is generated, and depending on the desired properties of the conjugated agent, including a spacer or linker between the regulatory nucleic acid and the binding moiety can be either advantageous or disadvantageous. If including a linker is advantageous, then the linker can have many different types and chemical compositions.

[0174] Typically, linkers are designated as “cleavable” or “non-cleavable.” Cleavable linkers are generally used when it is desired that the payload and its conjugated binding portion be released, allowing one or both to better fulfill their functions (e.g., U.S. Patent Nos. 10,808,039 and 9,463,252, each of which is incorporated herein by reference in its entirety). Non-cleavable linkers are generally used to maintain the desired activity, properties, and stability of conjugated pharmaceutical agents, such as enzymes attached to probes or (m)Abs, to facilitate ELISA assays, to increase affinity or bispecificity, etc. Cleavable linkers include chemically cleavable linkers, such as those by hydrolysis, pH alteration, reduction, or oxidation, and enzymatically cleavable linkers, such as those by the action of proteases, esterases, glucosidases, glucuronidases, galactosidases, phosphatases, phosphodiesterases, nucleases, lipases, or any enzyme capable of cleaving the linker to release the biomolecule from other compounds.

[0175] In some implementations, the cleavable linker is or contains a disulfide bond, ester, phosphate diester, sugar, or lipid.

[0176] In some implementations, the non-cleavable linker is chemically, enzymatically, or otherwise biochemically and physiologically stable. Therefore, the non-cleavable linker does not contain chemically, biochemically, enzymatically cleavable, or physiologically unstable bonds.

[0177] In some implementations, whether cleavable or non-cleavable, adapters can be assembled via a chemical linkage reaction between a regulatory nucleic acid and its conjugated binding moiety. The regulatory nucleic acid and binding moiety may or may not be modified initially to increase or promote their reactivity with each other. Such modifications can also increase or improve the specificity and degree of conjugation reaction, when desired. Adapters can be assembled in a single reaction or through a stepwise reaction until the desired adapter and regulatory nucleic acid have been prepared.

[0178] Non-limiting examples of chemical linking reactions that form conjugated pharmaceutical agents include reactions of various thiols to form disulfides, reactions of thiols to form thioethers with alkyl halides or maleimides, reactions of alkynes to form triazoles with azides (“click reactions”), reactions of aldehydes to form hydrazones, imines, and oxyimines with acylhydrazides, amines, or aminooxy compounds, and reactions of carboxylic acids to form amides, thioesters, and esters with amines, thiols, or alcohols (i.e., nucleophiles). Carboxylic acids can be formed in the presence of amines, thiols, or alcohols. In situActivation is necessary to make it reactive, or it can be pre-activated before the addition of a nucleophile, for example by conversion to an activated ester of N-hydroxysuccinimide (NHS) or sulfonated -NHS. Numerous reviews exist on chemical linking reactions, such as Spicer et al. (2018). Chem. Rev 2018, 118, 16, 7702-7743.

[0179] The reaction of thiols with maleimides is used very extensively, see, for example (Revasco et al. (2018) Chem. Eur. J. 10.1002 / chem.201803174), as are click reactions, see, for example (Fantoni et al. (2021) Chem. Rev., 121, 12, 7122-7154), and hydrazide formation (see HyNic Peptide Conjugation Protocol, Dirksen et al. (2006) J. Am. Chem. Soc., 128, 49, 15602-15603, Kozlov et al. (2004) Biopolymers73(5):621-30. doi: 10.1002 / bip.20009). Many companies sell chemical compounds and kits with protocols that enable the simple preparation of conjugates containing a variety of linkers.

[0180] As generally defined above and described in this article, a linker is a divalent group that connects / links a binding portion to a regulatory nucleic acid portion.

[0181] In some implementations, the connector is or contains a divalent straight chain or branched C 1-40 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 member saturated or partially unsaturated carbocyclic rings, phenyl, 3 to 6 member saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 member heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, in: R 1 It is an amino acid side chain; and R is selected from hydrogen or C with optional substitution. 1-6 Aliphatic, 3 to 6-membered saturated or partially unsaturated carbon rings, phenyl, 3 to 6-membered saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5 to 6-membered heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0182] In some implementations, the connector is or contains a divalent straight chain or branched C 1-35 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1 )2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0183] In some implementations, the connector is or contains a divalent straight chain or branched C1-30 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1 )2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0184] In some implementations, the connector is or contains a divalent straight chain or branched C 1-25 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1 )2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0185] In some implementations, the connector is or contains a divalent straight chain or branched C 1-20 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0186] In some implementations, the connector is or contains a divalent straight chain or branched C 1-15 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1 )2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0187] In some implementations, the connector is or contains a divalent straight chain or branched C 1-10 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1)2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0188] In some implementations, the connector is or contains a divalent straight chain or branched C 1-5 An aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by groups selected from: -CH(R 1 )-、-C(R 1 )2-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -O C(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, 3 to 6 saturated or partially unsaturated carbon rings, phenyl, 3 to 6 saturated or partially unsaturated heterocycles having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5 to 6 heteroaryl rings having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0189] In some implementations, the connector is or includes a structure selected from the following: , , or , Where X is NH or O.

[0190] In some embodiments, the cleavable linker is a cathepsin-cleavable linker. In some such embodiments, the linker is or contains a valine-citrulline (Val-Cit) motif. Where R is hydrogen or C 1-6 Lipids.

[0191] In some embodiments, the valine-citrulline linker is or contains .

[0192] In some embodiments, the valine-citrulline linker is or contains Where R is hydrogen or C 1-6 Lipids.

[0193] In some embodiments, the connector comprises a disulfide bond. In some embodiments, the connector comprises a polyethylene glycol moiety (e.g., -(CH2CH2O)). b -), where b is 1-50.

[0194] In some embodiments, the connector is or contains a group selected from the following:

[0195] Each of k, m, n, p, q, r, s, t, u, v, w, x, y, and z is 1-20; and R is hydrogen or C. 1-10 Lipids.

[0196] In some implementations, k is 3.

[0197] In some implementations, m is 3.

[0198] In some implementations, n is 2. In some implementations, n is 12.

[0199] In some implementations, p is 3.

[0200] In some implementations, each of m and p is 3.

[0201] In some implementations, q is 1.

[0202] In some implementations, r is 3. In some implementations, r is 4. In some implementations, r is 6.

[0203] In some implementations, s is 3. In some implementations, s is 4. In some implementations, s is 6.

[0204] In some implementations, each of r and s is 3. In some implementations, each of r and s is 4. In some implementations, each of r and s is 6.

[0205] In some implementations, t is 3. In some implementations, t is 5.

[0206] In some implementations, u is 3. In some implementations, u is 5.

[0207] In some implementations, each of t and u is 3. In some implementations, each of t and u is 5.

[0208] In some implementations, v is 3.

[0209] In some implementations, w is 4.

[0210] In some implementations, x is 8.

[0211] In some implementations, y is 2.

[0212] In some implementations, z is 1.

[0213] In embodiments, as shown in Formula I, certain polybasic structures of this disclosure may optionally be linked to one or more amino acids, which, in exemplary structures, are also linked to L-joints as defined herein. In such embodiments, the amino acid groups AA1, AA2, AA3 are each independently considered as chemical bonds (where no amino acid is present at that position) or selected from the following amino acids: .

[0214] In some embodiments, the specific L-groups of this disclosure include those wherein L is Or the group of the branch link, Where X is or , Or where X is: Where L is an optional connector, and Where M is Y = O, S or NSO2Me, and Z is the nucleic acid payload.

[0215] Nucleic acid payload In many embodiments, the payload portion used in this disclosure is or comprises an entity whose presence in relevant cells (e.g., cells of a tissue) achieves a specific effect (e.g., a specific detectable effect) (e.g., associated with it). In some embodiments, the associated effect is or comprises a specific biological and / or physiological effect. In some embodiments, the associated effect is or comprises an increase or decrease in the level or activity of a specific nucleic acid (or form thereof) in the cell.

[0216] In some embodiments, the payload portion is or contains nucleic acids. In some embodiments, the payload portion is or contains single-stranded nucleic acids. In other embodiments, the payload portion is or contains double-stranded nucleic acids. In some embodiments, the payload portion is or contains oligonucleotides.

[0217] In some embodiments, the regulatory nucleic acid of this disclosure has approximately 10-60 nucleotides, approximately 10-59 nucleotides, approximately 10-58 nucleotides, approximately 10-57 nucleotides, approximately 10-56 nucleotides, approximately 10-55 nucleotides, approximately 10-54 nucleotides, approximately 10-53 nucleotides, approximately 10-52 nucleotides, approximately 10-51 nucleotides, approximately 10-50 nucleotides, approximately 10-49 nucleotides, approximately 10-48 nucleotides, approximately 10-47 nucleotides, approximately 10-46 nucleotides, approximately 10-45 nucleotides, approximately 10-44 nucleotides, approximately 10-43 nucleotides, approximately 10-42 nucleotides, approximately 10-41 nucleotides, approximately 10-40 nucleotides, approximately 10-39 nucleotides, approximately 10-38 nucleotides, approximately 10-37 nucleotides, approximately 10-36 nucleotides, and approximately 10-3... The length and / or chain length (for multi-stranded regulatory nucleic acids, such as dsRNA) are within the range of 5 nucleotides, approximately 10-34 nucleotides, approximately 10-33 nucleotides, approximately 10-32 nucleotides, approximately 10-31 nucleotides, approximately 10-30 nucleotides, approximately 10-29 nucleotides, approximately 10-28 nucleotides, approximately 10-27 nucleotides, approximately 10-26 nucleotides, approximately 10-25 nucleotides, approximately 10-24 nucleotides, approximately 10-23 nucleotides, approximately 10-22 nucleotides, approximately 10-21 nucleotides, approximately 10-20 nucleotides, approximately 10-19 nucleotides, approximately 10-18 nucleotides, approximately 10-17 nucleotides, approximately 10-16 nucleotides, approximately 10-15 nucleotides, approximately 10-14 nucleotides, approximately 10-13 nucleotides, approximately 10-12 nucleotides, and approximately 10-11 nucleotides.In some embodiments, the regulatory nucleic acid of this disclosure has approximately 11-60 nucleotides, approximately 12-60 nucleotides, approximately 13-60 nucleotides, approximately 14-60 nucleotides, approximately 15-60 nucleotides, approximately 16-60 nucleotides, approximately 17-60 nucleotides, approximately 18-60 nucleotides, approximately 19-60 nucleotides, approximately 20-60 nucleotides, approximately 21-60 nucleotides, approximately 22-60 nucleotides, approximately 23-60 nucleotides, approximately 24-60 nucleotides, approximately 25-60 nucleotides, approximately 26-60 nucleotides, approximately 27-60 nucleotides, approximately 28-60 nucleotides, approximately 29-60 nucleotides, approximately 30-60 nucleotides, approximately 31-60 nucleotides, approximately 32-60 nucleotides, approximately 33-60 nucleotides, approximately 34-60 nucleotides, and approximately 35-60 nucleotides. The length and / or chain length (for multi-stranded regulatory nucleic acids, such as dsRNA) are approximately 36-60 nucleotides, 37-60 nucleotides, 38-60 nucleotides, 39-60 nucleotides, 40-60 nucleotides, 41-60 nucleotides, 42-60 nucleotides, 43-60 nucleotides, 44-60 nucleotides, 45-60 nucleotides, 46-60 nucleotides, 47-60 nucleotides, 48-60 nucleotides, 49-60 nucleotides, 50-60 nucleotides, 51-60 nucleotides, 52-60 nucleotides, 53-60 nucleotides, 54-60 nucleotides, 55-60 nucleotides, 56-60 nucleotides, 57-60 nucleotides, 58-60 nucleotides, and 59-60 nucleotides.

[0218] In some embodiments, the length of the regulatory nucleic acid disclosed herein is about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, and about... 34 nucleotides, approximately 35 nucleotides, approximately 36 nucleotides, approximately 37 nucleotides, approximately 38 nucleotides, approximately 39 nucleotides, approximately 40 nucleotides, approximately 41 nucleotides, approximately 42 nucleotides, approximately 43 nucleotides, approximately 44 nucleotides, approximately 45 nucleotides, approximately 46 nucleotides, approximately 47 nucleotides, approximately 48 nucleotides, approximately 49 nucleotides, approximately 50 nucleotides, approximately 51 nucleotides, approximately 52 nucleotides, approximately 53 nucleotides, approximately 54 nucleotides, approximately 55 nucleotides, approximately 56 nucleotides, approximately 57 nucleotides, approximately 58 nucleotides, approximately 59 nucleotides, approximately 60 nucleotides.

[0219] In some embodiments, the nucleic acid agent (e.g., an oligonucleotide agent) used according to this disclosure may comprise a single strand. In some embodiments, the nucleic acid may comprise more than one strand. In some embodiments, the nucleic acid may comprise one or more double-stranded portions. In some such embodiments, some or all of these portions may be formed by self-hybridization of sequences on a single strand; in some embodiments, some or all of these portions may be formed by hybridization of independent strands. In some embodiments, the nucleic acid comprising one or more double-stranded portions may comprise one or more gaps or notches and / or one or more protrusions or loops.

[0220] In some embodiments, the nucleic acid agents (e.g., oligonucleotide agents) used according to this disclosure may contain one or more structural features or properties associated with their mode of action. For example, those skilled in the art are familiar with a large body of literature on the structural features of oligonucleotides that trigger the degradation of their targets (e.g., by recruiting RNase H (such oligonucleotides are often referred to as “antisense” agents or “ASO”) and / or other elements of the Dicer and / or RNA-induced silencing complex (RISC) (such oligonucleotides are often referred to as “siRNA” agents), and / or regulate the splicing of target transcripts (e.g., making one splice form preferred over another) and / or act as guide RNA to recruit other mechanisms (e.g., nucleases, such as CRISPR / Cas or dsRNA-binding proteins or their conjugates) to specific nucleic acid sequences or as aptamers to bind to specific targets.

[0221] In some embodiments, the nucleic acid is or contains an interfering RNA (RNAi) agent. In some embodiments, the RNA is or contains a short interfering RNA (siRNA) agent. In some embodiments, the RNA is or contains a microRNA (miRNA) agent. In some embodiments, the nucleic acid is or contains a guide RNA (gRNA) agent.

[0222] In some embodiments, the nucleic acid is or contains a short interfering RNA (siRNA) agent. In some embodiments, the nucleic acid containing the siRNA agent may be linked to the target moiety at the sense strand (e.g., directly or indirectly). In some embodiments, the nucleic acid containing the siRNA agent may be linked to the target moiety at the antisense strand (e.g., directly or indirectly). In some embodiments, the nucleic acid containing the siRNA agent may be linked to the target moiety at the 5' end of the siRNA agent (e.g., directly or indirectly). In some embodiments, the nucleic acid containing the siRNA agent may be linked to the target moiety at the 3' end of the siRNA agent (e.g., directly or indirectly).

[0223] In some implementations, nucleic acids are or contain exon skippers, exon inclusion agents, or other splicing modulators.

[0224] In some implementations, nucleic acids are or contain aptamers.

[0225] In some embodiments, the nucleic acid agent is or contains an antisense oligonucleotide (ASO). In some embodiments, ASO regulates gene expression via an RNase H-mediated mechanism. In some embodiments, ASO regulates gene expression via steric hindrance.

[0226] In some implementations, the nucleic acid agent is or contains phosphoryldiamine morpholino oligonucleotide (PMO).

[0227] In some implementations, the nucleic acid agent is or contains peptide-nucleic acid (PNA).

[0228] In some implementations, the nucleic acid agent is or contains a nucleic acid analog, such as an RNA analog or a DNA analog, or a combination thereof.

[0229] In some embodiments, the nucleic acid may be linked to the target region at the sense strand (e.g., directly or indirectly). In some embodiments, the nucleic acid may be linked to the target region at the antisense strand (e.g., directly or indirectly). In some embodiments, the nucleic acid may be linked to the target region at the 5' end of the nucleic acid (e.g., directly or indirectly). In some embodiments, the nucleic acid may be linked to the target region at the 3' end of the nucleic acid (e.g., directly or indirectly).

[0230] For example, in some embodiments, the nucleic acid analog comprises one or more modified (as opposed to canonical DNA and / or RNA) nucleotides. In some embodiments, the modified nucleotide comprises one or more of the following: a modified backbone, a modified nucleotide, a modified sugar (e.g., modified ribose or modified deoxyribose), or a combination thereof. In some embodiments, the modified nucleotide may be or contains one or more naturally occurring modifications; in some embodiments, the modified nucleotide may be or contains one or more non-naturally occurring modifications.

[0231] In some implementations, the nucleic acid analog contains one or more bonds that are not phosphodiester bonds (e.g., are or contain thiophosphate bonds or phosphoryldiamine bonds).

[0232] In some embodiments, the nucleic acid analog comprises one or more morpholino subunits linked together by phosphorus-containing bonds. In some embodiments, one or more morpholino subunits in the oligonucleotide analog are linked by phosphoryldiamine bonds. The synthesis, structure, and binding properties of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, as well as PCT application No. PCT / US07 / 11435 (Cat Bond) and U.S. Serial No. 08 / 012,804 (Improved Synthesis), all of which are incorporated herein by reference. The morpholino subunits linked by phosphoryldiamine bonds are disclosed in U.S. Patent No. 11,071,749, the entire contents of which are incorporated herein by reference. In some embodiments, the nucleic acid agent is or comprises aPMO. In some implementations, the PMO is essentially uncharged, for example, having a neutral charge.

[0233] In some implementations, the nucleic acid agent has a negative charge.

[0234] In some implementations, the nucleic acid agent is essentially uncharged, for example, having a neutral charge.

[0235] Those skilled in the art will understand that, in some embodiments, the nucleic acid agent used according to this disclosure may include one or more DNA residues or analogs thereof, one or more RNA residues or analogs thereof, and / or combinations thereof. Furthermore, those skilled in the art will understand that, in some embodiments, the nucleic acid agent may include one or more phosphodiester bonds, thiophosphate bonds, or other suitable bonds.

[0236] In some implementations, nucleic acid agents contain natural residues, such as DNA residues and / or RNA residues.

[0237] In some implementations, the nucleic acid agent comprises one or more analogs, such as DNA analogs and / or RNA analogs.

[0238] In some implementations, the nucleic acid agent contains DNA residues and / or RNA residues, such as natural residues or analogs.

[0239] In some embodiments, the nucleic acid comprises one or more chiral centers (e.g., possibly present in, for example, phosphate thioester bonds). In some embodiments, a formulation of a nucleic acid having a chiral center is stereopure with respect to that center because it contains only one stereoisomer of that center. In some embodiments, both stereoisomers are present. In some embodiments, the formulation represents a racemic mixture of stereoisomers at that location. In some embodiments, a formulation of a nucleic acid having more than one chiral bond may be stereopure with respect to one or more centers and mixed with respect to one or more other centers (e.g., racemic). In some embodiments, the formulation may be stereopure at all chiral centers. In some embodiments, the formulation may be racemic (e.g., at all or all chiral centers).

[0240] In some embodiments, the nucleic acid comprises a structure containing a first wing sequence, a spacer sequence, and a second wing sequence. Nucleic acids containing such wing-space-wing sequences are generally referred to as spacer aggregates. In some embodiments, the spacer sequence is side-joined with the first and second wing sequences. In some embodiments, the spacer sequence comprises about 6-10 nucleotides. In some embodiments, the wing sequence comprises one or more nucleotides. In some embodiments, the wing sequence comprises one or more modified nucleotides, such as those disclosed herein. In some embodiments, the spacer aggregate functions by recruiting RNase H. In some embodiments, the spacer aggregate is a chimeric antisense oligonucleotide (ASO) containing a central sequence of phosphate-thioester DNA nucleotides (forming a “DNA spacer”) with a sequence of modified RNA residues side-joined at each end. Not wishing to be bound by theory, such wing-joint modified RNA residues are thought to protect the DNA spacer region from nuclease degradation, while the central DNA spacer region allows RNase-H-mediated cleavage of target RNA.

[0241] In some embodiments, the nucleic acid includes a protruding end. In some embodiments, the protruding end is a 3' protruding end or a 5' protruding end. In some embodiments, the protruding end is a 3' protruding end. In some embodiments, the protruding end includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the nucleic acid is double-stranded and includes a protruding end.

[0242] In some implementations, the nucleic acid contains at least one stem-loop structure.

[0243] The oligonucleotides disclosed herein typically contain at least one sequence element that hybridizes to a target sequence. In some embodiments, the nucleic acid agent (e.g., the oligonucleotide) is or contains an antisense sequence element. In some embodiments, the antisense sequence element is complementary to at least a portion of one or more of the following, for example, exons, introns, untranslated regions, splice sites, promoter regions, enhancer regions, or non-coding regions in a gene transcript. In some embodiments, the antisense sequence element is complementary to a portion of the target sequence in the sense strand.

[0244] In some embodiments, the nucleic acid comprises sequence elements that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target sequence in the sense strand. In some embodiments, the nucleic acid comprises sequence elements that are complementary to (i.e., 100% complementary to) the target sequence in the sense strand.

[0245] In some embodiments, the nucleic acid comprises sequence elements that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target sequence in the antisense strand. In some embodiments, the nucleic acid comprises sequence elements that are complementary to (i.e., 100% complementary to) the target sequence in the antisense strand.

[0246] In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 80% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 85% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 90% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 95% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 96% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 97% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 98% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides that are at least 99% complementary to a portion of the target sequence. In some embodiments, the nucleic acid comprises at least one sequence element having at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides that are 100% complementary to a portion of the target sequence.

[0247] In some implementations, the nucleic acid comprises two or more sequence elements having at least three, four, five, six, seven, eight, nine, or ten consecutive nucleotides that are at least 80% complementary to a portion of the target sequence.

[0248] In some embodiments, the nucleic acid binds to at least a portion of the target via Watson-Crick base pairing. In some embodiments, the nucleic acid binds to at least a portion of the target via Hoogsteen base pairing and / or other non-canonical base pairing.

[0249] In some embodiments, the nucleic acid (e.g., oligonucleotide) is characterized in that when the oligonucleotide, a composition containing an oligonucleotide, or a conjugate containing an oligonucleotide is delivered to a cell, tissue, or organism expressing the target, a decrease in the expression and / or activity of the target is observed compared to cells, tissues, or organisms that have not been delivered the oligonucleotide, the composition containing an oligonucleotide, or the conjugate containing an oligonucleotide.

[0250] In some embodiments, the nucleic acid (e.g., oligonucleotide) is characterized in that when the oligonucleotide, a composition containing an oligonucleotide, or a conjugate containing an oligonucleotide is delivered to a cell, tissue, or organism expressing the target, a decrease in target expression and / or activity is observed compared to a cell, tissue, or organism that does not express the target (e.g., no detectable target expression).

[0251] In some embodiments, the nucleic acid (e.g., oligonucleotide) is characterized in that, when the oligonucleotide, a composition containing an oligonucleotide, or a conjugate containing an oligonucleotide is delivered to a cell, tissue, or organism expressing the target, a change in the expression and / or activity of the target is observed relative to that observed with a suitable reference agent known to have a specific effect on the target. In some embodiments, the manner and / or extent to which the expression and / or activity of the target is altered is comparable to, or determined relative to, that observed with a suitable reference agent known to have a specific effect on the target. In some embodiments, the reference agent may be a positive control reference agent. In some embodiments, the reference may be a negative control reference agent.

[0252] In some implementations, the nucleic acid (e.g., oligonucleotide) is characterized in that, when delivered to cells, tissues, or organisms expressing the target, the expression and / or activity of the target are modulated (e.g., reduced) compared to cells, tissues, or organisms where the oligonucleotide was not delivered.

[0253] The regulatory nucleic acid agents disclosed herein can be programmed to target human target genes, including conserved portions of such genes in target orthologs of other mammalian species. Without being theoretically limited, it is believed that combinations or subcombinations of the aforementioned properties and specific target sites, or specific modifications in these regulatory nucleic acid agents, confer improved efficacy, stability, potency, durability, and safety to the regulatory nucleic acid agents of this disclosure.

[0254] Therefore, this disclosure provides methods for treating and / or preventing target gene and / or target RNA-related conditions using conjugates, such as glomerular diseases, tubular diseases, other kidney diseases, congenital metabolic disorders, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, or viral infections or combinations thereof, wherein the conjugates comprise a regulatory nucleic acid agent composition that affects RNase H-mediated degradation and / or RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the target gene.

[0255] In some embodiments, the conjugated regulatory nucleic acid agent of this disclosure comprises an RNA strand (antisense strand) having a length of up to about 30 nucleotides or less, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 2 A region of 0-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, which is substantially complementary to at least a portion of the mRNA transcript of the target gene.

[0256] In some embodiments, one or both strands of the antisense oligonucleotide (ASO) and / or double-stranded RNAi agent of this disclosure are up to 66 nucleotides in length, such as 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides, having a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the target gene. In some embodiments, such ASO and / or iRNA agents with a longer antisense strand preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense strand and the antisense strand form a doublet of 18-30 consecutive nucleotides.

[0257] The ASOs and / or iRNAs disclosed herein can target and inhibit and / or degrade the mRNA of corresponding genes (target genes) in mammals. In vitro assays demonstrate that ASOs and / or iRNAs targeting target genes can effectively mediate RNAi, leading to significant inhibition of target gene expression. Therefore, methods and compositions including these ASOs and / or iRNAs can be used to treat subjects suffering from conditions related to target genes and / or target RNAs, such as glomerular diseases, tubular diseases, other kidney diseases, congenital metabolic disorders, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, viral infections, etc.

[0258] Therefore, this disclosure provides methods and combination therapies for treating subjects with conditions that would benefit from regulation (e.g., inhibition or reduction) of the expression of target genes using conjugated regulatory nucleic acid agents, such as target gene and / or target RNA-related conditions, including glomerular diseases, tubular diseases, other kidney diseases, congenital metabolic disorders, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, viral infections, or combinations thereof, etc., wherein the conjugated regulatory nucleic acid agents are such as conjugated ASO and / or iRNA compositions that affect RNase H-mediated degradation and / or RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcripts of target genes.

[0259] This disclosure also provides a method for preventing at least one symptom in a subject suffering from a condition that would benefit from the suppression or reduction of target gene expression, such as glomerular disease, tubular disease, other kidney diseases, congenital metabolic defects, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, viral infections, combinations thereof, etc.

[0260] This specification discloses compositions, uses, and methods for preparing and using compositions containing regulatory nucleic acid agents (e.g., ASO and / or iRNA, etc.) to regulate (e.g., inhibit) the expression of target genes, and for treating subjects who will benefit from the regulation (e.g., inhibition and / or reduction) of target gene expression (e.g., subjects susceptible to or diagnosed with target gene and / or target RNA-related disorders).

[0261] In some aspects, this disclosure provides ASO and / or iRNA for inhibiting the expression of target genes. In some embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of target genes in cells such as cells in a subject's body, the subject being, for example, a mammal, such as a person susceptible to a condition related to the target gene and / or target RNA, such as a metabolic condition, including nephropathy and various other kidney diseases or disorders.

[0262] dsRNA agent The double-stranded region of the dsRNA disclosed herein can have any length that allows the desired target RNA to be specifically degraded via the RISC pathway, and the length can range from about 19 to 36 base pairs, for example, a length of about 19-30 base pairs, such as a length of about 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, or 36 base pairs, such as a length of about 1... 9-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In some embodiments, the length of the double-stranded region is 19-21 base pairs, for example, 21 base pairs. The ranges and lengths mentioned above, as well as the ranges and lengths between them, are also considered part of this disclosure.

[0263] The two strands forming a double-stranded structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of a larger molecule and are therefore linked by an unbroken nucleotide chain between the 3' end of one strand and the 5' end of the other strand forming the double-stranded structure, the linking RNA strands are called a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4-10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4-8 nucleotides.

[0264] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules do not need to be covalently linked, but can be covalently linked. When the two strands are covalently linked in a manner other than a continuous nucleotide chain between the 3' end of one strand and the 5' end of the other strand forming a duplex structure, the linker structure is called a "dsRNA adapter." The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi can contain one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides (e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides). In other embodiments, at least one strand of the RNAi agent contains a 5' overhang of at least one nucleotide. In some embodiments, at least one strand contains a 5' overhang of at least two nucleotides (e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides). In still other embodiments, one strand of the RNAi agent contains an overhang of at least one nucleotide at both the 3' and 5' ends.

[0265] In some embodiments, the iRNA agent disclosed herein is dsRNA, each strand of which contains 19-23 nucleotides and interacts with a target RNA sequence (e.g., a target gene) to guide the cleavage of the target RNA.

[0266] In some embodiments, the iRNA disclosed herein is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence (e.g., a target mRNA sequence) to guide the cleavage of the target RNA.

[0267] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain at least one nucleotide overhang; alternatively, the overhang may contain at least two, three, four, five, or more nucleotides. The nucleotide overhang may contain or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotide overhang may be present at the 5' end, 3' end, or both of the antisense or sense strand of the dsRNA.

[0268] In one embodiment, the antisense strand of the dsRNA has 1-10 nucleotide overhangs at either the 3' or 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In another embodiment, one or more nucleotides in the overhangs are replaced by nucleoside phosphate thioesters.

[0269] In some embodiments, the antisense strand of the dsRNA has 1-10 nucleotides at its 3' or 5' end, such as 0-3, 1-3, 2-4, 2-5, 4-10, or 5-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In one embodiment, the sense strand of the dsRNA has 1-10 nucleotides at its 3' or 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In another embodiment, one or more nucleotides in the overhangs are replaced by nucleoside phosphate thioesters.

[0270] In some embodiments, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the overhang on the sense strand or antisense strand, or both, may include an extension length longer than 10 nucleotides (e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides). In some embodiments, the extended overhang is on the sense strand of the duplex. In some embodiments, the extended overhang is located at the 3' end of the sense strand of the duplex. In some embodiments, the extended overhang is located at the 5' end of the sense strand of the duplex. In some embodiments, the extended overhang is located on the antisense strand of the duplex. In some embodiments, the extended overhang is located at the 3' end of the antisense strand of the duplex. In some embodiments, the extended overhang is located at the 5' end of the antisense strand of the duplex. In some embodiments, one or more nucleotides in the extended overhang are replaced by nucleoside phosphate thioesters. In some embodiments, the overhang includes a self-complementary portion, enabling the overhang to form a hairpin structure stable under physiological conditions.

[0271] "Blunt" or "blunt end" means that there are no unpaired nucleotides at the ends of a double-stranded RNA agent, i.e., no nucleotide overhangs. A "blunt-end" double-stranded RNA agent is double-stranded throughout its entire length, meaning there are no nucleotide overhangs at either end of the molecule. The RNAi agents disclosed herein include RNAi agents without a nucleotide overhang at one end (i.e., agents with one overhang and one blunt end) or RNAi agents without nucleotide overhangs at either end. Most commonly, such molecules are double-stranded throughout their entire length.

[0272] The term “antisense strand” or “guide strand” refers to the strand of iRNA, such as dsRNA, which includes regions that are substantially complementary to the target sequence (e.g., target mRNA).

[0273] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence, such as a target nucleotide sequence as defined herein). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most tolerant mismatches are in terminal regions, such as within 5, 4, or 3 nucleotides at the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNA agents of this disclosure include nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agents of this disclosure includes no more than 4 mismatches with the target mRNA, for example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense double-stranded RNA agents of this disclosure include no more than 4 mismatches with the sense strand, for example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agents of this disclosure include nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of this disclosure includes no more than four mismatches with the antisense strand; for example, the sense strand includes four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, within five, four, or three nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, within the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatches are not in the seed region.

[0274] Therefore, RNAi agents as described herein may contain one or more mismatches with the target sequence. In one embodiment, an RNAi agent as described herein contains no more than three mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, an RNAi agent as described herein contains no more than two mismatches. In one embodiment, an RNAi agent as described herein contains no more than one mismatch. In one embodiment, an RNAi agent as described herein contains zero mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch may optionally be restricted to the last five nucleotides from the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the target gene region typically contains no mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch with the target sequence effectively inhibits the expression of the target gene. It is important to consider the efficacy of mismatched RNAi agents in suppressing the expression of target genes, especially if specific complementary regions of the target gene are known to have polymorphic sequence variations in the population.

[0275] As used herein, the term “sense strand” or “passenger strand” refers to the strand of iRNA that includes regions substantially complementary to the regions of the antisense strand as defined herein.

[0276] As used in this article, “virtually all nucleotides are modified” means that most, but not all, nucleotides are modified, and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0277] As used herein, the term "cleavage region" refers to the region immediately adjacent to a cleavage site. A cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases located at either end of the cleavage site and immediately adjacent to it. In some embodiments, the cleavage region comprises two bases located at either end of the cleavage site and immediately adjacent to it. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region comprises nucleotides 11, 12, and 13.

[0278] As used herein, unless otherwise stated, the term "complementary" when used to describe a first nucleotide sequence associated with a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PPIES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions that may be encountered in vivo. Depending on the final application of the hybridized nucleotides, those skilled in the art will be able to determine a set of conditions most suitable for testing the complementarity of the two sequences.

[0279] Complementary sequences within iRNAs (e.g., within dsRNAs as described herein) comprise base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence along the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “perfectly complementary” to each other. However, when the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be perfectly complementary, or they may form one or more, but typically no more than five, four, three, or two mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to their final application, such as repressing gene expression via a RISC pathway. However, when two oligonucleotides are engineered to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches that determine complementarity. For example, for the purposes described herein, a dsRNA comprising one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide (where the longer oligonucleotide contains a 21-nucleotide sequence perfectly complementary to the shorter oligonucleotide) may still be referred to as “perfectly complementary.”

[0280] As used herein, “complementary” sequences may also include non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, or formed entirely from them, provided that the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing or Hoogstein base pairings.

[0281] The terms “complementary,” “fully complementary,” and “substantially complementary” are used herein for base matching between the sense and antisense strands of dsRNA or between the antisense strand of a double-stranded RNA agent and the target sequence, as will be understood from the context of their use.

[0282] As used herein, a polynucleotide that is substantially complementary to at least a portion of messenger RNA (mRNA) is a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding a target polypeptide). For example, a polynucleotide is complementary to at least a portion of the target mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the target polypeptide.

[0283] Technicians are well aware that dsRNAs with a double-stranded structure of approximately 20 to 23 base pairs (e.g., 21 base pairs) are considered particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures can also be effective (Chu and Rana (2007) RNA14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).

[0284] Typically, "iRNA" comprises chemically modified ribonucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications used in dsRNA molecules are covered by "iRNA".

[0285] In some embodiments of this disclosure, if a deoxynucleotide is present in the RNAi agent, the presence of the deoxynucleotide can be considered as constituting a modified nucleotide.

[0286] The dsRNA agents disclosed herein include those having an antisense strand comprising a complementary region complementary to at least a portion of the mRNA formed during the expression of the target gene. In embodiments, the complementary region is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length). When contacted with cells expressing the target gene, the iRNA inhibits the expression of the target gene (e.g., human, primate, non-primate, or rat target gene) by at least about 50%, as determined by, for example, PCR-based or branched-strand DNA (bDNA) methods, or by protein-based methods, such as by immunofluorescence analysis, using techniques such as Western blotting or flow cytometry. In some embodiments, the inhibition of expression is determined in a suitable somatic cell line using, for example, a concentration of 10 nM siRNA, by qPCR methods known in the art. In some embodiments, inhibition of in vivo expression is determined by knocking down the human gene in rodents expressing the human gene (e.g., mice expressing the human target gene or mice infected with AAV), for example when administered in a single dose, such as at the lowest point of RNA expression at 3 mg / kg.

[0287] dsRNA comprises two RNA strands that, under the conditions of dsRNA use, are complementary and hybridize to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary to, and usually perfectly complementary to, the target sequence. The target sequence may be derived from the mRNA sequence formed during the expression of the target gene. The other strand (the sense strand) contains a region complementary to the antisense strand, such that the two strands hybridize and form a double-stranded structure when combined under appropriate conditions. As described elsewhere herein and as is known in the art, the complementary sequence of dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide.

[0288] Typically, the length of a double-stranded structure is 15 to 30 base pairs, for example, lengths of 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 1... 9-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In some embodiments, the length of the double-stranded structure is 18 to 25 base pairs, for example, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs, for example, a length of 19-21 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

[0289] Similarly, the length of the region complementary to the target sequence is 15 to 30 nucleotides, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, for example, 19-23 nucleotides or 21-23 nucleotides in length. Ranges and lengths between the above ranges and lengths are also considered part of this disclosure.

[0290] In some implementations, the length of the double-stranded structure is 19 to 30 base pairs. Similarly, the length of the region complementary to the target sequence is 19 to 30 nucleotides.

[0291] In some embodiments, the dsRNA is about 19 to about 23 nucleotides long or about 25 to about 30 nucleotides long. Typically, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As those skilled in the art will also recognize, the region of RNA targeted for cleavage is typically a portion of a larger RNA molecule (typically an mRNA molecule). In relevant cases, the “portion” of the mRNA target is a sufficiently long contiguous sequence of the mRNA target to make it a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0292] Those skilled in the art will also recognize that the double-stranded region is the major functional part of the dsRNA, for example, about 19 to about 30 base pairs, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30. The regions are double-stranded regions of 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Therefore, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is dsRNA, in relation to a functional double-stranded region of, for example, 15-30 base pairs that is processed to target the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, the miRNA is dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another implementation, by cleaving larger dsRNAs, iRNAs that are useful for target gene expression are not generated in target cells.

[0293] As described herein, dsRNAs may further include one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang have better repressive properties compared to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The overhangs may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present at the 5' end, 3' end, or both of the antisense or sense strand of the dsRNA.

[0294] dsRNA can be synthesized using standard methods known in the art. The double-stranded RNAi compounds of this disclosure can be prepared using a two-step procedure. First, a single strand of the double-stranded RNA molecule is prepared separately. Then, the component strand is annealed. The single strand of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotide chains containing non-natural or modified nucleotides. Similarly, the single-stranded oligonucleotides of this disclosure can be prepared using liquid-phase or solid-phase organic synthesis, or both.

[0295] The modified regulatory nucleic acid of this disclosure In some embodiments, the conjugated regulatory nucleic acid agents of this disclosure comprise one or more modified (as opposed to canonical DNA and / or RNA) nucleotides. In some embodiments, the modified nucleotides comprise one or more of the following: a modified backbone, a modified nucleotide, a modified sugar (e.g., modified ribose or modified deoxyribose), or a combination thereof. In some embodiments, the modified nucleotides may be or contain one or more naturally occurring modifications; in some embodiments, the modified nucleotides may be or contain one or more non-naturally occurring modifications.

[0296] In some implementations, the regulatory nucleic acid agent contains one or more bonds that are not phosphodiester bonds (e.g., are or contain thiophosphate bonds or phosphoryldiamine bonds).

[0297] In some embodiments, the regulatory nucleic acid agent comprises one or more morpholino subunits linked together by phosphorus-containing bonds. In some embodiments, one or more morpholino subunits in the oligonucleotide agent are linked by phosphoryldiamine bonds. The synthesis, structure, and binding properties of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, as well as PCT application No. PCT / US07 / 11435 (Cat Bond) and U.S. Serial No. 08 / 012,804 (Improved Synthesis), all of which are incorporated herein by reference. The phosphoryldiamine-linked morpholino subunits are disclosed in U.S. Patent No. 11,071,749, the entire contents of which are incorporated herein by reference. In some embodiments, the regulatory nucleic acid agent is or comprises PMO. In some implementations, the PMO is essentially uncharged, for example, having a neutral charge.

[0298] In some implementations, the regulatory nucleic acid agent has a negative charge.

[0299] In some implementations, the regulatory nucleic acid agent is essentially uncharged, for example, having a neutral charge.

[0300] Those skilled in the art will understand that, in some embodiments, the regulatory nucleic acid agents of this disclosure may include one or more DNA residues or analogs thereof, one or more RNA residues or analogs thereof, and / or combinations thereof. Furthermore, those skilled in the art will understand that, in some embodiments, the regulatory nucleic acid agents of this disclosure may include one or more phosphodiester bonds, thiophosphate bonds, or other suitable bonds.

[0301] In some implementations, regulatory nucleic acid agents contain natural residues, such as DNA residues and / or RNA residues.

[0302] In some implementations, the regulatory nucleic acid agent comprises one or more analogs, such as DNA analogs and / or RNA analogs.

[0303] In some implementations, the regulatory nucleic acid agent contains DNA residues and / or RNA residues, such as natural residues or analogs.

[0304] In some embodiments, the regulatory nucleic acid of this disclosure comprises one or more chiral centers (e.g., possibly present in, for example, phosphate thioester bonds). In some embodiments, a formulation of a regulatory nucleic acid having a chiral center is stereopure with respect to that center because it contains only one stereoisomer of that center. In some embodiments, both stereoisomers are present. In some embodiments, the formulation represents a racemic mixture of stereoisomers at that location. In some embodiments, a formulation of a regulatory nucleic acid having more than one chiral bond may be stereopure with respect to one or more centers and mixed (e.g., racemic) with respect to one or more other centers. In some embodiments, the formulation may be stereopure at all chiral centers. In some embodiments, the formulation may be racemic (e.g., at all or all chiral centers).

[0305] In some embodiments, the regulatory nucleic acid of this disclosure comprises one or more modified nucleotides. In some embodiments, the modified nucleotides comprise one or more of the following: a modified backbone, modified nucleotides, modified ribose, modified deoxyribose, or combinations thereof.

[0306] In some embodiments, the modified nucleotide is selected from: 2'-O-methyl modified nucleotides, 5-methylcytidine, 5-methyluridine, nucleotides containing a 5'-thiophosphate group, morpholinonucleotides (e.g., PMO), terminal nucleotides linked to a cholesterol derivative or a dodecanoic acid bis(decylamide) group, 2'-deoxy-2'-fluorinated modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debased nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholinonucleotides (e.g., PMO), aminophosphates, non-natural bases containing nucleotides based on a phosphoryl guanidine (PN) backbone, or combinations thereof.

[0307] In some embodiments, the modified nucleobases comprise C7-modified denitroadenine, C7-modified denitroguanosine, C5-modified cytosine, C5-modified uridine, N1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, 5-methoxycytidine, or combinations thereof.

[0308] In some embodiments, the modified sugar (e.g., modified ribose or modified deoxyribose) comprises: 2'-fluorine modification, 2'-O-methyl (2'OMe) modification, locked nucleic acid (LNA), 2'-fluoroarabinose nucleic acid (FANA), hexitol nucleic acid (HNA), 2'-O-methoxyethyl (2'MOE) modification, or combinations thereof.

[0309] In some embodiments, the modified backbone comprises phosphate thioester (PS) modification, phosphoryl guanidine (PN) modification, borophosphate modification, alkyl phosphonate nucleic acid (phNA), peptide nucleic acid (PNA), or a combination thereof.

[0310] In some embodiments, the regulatory nucleic acid agents of this disclosure comprise, for example, one or more modifications to the 5' end of the oligonucleotide. In some embodiments, the regulatory nucleic acid agents of this disclosure comprise a 5' amino group modification.

[0311] In some embodiments, the regulatory nucleic acid agents of this disclosure are partially modified (e.g., at least 5%) for a specific modification along the entire sequence length.

[0312] In some implementations, the regulatory nucleic acid agents of this disclosure are fully modified for specific modifications along the entire sequence length.

[0313] In some implementations, at least 5% of a specific nucleotide (e.g., A, G, C, T, or U) in the oligonucleotide is modified.

[0314] In some implementations, all (e.g., 100%) of the specific nucleotides (e.g., A, G, C, T, or U) of the oligonucleotide are modified.

[0315] In some embodiments, the nucleotide sequence of the regulatory nucleic acid agent (e.g., ASO and / or dsRNA) of this disclosure is unmodified and does not include, for example, chemical modifications or conjugations known in the art and described herein. In other embodiments, the nucleotide sequence of the regulatory nucleic acid agent (e.g., ASO and / or dsRNA) of this disclosure is chemically modified to enhance stability or other beneficial properties. In some embodiments of this disclosure, substantially all nucleotides of the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) are modified. In other embodiments of this disclosure, all nucleotides of the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) or substantially all nucleotides of the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) are modified such that no more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in one strand of the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.).

[0316] The nucleic acids provided in this disclosure can be synthesized or modified using methods established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL register (ed.), JohnWiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse linkage) or 3' terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.); base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base paired with an expanded pool of partners, base removal (de-base nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified backbone or RNA without native nucleoside internucleotide bonds. RNA with a modified backbone includes RNA without phosphorus atoms in its backbone. For the purposes of this specification, and as sometimes mentioned in the art, RNA modified without a phosphorus atom in its internucleotide backbone may also be considered an oligonucleotide. In some embodiments, the modified regulatory nucleic acid (e.g., ASO, dsRNA, etc.) will have a phosphorus atom on its internucleotide backbone.

[0317] Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thiophosphatidyl esters, thioalkylphosphonates, thioalkyl phosphate triesters, and borophosphates having normal 3'-5' bonds, their 2'-5' linked analogs, and those having opposite polarities, wherein adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of this disclosure, the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agents of this disclosure are in free acid form. In other embodiments of this disclosure, the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agents of this disclosure are in salt form. In one embodiment, the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agent of this disclosure is in sodium salt form. In some embodiments, when the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agent of this disclosure is in sodium salt form, sodium ions are present in the agent as counterions for substantially all phosphodiester and / or thiophosphate groups in the agent. Agents in which substantially all phosphodiester and / or thiophosphate bonds have sodium counterions include no more than 5, 4, 3, 2, or 1 phosphodiester and / or thiophosphate bonds without sodium counterions. In some embodiments, when the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agent of this disclosure is in sodium salt form, sodium ions are present in the agent as counterions for all phosphodiester and / or thiophosphate groups in the agent.

[0318] Representative U.S. patents teaching the preparation of the aforementioned phosphorus-containing bonds include, but are not limited to, U.S. patent numbers 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, and 5,453,4 96、5,455,233、5,466,677、5,476,925、5,519,126、5,536,821、5,541,316、5,550,111、5,563,253、5,571,799、5,587,361、5,625,050、6,028,188、6,124,445、6,160,109、6,169,170、6,172,209、6, U.S. Patent RE39464, the entire contents of which are hereby incorporated herein by reference.

[0319] RNA backbones excluding phosphorus modifications have backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatom and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatom or heterocyclic nucleoside bonds. These include those having: morpholino bonds (partially formed by the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones having mixed N, O, S, and CH2 component moieties.

[0320] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. patent numbers 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, and 5,470,967. The entire contents of 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439 are hereby incorporated herein by reference.

[0321] Consider suitable RNA mimics for regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agents used in the conjugations provided herein, wherein the sugar and nucleoside internucleotide bonds (i.e., backbone) of the nucleotide units are replaced by novel groups. The base units are retained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound in which RNA mimics have been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone (specifically, an aminoethylglycine backbone). Nucleobases are retained and directly or indirectly bound to the aza-nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of which are hereby incorporated herein by reference. Other PNA compounds suitable for use in the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agents of this disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0322] In some embodiments, the regulatory nucleic acids of this disclosure include RNA having a phosphate thioester backbone and oligonucleotides having a heteroatom backbone, and particularly the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [referred to as methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [wherein the native phosphate diester backbone is represented as --O--P--O--CH2--] of U.S. Patent No. 5,602,240, and the amide backbone of U.S. Patent No. 5,602,240. In some embodiments, the RNA provided herein has the morpholino backbone structure of U.S. Patent No. 5,034,506.

[0323] The modified RNA may also contain one or more substituted sugar moieties. The regulatory nucleic acids (e.g., conjugated regulatory nucleic acid agents (e.g., ASO, dsRNA, etc.)) provided herein may include one of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; o-ynyl, O-ynyl, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Exemplary suitable modifications include O[(CH2)] n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to approximately 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl groups, O-alkylaryl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalating agents, groups for improving the pharmacokinetic properties of ASO and / or iRNA or groups for improving the pharmacodynamic properties of ASO and / or iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2. Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both the R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0324] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluorine (2'-F). Similar modifications can also be made at other sites on the RNA of the ASO and / or iRNA (particularly at the 3' terminal nucleotide or the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked dsRNA). The ASO and / or iRNA may also have sugar mimics such as a cyclobutyl moiety to replace the pentofuranose sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. patent numbers 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are common to this application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0325] ASO and / or iRNA may also include nucleobase (generally referred to as "bases" in the art) modifications or substitutions. As used herein, "unmodified" or "native" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and native nucleobases such as deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, and 6-dichlorouracil. Pyrimidines, cytosines and thymines, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine and 3-deadenine and 3-deadenine. Other nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in *Modified Nucleosides in Biochemistry, Biotechnology and Medicine*, edited by Herdewijn, P., Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia Of Polymer Science and Engineering*, pages 858-859, edited by Kroschwitz, J. L., John Wiley & Sons, 1990; those disclosed by Englisch et al., *Angewandte Chemie*, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, *dsRNA Research and Applications*, pages 289-302, edited by Crooke, ST. and Lebleu, B., CRC Press, 1993. Certain of these nucleosides are particularly useful for increasing the binding affinity of the oligomers provided in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6 °C–1.2 °C (Sanghvi, YS, Crooke, ST and Lebleu, B. eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and is an exemplary base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modification.

[0326] Representative U.S. patents teaching the preparation of certain modified nucleosides and other modified nucleosides include, but are not limited to, U.S. patent numbers 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, and 5,594,121. The entire contents of 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672 and 7,495,088 are hereby incorporated herein by reference.

[0327] The RNA of ASO or iRNA agents can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety containing an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-inner conformation. Adding locked nucleic acids to siRNAs has been shown to increase the stability of siRNAs in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0328] The RNA of an ASO or iRNA agent can also be modified to include one or more restricted ethyl nucleotides. As used herein, a “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid containing a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in the S conformation, referred to herein as “S-cEt”.

[0329] The ASO or iRNA agent disclosed herein may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. The CRN locks the ribose ring into a stable conformation and increases hybridization affinity with mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, thereby reducing ribose ring puckering.

[0330] Representative publications teaching the preparation of certain of the aforementioned CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, the entire contents of which are hereby incorporated herein by reference.

[0331] In some embodiments, the ASO or iRNA agent of this disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. A UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, forming an unlocked “sugar” residue. In one instance, the UNA also encompasses monomers in which the bond between C1' and C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another instance, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are hereby incorporated by reference). Representative U.S. publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publications Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of which are hereby incorporated herein by reference.

[0332] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetaminohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline) (Hyp-C6), N-(acetyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosyl-uridine-3"-phosphate, reverse base dT (idT), and others. Publications regarding such modifications can be found in PCT Publication WO 2011 / 005861.

[0333] Other modifications to the nucleotides of the ASO or iRNA agents disclosed herein include 5' phosphate or 5' phosphate mimics, such as the 5' terminal phosphate or phosphate mimics on the antisense strand of the ASO or iRNA agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0334] In one aspect of this disclosure, the agent used in the methods and compositions of this disclosure is a single-stranded antisense oligonucleotide (ASO) molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can stoichiometrically inhibit translation by base pairing with mRNA and physically blocking translation mechanisms, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule can be about 14 to about 30 nucleotides in length and has a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can contain a sequence of at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any of the antisense sequences described herein.

[0335] As used herein, the phrase “contacting cells with iRNA (such as dsRNA)” includes contacting cells by any possible means. Contacting cells with iRNA includes contacting cells with iRNA in vitro or in vivo. Contact can be direct or indirect. Thus, for example, an individual performing this method may physically contact iRNA with cells, or alternatively, may place iRNA in a state that permits or leads to its subsequent contact with cells.

[0336] In vitro cell contact can be performed, for example, by incubating cells with iRNA. In vivo cell contact can be performed, for example, by injecting iRNA into or near the tissue containing the cells, or by injecting iRNA into another area (e.g., the bloodstream or subcutaneous space), such that the agent subsequently reaches the tissue containing the cells to be contacted. For example, iRNA may contain or be conjugated to a ligand (e.g., macroprotein-binding, cuboprotein-binding, or other cell surface factor-binding moieties) that directs the iRNA to a site of interest (e.g., kidney cells). Combinations of in vitro and in vivo contact methods are also possible. For example, cells can be contacted with iRNA in vitro and then transplanted into a subject.

[0337] In some embodiments, contacting cells with an ASO or iRNA agent includes “introducing” or “delivering” the ASO or iRNA agent into cells by promoting or influencing cellular uptake or absorption. Absorption or uptake of the ASO or iRNA agent can occur through unassisted diffusion or active cellular processes, or via an adjuvant or device. The ASO or iRNA agent can be introduced into cells in vitro or in vivo. For example, for in vivo introduction, the ASO or iRNA agent can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipid transfection. Further methods, or those known in the art, are described below.

[0338] The term "lipid nanoparticle" or "LNP" refers to a vesicle containing a lipid layer that encapsulates a drug-active molecule, such as a nucleic acid molecule, like iRNA or a plasmid from which iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

[0339] Regulatory nucleic acids conjugated with ligands Another modification of the RNA of the conjugated regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agent disclosed herein involves chemically linking one or more ligands, portions, or conjugates to the conjugated regulatory nucleic acid agent, said ligands, portions, or conjugates enhancing, for example, the activity, cellular distribution, or cellular uptake of the conjugated regulatory nucleic acid agent in cells. Such portions include, but are not limited to, lipid portions, such as cholesterol portions (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556).In other embodiments, the ligands are bile acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers (e.g., beryl-S-triphenylmethylthiol) (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains (e.g., dodecyldiol or undecyl residues) (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990). 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids (e.g., hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphonate) (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0340] In some embodiments, the ligand alters the distribution, targeting, or lifetime of the conjugated regulatory nucleic acid agent into which it is incorporated. In some embodiments, for example, compared to species lacking such ligands, the ligand provides enhanced affinity for selected targets (e.g., molecules, cells, or cell types), compartments (e.g., cellular or organ compartments), tissues, organs, or body regions. Some ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0341] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylglucosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-mimicking polyamine, dendritic polymer polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0342] The ligand may also include a targeting group, such as a cell or tissue target, such as a lectin, glycoprotein, lipid, or protein, like an antibody, that binds to a specific cell type, such as kidney cells (the kidney cell targeting portion is considered in detail elsewhere in this document). The targeting group may be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polygalactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics. In some embodiments, the ligand is a polygalactose, such as N-acetyl-galactosamine.

[0343] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin)), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O-hexadecylglycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl or phenoxazine), and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, thiol groups, PEG. (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiomarkers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraaza-macrocyclic Eu3+ complexes), dinitrophenyl, HRP, or AP.

[0344] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for the coligand, or antibodies, such as antibodies that bind to specific cell types (e.g., hepatocytes). Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0345] The ligand can be a substance, such as a drug, that can increase the uptake of conjugated regulatory nucleic acid agents into the cell, for example, by disrupting the cell's cytoskeleton, such as by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0346] In some embodiments, ligands linked to regulatory nucleic acid agents as described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acids, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing a number of thiophosphate bonds are also known to bind to serum proteins, therefore short oligonucleotides (e.g., oligonucleotides containing a number of thiophosphate bonds in their backbone of about 5, 10, 15, or 20 bases) are also suitable as ligands (e.g., as PK-regulating ligands) in this disclosure. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in the embodiments described herein.

[0347] The ligand-conjugated nucleic acid agents of this disclosure can be synthesized using oligonucleotides with reactive side-chain functional groups, such as those derived from linker molecules attached to the oligonucleotide. Such reactive oligonucleotides can react directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands having a linker motif.

[0348] The oligonucleotides used in the conjugates disclosed herein can be conveniently and routinely prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, Calif.). Alternatively or alternatively, any other methods known in the art for such synthesis can be employed. Similar techniques are also known for the preparation of other oligonucleotides, such as phosphate thioesters and alkylated derivatives.

[0349] In the ligand-conjugated nucleic acid agents and ligand molecules with sequence-specific links of nucleosides disclosed herein, oligonucleotides and oligonucleotides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already containing linking portions, ligand-nucleotide or nucleoside conjugate precursors already containing ligand molecules, or structural units containing non-nucleoside ligands.

[0350] When using a nucleotide conjugate precursor that already contains a linker, the synthesis of the sequence-specific linked nucleoside is typically already complete, and then the ligand molecule reacts with the linker to form the ligand-conjugated oligonucleotide. In some embodiments, in addition to commercially available standard and non-standard phosphorus amides routinely used in oligonucleotide synthesis, the oligonucleotides or linked nucleosides of this disclosure are synthesized using phosphorus amides derived from the ligand-nucleoside conjugate via an automated synthesizer.

[0351] carbohydrate conjugates In some embodiments of the compositions and methods disclosed herein, the targeted partially conjugated nucleic acid agents (e.g., ASO or iRNA agents) also comprise carbohydrates. Carbohydrate-conjugated nucleic acid agents facilitate in vivo delivery of nucleic acids and are suitable compositions for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each carbon atom bonded to an oxygen, nitrogen, or sulfur atom; or a compound having a carbohydrate moiety as part of it, the carbohydrate moiety being composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each carbon atom bonded to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and above sugars (e.g., C5, C6, C7 or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0352] Longer carbohydrate modifications (e.g., C16 or C20 modifications) have also been described as effective for delivering regulatory nucleic acid agents to the central nervous system (CNS).

[0353] In some embodiments, the carbohydrate conjugates used in the compositions and methods of this disclosure are monosaccharides.

[0354] In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US 8,106,022, the entire contents of which are hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate acts as a ligand for targeting regulatory nucleic acid agents to specific cells. In some embodiments, the GalNAc conjugate targets regulatory nucleic acid agents to hepatocytes, for example, by acting as a ligand for the desialylate glycoprotein receptor of hepatocytes (e.g., hepatocytes).

[0355] Kidney cell targeting components and conjugates In some embodiments, a targeting portion of a factor is employed that specifically binds to the surface of a target cell of interest (e.g., kidney-associated cells). In some embodiments, the provided techniques enable targeted delivery of the payload portion to the target cell, tissue, organ, or organism of interest, with minimal off-target effects. In some embodiments, the targeting portion, as described herein, specifically binds to a factor preferentially present on the surface of the target cell or tissue of interest (e.g., relative to one or more non-target cells or tissues). In some embodiments, the targeting portion, as described herein, specifically binds to a factor that is specific to the target cell or tissue of interest.

[0356] In some embodiments, the targeting moiety disclosed herein is associated (e.g., conjugated or otherwise linked) with the regulatory nucleic acid agent of this disclosure to target macroproteins and / or cuboproteins (renal cell surface factor receptors). Certain examples of such targeting moieties have been previously described (see PCT / US23 / 16319) as particularly suitable for delivering nucleic acid agents into cells, especially into kidney-associated cells (e.g., renal cells). Conjugated agents including a macroprotein-binding moiety conjugated (optionally via a linker) with a nucleic acid agent are particularly useful for delivering such nucleic acid agents into cells expressing macroproteins. Such conjugated agents are particularly useful for delivering nucleic acid agents into renal cells.

[0357] The targeting portions disclosed herein may bind to (e.g., selectively bind to) surface factors (e.g., portions or parts thereof, and / or specific forms thereof, such as their disease-related forms) present on the surface of target cells of interest (e.g., kidney cells) disclosed herein.

[0358] Not wishing to be bound by theory, certain embodiments of this disclosure provide for the binding of a targeting portion associated with the regulatory nucleic acid agent of this disclosure to cell surface factors present on the surface of relevant (e.g., kidney) cells (e.g., tissue cells) to achieve internalization of the cell surface factors, as well as the internalization of the bound targeting portion (as part of a conjugate agent comprising the targeting portion disclosed herein and one or more regulatory nucleic acid compounds). In some embodiments, such internalization may mean that the relevant cell surface factors are no longer (at least for a period of time) present on the surface of cells (e.g., tissue cells) for, for example, signal transduction and / or binding to ligands.

[0359] In some embodiments, renal cell surface factors are present on the surface (e.g., detectable thereon) of kidney-related tissues (e.g., tissues that are part of or can be found in the kidney, such as during development, during tissue homeostasis, and / or during disease or condition). In some embodiments, renal cell surface factors are present on proximal tubular epithelial cells, podocytes, and / or renal cyst cells (e.g., in polycystic kidney disease). In some embodiments, an internalized conjugate agent (e.g., a portion thereof, such as a payload portion) is delivered to vesicles (e.g., lysosomes, endosomes, clathrin-coated pits, or intracellular membrane organelles, or combinations thereof) within the cell. In some embodiments, an internalized conjugate agent (e.g., a portion thereof, such as a payload portion) is delivered to compartments within the cell, such as the cytoplasm, mitochondria, ribosomes, nucleus, nucleolus, or any other compartment within the cell, or combinations thereof. In some implementations, an internalized conjugate agent (e.g., a portion thereof, such as a regulatory nucleic acid agent associated with a target portion) within a cell (e.g., in a vesicle or compartment of the cell) can reduce the expression and / or activity of the target of the regulatory nucleic acid agent.

[0360] In some embodiments, internalizing the conjugated agent (e.g., a portion thereof, such as a regulatory nucleic acid agent associated with the target moiety) into a cell (e.g., a vesicle or compartment within the cell) uncouples (e.g., segregates) the target moiety from the regulatory nucleic acid agent. In some embodiments, the target moiety is uncoupled (e.g., segregates) from the regulatory nucleic acid agent by chemical reaction and / or mechanical separation. In some embodiments, the chemical reaction includes an enzymatic reaction to cleave the linker that connects the target moiety to the regulatory nucleic acid agent.

[0361] In some implementations, the conjugated agent (e.g., a portion thereof, such as a regulatory nucleic acid agent associated with the target moiety) is internalized into cells (e.g., vesicles or compartments within the cell) to uncouple the target moiety from the regulatory nucleic acid agent associated with the target moiety.

[0362] In some embodiments, the conjugated agents disclosed herein can be filtered by glomerular capillaries into, for example, Bowman's capsule. In some embodiments, the conjugated agents disclosed herein have size, charge, conformation, and / or other properties that allow them to be filtered by glomerular capillaries. In some embodiments, the glomerular filtration threshold is in the range of 30-50 kDa.

[0363] In some implementations, cell surface factors (e.g., renal cell surface factors) are or contain receptors selected from macroproteins, cuboproteins, or both.

[0364] Giant protein Macroprotein is a receptor of approximately 600 kDa (about 4655 amino acids) and belongs to the low-density lipoprotein receptor family (as disclosed in Nielsen R. et al. (2016), Kidney Int. 89(1):58-67). Macroprotein is also known as LDL receptor-associated protein 2 (LRP2), glycoprotein 330 (Gp330), calcium sensor protein, or Heyman nephritis antigen homolog.

[0365] Human giant protein sequence: The extracellular domains of the macroprotein include clusters of cysteine-rich complement-type repeat sequences. These repeat sequences are separated by a β-propeller domain containing a YWTD motif and an EGF-type repeat sequence. The macroprotein possesses a transmembrane domain that localizes it to portions of the cell membrane including cholesterol and / or glycosphingolipids. The macroprotein also possesses an intracellular C-terminal cytoplasmic domain that regulates receptor transport and / or endocytosis. The cytoplasmic domains of the macroprotein contain an NPXY motif and several other domains, such as proline-rich sequences and a PDZ motif. The cytoplasmic domains of the macroprotein are involved in receptor internalization. A typical structure of the macroprotein is disclosed in Marzolo and Farfan (2011). Biol Res 44: Figure 1, pp. 89-105, is hereby incorporated by reference in its entirety. Extracellular domains of macroproteins may also include one or more post-translational modifications, such as glycosylation.

[0366] Macroproteins have been described to interact with co-receptor cuboproteins at least in certain conditions. Macroproteins have been identified on the surface of one or more of the following tissues and / or cells: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils, and / or platelets); the nervous system (e.g., brain tissue, cortex, cerebellum, retinal cells (e.g., retinal pigment epithelial cells (RPE)), spinal cord cells, nerve cells, neurons, and / or supporting cells); endothelial cells; and muscles (e.g., cardiac muscle, smooth muscle, and / or...). The kidneys are located in the following tissues: skeletal muscle; small intestine; colon; adipocytes; kidneys; liver; lungs; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; pituitary gland; breast; skin; ovary; uterus; placenta; prostate; and testes. In kidney tissue, macroproteins have been reported to be present on the surface of proximal tubular epithelial cells and podocytes. Macroprotein expression has been observed in the brush border, endocytic vesicles, dense apical tubules, and / or lysosomes of the proximal tubular epithelial cells of the kidney. Several ligands for macroproteins have been identified, some of which are disclosed in Nielsen et al., 2016. The polybasic targeting moieties disclosed herein (e.g., polymyxin B, colistin, and related structures) are particularly described as high-affinity macroprotein ligands.

[0367] In some embodiments, the target renal cell surface factor used to deliver the target-related regulatory nucleic acid compound of this disclosure is a macroprotein or a fragment or variant thereof.

[0368] In some embodiments, the targeting portion is or comprises a macroprotein-binding portion. In a particular embodiment, the targeting portion binds to the extracellular domain of the macroprotein (e.g., a site that binds to the extracellular domain, such as a site exposed when the macroprotein is on a cell surface). In other particular embodiments, the conjugate comprises a targeting portion that binds to the extracellular domain of the macroprotein (e.g., a site that binds to the extracellular domain, such as a site exposed when the macroprotein is located on a cell surface) and, upon binding to the macroprotein, causes internalization of the macroprotein.

[0369] Cubic protein Cubic protein is a receptor of approximately 460 kDa. It is also known as IFCR, Gp280, intrinsic factor-vitamin B12 receptor, MGA1, or IGS1. As an extracellular protein, cubic protein can interact with other membrane proteins, such as macroproteins. One of its functions is as a receptor for the intrinsic factor-vitamin B12 complex.

[0370] Human cubic protein sequence: The extracellular domains of the cuboprotein include repeat sequences of the CUB domain (complement C1r / C1s, Uegf [epidermal growth factor-associated sea urchin protein], and bone morphogenetic protein 1) and EGF-type repeat sequences. The typical structure of the cuboprotein is disclosed in Marzolo and Farfan (2011). Biol Res 44: Figure 1, pp. 89-105, is hereby incorporated by reference in its entirety. Extracellular domains of cubic proteins may also include one or more post-translational modifications, such as glycosylation.

[0371] Cubic proteins have been reported to be found on the surface of one or more of the following tissues and / or cells: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils, and / or platelets); the nervous system (e.g., brain tissue, cortex, cerebellum, retinal cells, spinal cord cells, nerve cells, neurons, and / or supporting cells); endothelial cells; muscles (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle); small intestine; colon; adipocytes; kidneys; liver; lungs; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; pituitary gland; breast; skin; ovaries; uterus; placenta; prostate; and testes. In kidney tissue, cubic proteins have been reported to be present on the surface of proximal tubular epithelial cells and podocytes. Several ligands for cubic proteins have been identified, some of which are disclosed in Nielsen et al., 2016.

[0372] Other exemplary cubic protein binding portions or ligands are disclosed in U.S. Patent No. 10,065,993, International Patent Application WO 2017 / 100700, International Patent Application WO 2018 / 232122 and International Patent Application WO 2015 / 027205, the entire contents of which are hereby incorporated by reference.

[0373] In some implementations, the renal cell surface factor is a cuboprotein or a fragment or variant thereof.

[0374] In some implementations, the targeting portion is or includes a cuboprotein-binding portion.

[0375] In some embodiments, the targeting portion (e.g., a macroprotein-binding portion) is or comprises a xenobiotic, such as those disclosed elsewhere herein. In some embodiments, the xenobiotic is selected from polymyxin, aprotinin, pollenin, or combinations thereof.

[0376] Other nucleic acid modifications In some respects, the renal cell-targeting portions disclosed herein are intended for the delivery of nucleic acid payloads. It is expressly contemplated that such renal cell-targeting portions may be used in combination with one or more of a variety of other nucleic acid modifications known in the art. Specific nucleic acid modifications expressly contemplated for use in combination with the renal cell-targeting portions of this disclosure include, but are not limited to, “extended nucleic acid” (“exNA”) modifications and a series of phosphoryl guanidine skeletal bonds (“PN Chemistry”).

[0377] "Extended nucleic acid" ("exNA") modification generally refers to a modification in which a methyl group is inserted between the 3'-phosphate group of the first nucleoside and the 5'-carbon of the ribose of the next nucleoside in the oligonucleotide chain (progressing from 5' to 3'). Therefore, such a canonical "exNA" modification has the following general structure: In some embodiments, optional "exNA" modifications, which are either baseless and / or have an extension longer than methyl and / or optionally substituted on one or more of the base, 2'-substituent, and / or extended carbon chain, are also explicitly considered. Therefore, in some embodiments of this disclosure, the term "exNA" may refer to any portion having the following structure: , where: R 1 It is a nucleobase or a debasement structure (e.g., H, CH3, or other modified structures, which are optionally substituted); R 2 It is a 2' substituent, including, for example but not limited to, 2'-O-alkyl (e.g., 2'-O-methyl, etc.), 2'-F, etc.; and C 1-10 It refers to a chain of 1 to 10 carbons, which may be optionally saturated or unsaturated, and contains optionally substituted alkyl (e.g., optionally substituted methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl), alkenyl (e.g., optionally substituted methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, heptylene, octylene, nonylene, or decylene) or ynylene (e.g., ethynyl, optionally substituted propynyl, butynyl, pentynyl, hexynyl, heptynyl, ocynyl, nonynyl, or decynyl) chains.

[0378] In the relevant implementation scheme, C shown above 1-10 Branches in the region are also considered to be modified by "exNA". For example, in some implementations, the "exNA" modification of this disclosure can also be represented by the following formula: , where R 1 It is a nucleobase or a debasement structure (e.g., H, CH3, or other modified structures, which are optionally substituted); R 2It is a 2' substituent, including, but not limited to, 2'-O-alkyl (e.g., 2'-O-methyl, etc.), 2'-F, etc.; and the side substitution on the 5' of the ribose can be 2-11 atoms, with different amounts of substituents at X and Y (from X=Y=H to long n- or branched alkyl structures).

[0379] In some implementations, “exNA” and / or other internucleotide modifications may be located at or near the 3' end of the oligonucleotide therapeutic agent (e.g., at or near the 3' end of the siRNA guide strand, at or near the 3' end of the antisense agent, etc.), where, without being bound by theory, the exNA modification may confer resistance of the exNA-modified nucleic acid payload to 3'-exonuclease-mediated digestion (Yamada, K. et al.). Nature Biotechnology 2024). In some embodiments, the exNA-phosphothioester nucleoside internucleotide bond may be located at the last nucleoside internucleotide bond at the 3' end of the nucleic acid payload, and optionally at the penultimate nucleoside internucleotide bond (e.g., exNA-PS modification at the 3' end region of the siRNA guide strand and / or ex-NA-PS modification at the 3' end region of the antisense oligonucleotide). As disclosed in WO 2021 / 195533, exNA modification may be used in combination with a range of other commonly used oligonucleotide modifications, including but not limited to the following: .

[0380] In such implementations, it is also explicitly considered that such exNA modifications can be combined with the aforementioned "base" or R. 1 Any bases at the position that are recognized in the art can be used effectively in synergy, including but not limited to the following representative naturally occurring and artificially modified bases known in the art: .

[0381] A phosphorylguanidine-containing backbone (“PN backbone”) bond is also explicitly considered for use in combination with the renal cell-targeting portion of this disclosure. Such PN backbone modifications were initially identified as members of the following class in WO 2023 / 201095: Exemplary forms of PN skeleton modification include, but are not limited to, the following: , , , , , , , , , and , where W is O or S. In a particular embodiment, the PN skeleton modification is selected from the following: , and (The latter structure is referred to as the “n001” modification of WO2023 / 201095). Exemplary PN skeleton bonds are also disclosed, for example, in U.S. Patent No. 11,208,430.

[0382] In addition to the use of the aforementioned phosphorylguanidine structure to modify the nucleoside internucleotide bonds of the nucleic acid agents disclosed herein, a number of other guanidine-based moieties are also explicitly considered herein for attachment to linking phosphorus groups. Such other guanidine-based moieties include, but are not limited to, the following: , where R LS It can be -Cl, -Br, -F, N(Me)2 or NHCOCO3 independently.

[0383] Other skeletal modifications known in the art have also been considered for use with the targeting moieties and payloads of this disclosure. In particular, modifications including one or more methanesulfonylaminophosphate modifications and / or butanesulfonylaminophosphate modifications (and / or other structure-related modifications) have also been explicitly considered for use in the nucleic acid agents of this disclosure. Compared to thiophosphate modifications, methanesulfonylaminophosphate and butanesulfonylaminophosphate modifications have the following structures (see Sergeeva et al. (2024)). Front Chem (., 12-2024 doi.org / 10.3389 / fchem.2024.1342178): In some embodiments, the oligomer (including, for example, antisense oligonucleotides, siRNA, etc., or portions thereof) comprises or is composed of oligonucleotides, said oligonucleotides being composed of linked nucleosides and having at least one inter-nucleoside linker group of the following formula: Wherein X is selected from O or S, and R is selected from aryl, substituted aryl, heterocyclic, substituted heterocyclic, aromatic heterocyclic, substituted aromatic heterocyclic, diazole, substituted diazole, C1-C6 alkoxy, C1-C20 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C20 alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkynyl, and conjugate groups. In some embodiments, X is O, and R is methyl, and the internucleotide linker of the above formula is the same as the internucleotide linker of the following formula.

[0384] In some embodiments, the oligomer (including, for example, antisense oligonucleotides, siRNA, etc., or portions thereof) comprises or is composed of oligonucleotides, said oligonucleotides being composed of linked nucleosides and having at least one inter-nucleoside linker group of the following formula: (Methanesulfonylaminophosphate nucleoside internucleotide bond; see, for example, WO 2023 / 278589).

[0385] In some embodiments, the oligomer (including, for example, antisense oligonucleotides, siRNA, etc., or portions thereof) comprises or is composed of oligonucleotides, said oligonucleotides being composed of linked nucleosides and having at least one inter-nucleoside linker group of the following formula: .

[0386] In some embodiments, the oligomer (including, for example, antisense oligonucleotides, siRNA, etc., or portions thereof) comprises or is composed of oligonucleotides, said oligonucleotides being composed of linked nucleosides and having at least one inter-nucleoside linker group of the following formula: Modifications to the phosphate backbone can be located anywhere in the oligonucleotide agents disclosed herein that is known in the art and / or disclosed herein.

[0387] Compared to unmodified oligonucleotides, the modified oligonucleotides of this disclosure comprise at least one modification (i.e., comprising at least one modified nucleoside (comprising a modified sugar moiety, a stereonon-standard nucleoside, and / or a modified nucleobase) and / or at least one modified internucleotide bond). In some embodiments, the modified internucleotide bond is a modified internucleotide linker having any of the above formulas. In some embodiments, the compounds described herein are oligomers (including oligomers as antisense agents, siRNAs, etc., or portions thereof) having at least one modified internucleotide linker having any of the above formulas.

[0388] Measurement of expression regulation Regulation of target RNA and / or target polypeptide expression can be determined using a variety of methods known in the art. For example, target mRNA levels can be quantified by, for instance, Northern blotting, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed using methods known in the art on total cellular RNA or poly(A) expression. + RNA isolation is performed on mRNA. Methods for RNA isolation are taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Vol. 1, pp. 4.1.1–4.2.9 and 4.5.1–4.5.3, John Wiley & Sons, Inc., 1993.

[0389] Northern blot analysis is a standard method in the field and is taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Vol. 1, pp. 4.2.1–4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative PCR can be conveniently performed using the commercially available ABI PRISM™ 7700 sequence detection system, which is available from PE-Applied Biosystems, Foster City, Calif., and used according to the manufacturer's instructions. The analytical methods for regulating RNA levels are not limited to this disclosure.

[0390] The level of target proteins encoded by target mRNA or DNA can be quantified using a variety of methods well-known in the art, such as immunoprecipitation, Western blotting, ELISA, or fluorescence activated cell sorting (FACS). Antibodies against target proteins encoded by target mRNA can be identified and obtained from a variety of sources, such as the MSRS Antibody Catalog (Aerie Corporation, Birmingham, Mich.) or can be prepared using conventional antibody production methods. Methods for preparing polyclonal antisera are taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Vol. 2, pp. 11.12.1–11.12.9, John Wiley & Sons, Inc., 1997. Methods for preparing monoclonal antibodies are taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Vol. 2, pp. 11.4.1–11.11.5, John Wiley & Sons, Inc., 1997.

[0391] Immunoprecipitation is the standard method in this field and can be found, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Vol. 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot analysis is also standard in this field and can be found, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Vol. 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997.

[0392] Active target segment A location on a target nucleic acid defined by one or more active regulatory RNA compounds is referred to as an "active target region." Within the active target region, the activity of the regulatory RNA compound (e.g., as defined by the percentage of inhibition) may vary substantially. Active regulatory RNA compounds are those compounds identified as regulating the expression of their target RNA. In some embodiments, the active regulatory RNA compound is repressive, optionally inhibiting the expression of its target RNA by at least about 50%, optionally at least about 70%, and optionally at least about 80% or more. In some embodiments, the required level of inhibition for defining an active repressive RNA compound is defined based on the results of screening used to define the active target region. Those skilled in the art will understand that the percentage of inhibition of the target mRNA by the repressive RNA compound will vary in different assays due to factors related to assay conditions.

[0393] Hybridization As used herein, “hybridization” refers to the pairing of the complementary strand of an antisense compound with its target sequence and / or the pairing of the complementary strands of a double-stranded nucleic acid molecule with each other. While not limited to specific mechanisms, the most common mechanism of pairing involves hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between complementary nucleosides or nucleotide bases (nucleobases). For example, the natural base adenine is complementary to the natural nucleobases thymidine and uracil, which pair by forming hydrogen bonds. The natural base guanine is complementary to the natural base 5-methylcytosine and an artificial base called a G-clamp. Hybridization can occur under various conditions.

[0394] Regulatory RNA compounds are specifically hybridizable when there is sufficient complementarity to prevent nonspecific binding of the regulatory RNA compound to nontarget nucleic acid sequences under conditions requiring specific binding (i.e., under physiological conditions in in vivo assays or therapeutic applications, and under the conditions under which the assay is performed in vitro).

[0395] As used herein, “strict hybridization conditions” or “strict conditions” refer to conditions under which a regulatory RNA compound will hybridize with its target sequence but with a minimum number of other sequences. Strict conditions are sequence-dependent and vary under different conditions, and the “strict conditions” for a regulatory RNA compound to hybridize with its target sequence are determined by the properties and composition of the regulatory RNA compound and the assays studied.

[0396] Complementarity As used herein, “complementarity” refers to the ability of two nucleobases on two oligomeric or antisense compounds to precisely pair with their target nucleic acid. For example, if a nucleobase at a certain position in an antisense compound can hydrogen-bond with a nucleobase at a certain position in a target nucleic acid, the hydrogen-bonded position between the oligonucleotide and the target nucleic acid is considered a complementary position. When a sufficient number of complementary positions in each molecule are occupied by nucleobases that can hydrogen-bond with each other, the antisense compound and the other DNA or RNA are complementary to each other. Therefore, “specifically hybridizable” and “complementary” are terms used to indicate a sufficient degree of precise pairing or complementarity on a sufficient number of nucleobases, such that stable and specific binding occurs between the antisense compound and the target nucleic acid, or between the corresponding sense and antisense strands (or subsequences) of a double-stranded nucleic acid molecule.

[0397] identity Double-stranded RNA compounds, antisense compounds, or portions thereof may have a defined percentage of identity with the target sequence and / or its complement. As used herein, a sequence is considered identical to a sequence disclosed herein if it has the same nucleobase pairing ability. For example, RNAs containing uracil instead of thymidine in a disclosed sequence would be considered identical because they both pair with adenine. This identity can be along the entire length of the oligomer or in a portion of a given strand, such as in a portion of the antisense compound (e.g., comparing nucleobases 1-20 of a 27-mer with that of a 20-mer to determine the percentage of identity of the 27-mer at residues 1-20 with the comparison 20-mer (or with a defined 20-nucleotide sequence within the target nucleic acid molecule). Those skilled in the art will understand that regulatory RNA compounds (e.g., siRNA compounds or antisense compounds) do not need to have the same sequence as those described herein to function similarly to the regulatory RNA compounds described herein. Shortened or extended forms of the regulatory RNA compounds taught herein (on one or both strands of the siRNA compound) or those taught herein... Dissimilar forms of regulatory RNA compounds are all within the scope of this disclosure. A dissimilar form is one in which each base does not have the same pairing activity as the regulatory RNA compounds disclosed herein. The base may be shorter or have at least one abase debasement site, thus lacking the same pairing activity. Alternatively, a dissimilar form may include at least one base replaced by a different base with a different pairing activity (e.g., G may be replaced by C, A, or T). The percentage of identity is calculated based on the number of bases having the same base pairing corresponding to a reference sequence with which it is compared (which may be a target sequence, the complementary strand of dsRNA, an antisense oligonucleotide, and / or an antisense strand sequence, etc.). Dissimilar bases may be adjacent to each other, scattered throughout the oligonucleotide, or both.

[0398] For example, a 16-mer having the same sequence of nucleobases 2-17 as the 20-mer has 80% identity with the 20-mer over its entire length. Alternatively, a 20-mer containing four nucleobases different from the 20-mer also has 80% identity with the 20-mer. A 14-mer having the same sequence of nucleobases 1-14 as the 18-mer has 78% identity with the 18-mer. Such calculations are entirely within the capabilities of those skilled in the art.

[0399] The identity percentage is based on the percentage of nucleosides in the original sequence present in a portion of the modified sequence. Therefore, a 30-nucleobase antisense compound comprising the complete sequence of complement containing a 20-nucleobase active target region will have a portion with 100% identity to complement containing the 20-nucleobase active target region, while also containing an additional 10-nucleobase portion. The complement containing the active target region can constitute a single portion. In some embodiments, the oligonucleotide has at least about 80%, optionally at least about 85%, even more preferably at least about 90%, and most preferably at least 95% identity to at least a portion of the complement containing the active target region provided herein.

[0400] It is well known to those skilled in the art that it is possible to increase or decrease the length of regulatory RNA compounds and / or introduce mismatched bases (with the target sequence, the complementary strand of the double-stranded RNA, or both) without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992, incorporated herein by reference), the ability of a series of ASOs of 13-25 nucleotides in length to induce target RNA cleavage was tested. ASOs of 25 nucleotides in length with 8 or 11 mismatched bases near the end of the ASO were able to direct specific cleavage of the target mRNA, although to a lesser extent than ASOs without mismatches. Regulatory RNA compounds having shorter or longer (independently on either or both strands of the double-stranded nucleic acid compound) or containing a series of mismatched nucleotides are considered in this disclosure, provided that regulatory RNA activity against the target sequence is maintained.

[0401] treat The conjugated regulatory nucleic acid compounds disclosed herein can be used to regulate the expression of target RNAs and / or target peptides in animals (such as humans). In one non-limiting embodiment, the method includes administering an effective amount of a regulatory RNA compound that inhibits the expression of target RNAs and / or target peptides to said animal requiring treatment for a disease or disorder associated with the target RNAs and / or target peptides. Diseases or disorders associated with target RNAs and / or target peptides include, but are not limited to, glomerular diseases, tubular diseases, other kidney diseases, congenital metabolic defects, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, or viral infections, or combinations thereof. In one embodiment, the conjugated regulatory RNA compound effectively inhibits the level or function of the target mRNA. Because a decrease in the level of the target mRNA can also lead to an alteration in the level of the protein product encoded by the mRNA expression, such alterations can also be measured. Regulatory RNA compounds that effectively inhibit the level or function of the target RNA or target protein expression product are considered to be active repressive RNA compounds. In one embodiment, the regulatory RNA compound of this disclosure inhibits the expression of target RNA, resulting in a reduction of RNA by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0402] For example, a reduction in the expression of target RNA and / or target peptides can be measured in the body fluids, tissues, or organs of an animal. Methods for obtaining samples (such as body fluids (e.g., blood, plasma, urine, saliva, etc.), tissues (e.g., biopsies), or organs) for analysis and sample preparation methods that permit analysis are well known to those skilled in the art. Methods for analyzing RNA and protein levels have been discussed above and are well known to those skilled in the art. The effectiveness of treatment can be assessed by measuring biomarkers related to the expression of target RNA and / or target peptides in the aforementioned body fluids, tissues, or organs collected from animals exposed to one or more compounds using conventional clinical methods known in the art.

[0403] The regulatory nucleic acid agent disclosed herein can be used in pharmaceutical compositions by adding an effective amount of the compound to a suitable pharmaceutically acceptable diluent or carrier. Acceptable carriers and diluents are well known to those skilled in the art. The selection of a diluent or carrier is based on a number of factors, including but not limited to the solubility of the compound and the desired route of administration. Such considerations are well known to those skilled in the art. In one aspect, the compound inhibits the expression of target RNA and / or target peptides. The regulatory nucleic acid agent can also be used to manufacture medicaments for treating diseases and disorders associated with the expression of target RNA and / or target peptides.

[0404] Methods for contacting bodily fluids, organs, or tissues with effective amounts of one or more regulatory RNA compounds or compositions are also considered. Bodily fluids, organs, or tissues may be contacted with one or more compounds disclosed herein, resulting in the regulation of target RNA and / or target polypeptide expression in the cells of the bodily fluids, organs, or tissues.

[0405] Therefore, this document provides the use of isolated single-stranded or double-stranded regulatory nucleic acid agents targeting target RNAs and / or target peptides in the manufacture of medicaments for treating diseases or conditions by the methods described above. In some embodiments, the regulatory nucleic acid agent is a single-stranded antisense compound. In other embodiments, the regulatory nucleic acid agent is a double-stranded repressive RNA compound.

[0406] In some embodiments, the regulatory nucleic acid agent (e.g., oligonucleotide) of this disclosure is characterized in that, when the regulatory nucleic acid agent of this disclosure, a composition containing the regulatory nucleic acid agent of this disclosure, or a conjugate agent containing the regulatory nucleic acid agent of this disclosure is delivered to cells, tissues, or organisms expressing a target, a decrease in target expression and / or activity is observed compared to cells, tissues, or organisms that have not been delivered the regulatory nucleic acid agent of this disclosure, a composition containing the regulatory nucleic acid agent, or a conjugate agent containing the regulatory nucleic acid agent.

[0407] In some embodiments, the regulatory nucleic acid agent (e.g., oligonucleotide) of this disclosure is characterized in that, when the regulatory nucleic acid agent of this disclosure, a composition comprising the regulatory nucleic acid agent of this disclosure, or a conjugate agent comprising the regulatory nucleic acid agent of this disclosure is delivered to cells, tissues, or organisms expressing a target, a decrease in target expression and / or activity is observed compared to cells, tissues, or organisms that do not express a target (e.g., no detectable target expression).

[0408] In some embodiments, the regulatory nucleic acid agent (e.g., oligonucleotide) of this disclosure is characterized in that, when the regulatory nucleic acid agent of this disclosure, a composition comprising the regulatory nucleic acid agent of this disclosure, or a conjugate comprising the regulatory nucleic acid agent of this disclosure is delivered to cells, tissues, or organisms expressing a target, an alteration in target expression and / or activity is observed relative to that observed with a suitable reference agent known to have a specific effect on the target. In some embodiments, the manner and / or extent to which target expression and / or activity is altered is comparable to, or determined relative to, that observed with a suitable reference agent known to have a specific effect on the target. In some embodiments, the reference agent may be a positive control reference agent. In some embodiments, the reference may be a negative control reference agent.

[0409] In some embodiments, the regulatory nucleic acid agents of this disclosure (e.g., oligonucleotides, dsRNA, etc.) are characterized in that, when delivered to cells, tissues, or organisms expressing a target, the expression and / or activity of the target are modulated (e.g., reduced) compared to cells, tissues, or organisms not delivered with the regulatory nucleic acid agents of this disclosure.

[0410] In some embodiments, the targeting moiety disclosed herein (e.g., polymyxin or other compounds) may be conjugated to the regulatory nucleic acid agents disclosed herein (e.g., oligonucleotides, dsRNA, etc.).

[0411] This disclosure also provides pharmaceutical compositions comprising or delivering the conjugated pharmaceutical agents disclosed herein. In some embodiments, the pharmaceutical compositions are formulated for intravenous, subcutaneous, intramuscular, parenteral, or oral delivery. In some embodiments, the pharmaceutical compositions comprise one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical compositions comprise less than 5% impurities. In some embodiments, the impurities comprise one or more of the following: endotoxins, cellular components, or aggregates.

[0412] In some embodiments, this document provides cells comprising the conjugated pharmaceutical agents disclosed herein. In some embodiments, the cells are in tissues, organs, or organisms.

[0413] This disclosure provides a payload portion comprising a nucleic acid agent that recognizes a target, which is attached to a first portion of a cleavage of a linker. In some embodiments, the payload portion is located in a cell in the presence of cell surface factors. In some embodiments, the cell also comprises a targeting portion attached to a second portion of a cleavage of the linker.

[0414] This article provides a method for delivering a conjugated drug to a subject, the method comprising the steps of administering to the subject a conjugated drug containing a target portion directly or indirectly connected to a payload portion, or a pharmaceutical composition containing such a drug.

[0415] This article also discloses a method for treating a disease or condition, the method comprising the steps of administering to a subject a conjugate agent or a pharmaceutical composition comprising a target portion directly or indirectly connected to the payload portion.

[0416] This article also discloses a method for treating diseases with nucleic acid agents, an improvement of which includes the following steps: administering a nucleic acid agent as a conjugate having a targeting moiety, for example, as disclosed herein.

[0417] In some embodiments, this disclosure provides improved delivery of a pharmaceutical agent to cells, a method comprising contacting a system or subject containing at least one cell with the conjugated pharmaceutical agent disclosed herein or a pharmaceutical composition containing therefrom.

[0418] In some embodiments of any of the methods disclosed herein, the conjugated agent is delivered to cells expressing cell surface factors. In some embodiments, the cell surface factor is a renal cell surface factor. In some embodiments, the renal cell surface factor is selected from macroproteins and / or cuboproteins.

[0419] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered to a tissue, organ, or fluid compartment.

[0420] In some embodiments of the methods, conjugated agents, compositions, or cells disclosed herein, the conjugated agent is internalized after binding to cell surface factors. In some embodiments, the internalization of the conjugated agent delivers a portion of the payload into an internal compartment or vesicle of the cell.

[0421] In some embodiments of the methods, conjugate agents, compositions or cells disclosed herein, the payload reduces the expression and / or activity of the targets provided in any of or in combination of Tables 1 to 4.

[0422] In some embodiments of any of the methods disclosed herein, contact includes applying a conjugated agent to: cells; tissues containing cells; or organisms containing cells.

[0423] In some embodiments of any of the methods disclosed herein, the application of the conjugated agent to the cells, tissues, or organisms delivers the payload portion to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the target cells, compared to other similar cells, tissues, or organisms that deliver the unconjugated payload portion.

[0424] In some embodiments of any of the methods disclosed herein, application of the conjugate agent to cells, tissues, or organisms delivers a portion of the payload to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the target cells, compared to non-target cells.

[0425] In some embodiments of the methods, conjugated pharmaceutical agents, compositions, or cells disclosed herein, the target cell is or comprises kidney cells.

[0426] In some embodiments of the methods, conjugated pharmaceutical agents, compositions, or cells disclosed herein, the target cell is or contains a cell expressing (e.g., detectably expressed) a cell surface factor. In some embodiments, the cell surface factor is or contains a renal cell surface factor. In some embodiments, the renal cell surface factor is a macroprotein or a variant or fragment thereof. In some embodiments, the renal cell surface factor is a cuboprotein or a variant or fragment thereof.

[0427] In some embodiments of the methods, conjugated pharmaceutical agents, compositions, or cells disclosed herein, the target cell is or contains the expression of one or more targets selected from the targets provided in any of Tables 1 to 4.

[0428] In some embodiments of the methods, conjugated pharmaceutical agents, compositions, or cells disclosed herein, non-target cells are or contain cells that do not express (e.g., undetectable expression) cell surface factors. In some embodiments, non-target cells are or contain cells that do not express (e.g., undetectable expression) renal cell surface factors (e.g., macroprotein and / or cuboprotein).

[0429] In some embodiments of any of the methods disclosed herein, administration of the conjugated agent to a cell, tissue, or organism reduces the expression and / or activity of the target portion by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to other similar cells, tissues, or organisms that deliver the unconjugated payload portion.

[0430] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered to cells expressing cell surface factors, such as those described herein. In some embodiments, the cell surface factors are selected from macroproteins and / or cuboproteins.

[0431] In some implementations, the cells are selected from: immune cells; nervous system cells; muscle cells; small intestinal cells; colon cells; adipocytes; kidney cells; liver cells; lung cells; spleen cells; stomach cells; esophageal cells; bladder cells; pancreatic cells; thyroid cells; salivary gland cells; adrenal gland cells; pituitary gland cells; mammary gland cells; skin cells; ovarian cells; uterine cells; placental cells; prostate cells; or testicular cells, or combinations thereof.

[0432] In some implementations, the cells are selected from: kidney cells, thyroid cells, parathyroid cells, inner ear cells, or nervous system cells, or combinations thereof.

[0433] In some implementations, the cells are selected from proximal tubule epithelial cells and / or podocytes.

[0434] In some embodiments of any of the methods disclosed herein, the disease is a disease associated with the expression of cell surface receptors. In some embodiments, the disease is a disease comprising cells in which both cell surface receptors and a target recognized by the payload portion are present.

[0435] In some embodiments of any of the methods disclosed herein, the disease or condition is selected from: glomerular diseases, tubular diseases, other kidney diseases, congenital metabolic defects, systemic metabolic diseases, thyroid diseases, parathyroid diseases, inner ear diseases, neurological diseases, viral infections, or combinations thereof.

[0436] In some embodiments of any of the methods disclosed herein, the conjugated drug is delivered intravenously, subcutaneously, intramuscularly, parenterally, or orally.

[0437] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered in one or more doses.

[0438] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered in combination with one or more other conjugate agents. In some embodiments, the one or more other conjugate agents comprise different payload portions, different connectors, different target portions, or combinations thereof.

[0439] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered in combination with one or more other therapeutic forms.

[0440] Nucleic acid payload target This document discloses, in particular, conjugate agents comprising a payload portion capable of acting on one or more targets, such as those disclosed herein.

[0441] In some embodiments, the target is present in cells or tissues selected from: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils and / or platelets); the nervous system (e.g., brain tissue, cortex, cerebellum, retinal cells, spinal cord cells, nerve cells, neurons and / or supporting cells; endothelial cells; muscle (e.g., cardiac muscle, smooth muscle and / or skeletal muscle); small intestine; colon; adipocytes; kidney; liver; lung; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; pituitary gland; breast; skin; ovary; uterus; placenta; prostate; or testis, or combinations thereof.

[0442] In some implementations, the target is present in tissues or cells selected from various sources: kidney tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissue.

[0443] In some implementations, the target (e.g., at a relatively high level) is present on kidney cells, such as proximal tubular epithelial cells and / or podocytes.

[0444] In some embodiments, the target is present in kidney-related cells (e.g., renal cells or cells that can be found in the kidney, such as during development, during tissue homeostasis, or during the course of a disease or condition). In some embodiments, the target is present in kidney-related tissues (e.g., tissues that are part of the kidney, such as during development, during tissue homeostasis, or during the course of a disease or condition).

[0445] In some implementations, cells expressing the target (e.g., cells of a tissue) also express the target portion, for example, as described herein.

[0446] In some implementations, the cells expressing the target (e.g., cells of a tissue) also express the kidney-specific targeting portion, for example, as disclosed herein.

[0447] In some embodiments, the expression and / or activity of the target can be demodulated in a disease or condition. In some embodiments, delivery of the conjugate agent to cells expressing the target (e.g., cells of a tissue) reduces the expression and / or activity of the target.

[0448] In some embodiments, conjugated agents are delivered to organisms with abnormal expression and / or activity of a target in cells (e.g., cells of a tissue) to treat a disease or condition and / or improve symptoms of the disease or condition in the organism.

[0449] In some implementations, the target is selected from any of the targets provided in Tables 1 to 4 or a combination thereof.

[0450] In some implementations, the target is or is contained in a gene product (e.g., a transcript) expressed in a specific cell (e.g., cell type) and / or tissue as described herein.

[0451] In some embodiments, the target is or is contained in a non-coding RNA (or other regulatory RNA species) expressed in a specific cell (e.g., cell type) and / or tissue as described herein. In some embodiments, the target is or contains long non-coding RNA (lncRNA), microRNA, Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), or a combination thereof.

[0452] In some embodiments, the target is expressed in cells and / or tissues having internalization receptors on its surface. In some embodiments, the target is expressed in cells and / or tissues having macroproteins on its surface. In some embodiments, the target is expressed in cells and / or tissues having cuboproteins on its surface. In some embodiments, the target is expressed in kidney cells. In some embodiments, the target is expressed in one or more of the following: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils and / or platelets); nervous system cells (e.g., brain tissue, cortex, cerebellum, retinal cells (e.g., retinal pigment epithelial cells (RPE)), spinal cord cells, nerve cells, neurons and / or supporting cells); endothelial cells; muscle (e.g., cardiac muscle, smooth muscle and / or skeletal muscle); small intestinal cells; colon cells; adipocytes; kidney cells; hepatocytes; lung cells; spleen cells; gastric cells; esophageal cells; bladder cells; pancreatic cells; thyroid cells; salivary gland cells; adrenal cells; pituitary cells; mammary gland cells; skin cells; ovarian cells; uterine cells; placental cells; prostate cells; or testicular cells, or combinations thereof. In some implementations, the target is expressed in proximal renal tubular epithelial cells (RPTEC), podocytes, and / or combinations thereof.

[0453] In some embodiments, the target is or is contained in a gene expressed in proximal renal tubular epithelial cells (RPTEC). In some embodiments, the target is selected from the RPTEC genes provided in Table 1 or combinations thereof. In some embodiments, the target has one or more of the characteristics and / or functions provided in Table 2, or combinations thereof. In some implementations, the target has one or more properties and / or functions selected from the following: A-kinase anchoring proteins; acyl-CoA dehydrogenase family; acyl-CoA thioesterases; aldehyde-ketone reductases; proteins containing ankyrin repeating domains; apolipoproteins; basic helical-loop-helical proteins; basic leucine zipper proteins; β-γ crystal proteins; blood group antigens; proteins containing BPI folds; proteins containing C-type lectin domains; C1q and TNF-related proteins; proteins containing C2 domains; cadherins; the CAP superfamily; CD molecules; chemokine ligands; claudin; collagen; complement system; the CTAGE family; cytochrome P450; Dbl family RhoGEF; proteins containing EF-hand domains; and erythrocyte membrane protein band 4.1; Contains F-BAR domain; Fatty acid-binding protein family; Fatty acid desaturase; Contains fibronectin type III domain; G protein-coupled receptor; Galactose agglutinin; Globolipid / villous protein; Glycoside hydrolase family 31; Contains GOLD domain; Contains GRAM domain; Contains halogen dehalogenase-like hydrolase domain; Heat shock protein; Histone; Homeobox; Contains I-BAR domain; Contains immunoglobulin superfamily domain; Interleukin receptor; Intermediate filament; Ion channel; Kinesin; Late keratinization envelope protein; Ligand-gated ion channel; Low-density lipoprotein receptor; M14 carboxypeptidase; Contains Maestro thermal repeat; Membrane spanning 4 domains; Metallothionein methyltransferase family; Mitochondrial respiratory chain complex assembly factor; Mitochondrial respiratory chain complex; Mucin; Myosin heavy chain; Contains N-BAR domain; N Terminal EF hand calcium-binding proteins; Na+ / K+ transporter ATPase interactions; NLR family; non-coding RNA; regulatory RNA; oxysterol-binding proteins; paraneoplastic Ma antigens; proteins containing PDZ domains; phospholipases; proteins containing Pleckstrin homology domains; protein phosphatase 1 regulatory subunits; proteins containing PWWP domains; Ras-associated domain family; Ras small GTPase superfamily; receptor helper proteins; receptor kinases; receptor ligands; proteins containing RNA-binding motifs; serine proteases; serine protease inhibitors; peptidase inhibitors; proteins containing SH2 domains; short-chain dehydrogenase / reductase superfamily; sideroflexin; signal transduction and RNA metabolism activation family; solute carriers; sorting linkers. nexin; proteins containing a sterile α-motif domain; STRIPAK complex; sulfatase; proteins containing a sushi domain; synaptic proteins; synaptotagmin; tetraspanin; proteins containing a 34-peptide repeat domain; proteins containing a tri-domain motif; the tubulin tyrosine ligase family; tubulin; proteins containing a WD repeat domain; zinc fingers; the ZYG11 cell cycle regulator family; or combinations thereof.

[0454] In some embodiments, the target is or is contained in a gene expressed in podocytes. In some embodiments, the target is selected from podocyte genes or combinations thereof provided in Table 3. In some embodiments, the target has one or more properties and / or functions, or combinations thereof, provided in Table 4. In some embodiments, the target has one or more properties and / or functions selected from proteins containing α / β hydrolase domains; ADAM metallopeptidase and thrombin-sensitive protein type 1 motifs; proteins containing ankyrin repeat domains; apolipoproteins; proteins containing Armadillo-like helical domains; basic leucine zipper proteins; blood group antigens; bone morphogenetic proteins; proteins containing C-type lectin domains; C1q and TNF-related proteins; carbonic anhydrase; CD molecules; chitinases; cilia and flagella-related proteins; Crumbs complexes; Dbl family Rho GEF; Contains EF-hand domain; Contains F-BAR domain; Contains fibronectin type III domain; Forkhead box; Formin; G protein-coupled receptor; Contains Gla domain; Glycosyltransferases; Homology box; Contains immunoglobulin superfamily domain; Ion channel; Junctophilin; Kallikrein; Ligand-gated ion channel; Lipid transport protein; Myosin light chain kinase family; Netrin; Contains PDZ domain; Phospholipase; Contains Plek substrate protein homology domain; Potassium voltage-gated channel regulatory subunit; Protein phosphatase; Ras small GTPase superfamily; Receptor kinase; Receptor ligand; rho GTPase-activating proteins; proteins containing RNA-binding motifs; semaphorins; serine proteases; serine protease inhibitors and peptidase inhibitors; members of the Shisa family; solute carriers; proteins containing sterile α-motif domains; the Stomatin family of erythrocyte membrane proteins; T-cell receptors; the four-span linker complex superfamily; transcription elongation factor A-like family; troponin complex subunits; microtubule polymerization-promoting proteins; proteins containing WD repeat domains; the Wnt family; zinc fingers; or combinations thereof.

[0455] Table 1. Exemplary RPTEC genes

[0456] Table 2. Exemplary characteristics and / or functions of RPTEC genes

[0457] Table 3. Exemplary podocyte genes

[0458] Table 4. Exemplary characteristics and / or functions of podocyte genes

[0459] Characterization of conjugate drugs In some embodiments, the conjugate agents provided and / or utilized according to this disclosure are characterized, for example, when they are provided to an associated system (e.g., comprising one or more cells, tissues, organs, or organisms), they affect the expression and / or activity of one or more targets or their forms.

[0460] In some embodiments, the agent is characterized by its effect on the RNA (e.g., mRNA) and / or protein (e.g., encoded by mRNA) targeted by the nucleic acid payload. In some such embodiments, such effects are assessed in vivo (i.e., in an organism). Alternatively or additionally, in some such embodiments, the effects are assessed in vitro (e.g., in a cell line).

[0461] In some embodiments, the conjugated pharmaceutical agent described and / or utilized according to this disclosure is characterized relative to an unconjugated nucleic acid agent (as a payload). In some embodiments, when evaluated under comparable conditions, the observed effects when a suitable in vivo or in vitro system is contacted with the conjugated pharmaceutical agent described herein are significantly greater than the effects observed when the system is contacted with the unconjugated nucleic acid agent under other comparable conditions.

[0462] Pharmaceutical Composition This disclosure provides, in particular, pharmaceutical compositions comprising or otherwise delivering a conjugated regulatory nucleic acid agent; typically, such pharmaceutical compositions comprise an active agent (e.g., an ASO or iRNA agent or a composition comprising such an agent) conjugated to a target agent disclosed herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0463] Pharmaceutical compositions containing iRNA can be used to prevent or treat target RNA-related conditions, such as metabolic disorders, including nephropathy and various other kidney diseases or conditions. Such pharmaceutical compositions are formulated according to a delivery mode. One example is a composition formulated for systemic administration via parenteral delivery, such as subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit the expression of the target gene.

[0464] In some embodiments, the pharmaceutical compositions of this disclosure are sterile. In another embodiment, the pharmaceutical compositions of this disclosure are pyrogen-free.

[0465] The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit the expression of the target gene. Typically, a suitable dose of the ASO or iRNA agent disclosed herein will be in the range of about 0.001 mg to about 200.0 mg per kilogram of body weight per day for the recipient, and generally in the range of about 1 mg to 50 mg per kilogram of body weight per day. Typically, a suitable dose of the ASO or iRNA agent disclosed herein will be in the range of about 0.1 mg / kg to about 5.0 mg / kg, optionally about 0.3 mg / kg to about 3.0 mg / kg. Repeated dosing regimens may include periodic administration of therapeutic doses of the ASO or iRNA agent, such as once a month, every 3-6 months, or once a year. In some embodiments, the ASO or iRNA agent is administered about once a month to about once every six months.

[0466] Following the initial treatment plan, treatment can be administered at a lower frequency. The duration of treatment can be determined based on the severity of the disease.

[0467] In other embodiments, the single-dose pharmaceutical composition may be long-acting, such that the dose is administered at intervals not exceeding 1 month, 2 months, 3 months, or 4 months. In some embodiments of this disclosure, a single-dose pharmaceutical composition of this disclosure is administered approximately once a month. In other embodiments of this disclosure, a single-dose pharmaceutical composition of this disclosure is administered quarterly (i.e., approximately every three months). In other embodiments of this disclosure, a single-dose pharmaceutical composition of this disclosure is administered twice a year (i.e., approximately every six months).

[0468] Those skilled in the art will understand that certain factors can affect the dosage and duration required for effective treatment of a subject, including but not limited to mutations present in the subject, prior treatment, the subject's overall health or age, and any other pre-existing conditions. Furthermore, treatment of a subject with an appropriate preventative or therapeutically effective amount of the composition may include a single treatment or a series of treatments.

[0469] iRNA can be delivered in a manner that targets specific tissues (e.g., kidney cells).

[0470] The pharmaceutical compositions disclosed herein include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, pre-formulated liquids, self-emulsifying solids, and self-emulsifying semi-solids. Formulations include those targeted at the kidneys.

[0471] The pharmaceutical formulations of this disclosure, which can be conveniently presented in unit dosage forms, can be prepared according to conventional techniques well-known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a drug carrier or excipient. Typically, the formulation is prepared by uniformly and tightly associating the active ingredient with a liquid carrier.

[0472] In some embodiments, the pharmaceutical compositions described herein may comprise buffer solutions, including neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextran, or mannitol; proteins, peptides, or amino acids (e.g., glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, such as the absence of detectable levels of contaminants (e.g., endotoxins).

[0473] In some embodiments, the pharmaceutical compositions described herein may be administered in a manner suitable for treating or preventing a disease, condition, or ailment. In some embodiments, the amount and / or frequency of administration may be determined by factors such as the patient's condition, and / or the type and / or severity of the patient's disease, condition, or ailment, although the appropriate dosage may be determined through clinical trials.

[0474] In some embodiments, the pharmaceutical compositions provided herein may be in the form of, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and non-infusionable solutions), dispersions or suspensions, liposomes, and suppositories. Typically, pharmaceutical compositions containing or delivering antibody agents are injectable or non-infusionable solutions; in some such embodiments, such compositions may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranasal, intramedullary, intramuscular, transarterial, sublingual, intranasal, local, or intraperitoneal administration. In some embodiments, the provided pharmaceutical compositions are formulated for intravenous administration. In some embodiments, the provided pharmaceutical compositions are formulated for subcutaneous administration.

[0475] The pharmaceutical compositions described herein can be formulated for administration using infusion techniques known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, the entire contents of which are hereby incorporated by reference).

[0476] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously with, or after) other therapies targeting the symptoms, disease, or condition (e.g., SOC therapy targeting the symptoms, disease, or condition). In some embodiments, the pharmaceutical compositions described herein may be administered before or after surgery.

[0477] In some implementations, the dosage of any of the aforementioned therapies to be administered to the subject will vary depending on the disease, condition, or ailment being treated and will be based on the specific subject. Dosage scaling for human administration may be performed according to practices generally accepted in the art.

[0478] Uses of conjugated regulatory nucleic acid compounds Cell types delivered In some embodiments, the regulatory nucleic acid agent of this disclosure (e.g., a conjugate agent comprising the target portion disclosed herein and one or more regulatory nucleic acid compounds) is delivered to cells (e.g., cells of a tissue), optionally wherein cell surface factors are present.

[0479] In some embodiments, the cells are or comprise cells selected from the following (e.g., cells of tissues): kidney cells; immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., bone marrow and / or lymphocytes], erythrocytes, eosinophils, neutrophils and / or platelets); nervous system cells (e.g., brain tissue, cortex, cerebellum, retinal cells (e.g., retinal pigment epithelial cells (RPE)), spinal cord cells, nerve cells, neurons and / or supporting cells; endothelial cells; muscle (e.g., cardiac muscle, smooth muscle and / or skeletal muscle); small intestinal cells; colon cells; adipocytes; hepatocytes; lung cells; spleen cells; stomach cells; esophageal cells; bladder cells; pancreatic cells; thyroid cells; salivary gland cells; adrenal cells; pituitary cells; mammary gland cells; skin cells; ovarian cells; uterine cells; placental cells; prostate cells; or testicular cells, or combinations thereof.

[0480] In some implementations, the cells are or comprise cells selected from the following (e.g., cells of tissues): kidney cells, thyroid cells, parathyroid cells, inner ear cells, or nervous system cells.

[0481] In some embodiments, the cells are or comprise kidney cells, such as those described herein. In some embodiments, the cells are kidney cyst cells (e.g., in polycystic kidney disease (PKD)). In some embodiments, the cells are or comprise proximal tubular epithelial cells, podocytes, or both.

[0482] In some embodiments, the cell delivering the conjugate disclosed herein expresses both cell surface factors (e.g., macroproteins and / or cuboproteins) and the target of the payload portion.

[0483] In some embodiments, the regulatory nucleic acid agents of this disclosure (e.g., conjugates comprising the target portions disclosed herein and one or more regulatory nucleic acid compounds) are administered to a subject suffering from a disease or condition, such as those disclosed herein. In some embodiments, the disease or condition includes cells in which surface cytokines (e.g., macroproteins and / or cuboproteins) and / or target RNAs are present.

[0484] Indications In some embodiments, the regulatory nucleic acid agents of this disclosure (e.g., conjugates of the target portions disclosed herein and one or more regulatory nucleic acid compounds) are used to treat and / or prevent symptoms of the diseases or conditions disclosed herein.

[0485] In some implementations, the diseases or conditions for which the regulatory nucleic acid compounds disclosed herein are provided have elevated or abnormal expression of cell surface factors such as macroprotein and / or cuboprotein.

[0486] In some embodiments, macroprotein expression has been reported, particularly in the following tissues and / or cells: kidney tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissues. In some embodiments, macroprotein is expressed (e.g., at relatively high levels) on the surface of kidney cells, such as proximal tubular epithelial cells and podocytes.

[0487] In some implementations, the disease or condition is selected from: glomerular disease, tubular disease, other kidney disease, congenital metabolic defect, systemic metabolic disease, thyroid disease, parathyroid disease, inner ear disease, neurological disease, or viral infection or a combination thereof.

[0488] In some implementations, the disease or condition is or includes a glomerular condition. In some implementations, a glomerular condition is selected from: lupus nephritis, Goodpasture syndrome, IgA nephropathy, Alport syndrome, glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, ApoL1 nephropathy, post-infectious glomerulonephritis, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis syndrome, anti-LRP2 nephropathy, C3 glomerulonephropathy, or combinations thereof.

[0489] In some implementations, the disease or condition is or includes a renal tubular condition. In some implementations, a renal tubular condition is selected from: Fanconi syndrome, cystinuria, Lowe syndrome, Dent syndrome, light chain proximal tubular disease, Gitelman syndrome, renal tubular acidosis, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, hereditary aminoaciduria, hereditary salt wasting disease, hereditary phosphate wasting disease, porphyria-associated nephropathy, nephrotic cystinuria, autosomal dominant tubulointerstitial nephropathy, or combinations thereof.

[0490] In some implementations, the disease or condition is or includes other kidney conditions. In some implementations, other kidney conditions are selected from: ADPKD, ARPKD, nephronophthisis, chronic kidney disease, kidney stones, acute kidney injury, Alagille syndrome, cardiorenal syndrome, renal cell carcinoma, renal osteodystrophy, or combinations thereof.

[0491] In some embodiments, the disease or condition is or includes a congenital metabolic defect. In some embodiments, the congenital metabolic defect is selected from: phenylketonuria, urea cycle disorder, maple syrup urine disease, galactosemia, hereditary tyrosinemia, glutamateemia, isovaleric acidemia, very long / long / medium / short chain acyl-CoA dehydrogenase deficiency, methylmalonic acidemia, primary hyperoxaluria, propionic acidemia, porphyria, Wilson's disease, pyruvate dehydrogenase deficiency, homocystinuria, hereditary fructose intolerance, nonketotic hyperglycinemia, or combinations thereof.

[0492] In some implementations, the disease or condition is or includes a systemic metabolic disorder. In some implementations, a systemic metabolic disorder is selected from: diabetes, obesity, hypertension, gout, polyneuropathy, hypoglycemia, vitamin B deficiency, cirrhosis, coronary heart disease, stroke, lipodystrophy, or a combination thereof.

[0493] In some implementations, the disease or condition is or includes a thyroid condition. In some implementations, a thyroid condition is selected from: Hashimoto disease, Graves' disease, hypothyroidism, hyperthyroidism, goiter, thyroid nodules, thyroiditis, thyroid cancer, thyroid-stimulating hormone tumor, thyroid hormone resistance, MCT8 deficiency, Riedel's thyroiditis, Pendred syndrome, sarcoidosis, McCune-Albright syndrome, familial abnormal albuminemia, thyroxine-binding globulin (TBG) deficiency, or combinations thereof.

[0494] In some implementations, the disease or condition is or includes parathyroid disorders. In some implementations, parathyroid disorders are selected from: hyperparathyroidism / hypercalcemia, hypoparathyroidism / hypocalcemia, nephrolithiasis (kidney stones), pancreatitis, granulomatous diseases, Addison's disease, and pernicious anemia (many of which fall under hyperparathyroidism and hypoparathyroidism).

[0495] In some implementations, the disease or condition is or includes an inner ear condition. In some implementations, an inner ear condition is selected from: hereditary sensorineural hearing loss, vestibular neuritis, Meniere's syndrome, benign paroxysmal positional vertigo, tinnitus, age-related hearing loss, bilateral vestibular loss, perilymphatic fistula (PLF), superior semicircular canal dehiscence syndrome (SCD), drug-induced ototoxicity, herpes zoster of the ear, purulent labyrinthitis, and vestibular schwannoma.

[0496] In some implementations, the disease or condition is or includes a neurological condition, such as a neurodegenerative disease. In some implementations, the neurological condition is selected from: Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, neuro-AIDS, brain cancer, stroke, brain injury, spinal cord injury, autism, lysosomal storage diseases, Fragile X syndrome, hereditary intellectual disability, hereditary ataxia, blindness, paralysis, stroke, traumatic brain injury and spinal cord injury, and lysosomal storage diseases such as MPS I, MPS II, MPS III A, MPS III B, metachromatic leukodystrophy, Gaucher, Krabbe, Pompe, CLN2, Niemann-Pick and Tay-Sachs diseases, and combinations thereof.

[0497] In some implementations, the disease or condition is or includes a viral infection. In some implementations, the viral infection includes polyomavirus (e.g., BK virus)-mediated nephropathy.

[0498] Delivery of regulatory nucleic acid agents The conjugated regulatory nucleic acid agent of this disclosure can be delivered to cells, such as those of a subject, including human subjects (e.g., subjects in need, such as subjects susceptible to or diagnosed with target RNA-related conditions (e.g., kidney disease, metabolic disorders, including nephropathy and various other kidney diseases or conditions)). For example, delivery can be performed by contacting cells with the conjugated regulatory nucleic acid agent of this disclosure in vitro or in vivo. In vivo delivery can also be performed directly by administering to a subject a composition comprising the conjugated regulatory nucleic acid agent (e.g., polymyxin-linked dsRNA). Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors encoding and directing the expression of the conjugated regulatory nucleic acid agent.

[0499] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be used in conjunction with the conjugated regulatory nucleic acid agents of this disclosure (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors that need to be considered for delivering conjugated regulatory nucleic acid agents include, for example, the biostability of the delivered molecule, the prevention of nonspecific effects, and the accumulation of the delivered molecule in the target tissue. RNA interference has also demonstrated success in local delivery to the CNS via direct injection (Dorn, G. et al. (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT. et al. (2004) Neuroscience 129:521-528; Thacker, ER. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602). Modification of RNA or drug carriers can also allow conjugated regulatory nucleic acid agents to target tissues and avoid undesirable off-target effects. Conjugated regulatory nucleic acid agents can be further modified by chemical conjugation with lipophilic groups (such as cholesterol) to enhance cellular uptake and prevent degradation. For example, systemic injection of an iRNA agent targeting ApoB conjugated to the lipophilic cholesterol moiety into mice resulted in knockdown of apoB mRNA in the liver and jejunum (Soutschek, J. et al. (2004) Nature 432:173-178).

[0500] In alternative implementations, the conjugated regulatory nucleic acid agent can be delivered using drug delivery systems such as nanoparticles, dendritic polymers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of the conjugated regulatory nucleic acid agent (the negatively charged regulatory nucleic acid) and also enhance interactions at the negatively charged cell membrane to allow for efficient cellular uptake of the conjugated regulatory nucleic acid agent. Cationic lipids, dendritic polymers, or polymers can bind to the conjugated regulatory nucleic acid agent or be induced to form vesicles or micelles encapsulating the conjugated regulatory nucleic acid agent (see, for example, Kim SH et al. (2008) Journal of Controlled Release 129(2):107-116). When administered systemically, the formation of vesicles or micelles further prevents the degradation of the conjugated regulatory nucleic acid agent. Methods for preparing and administering cationically conjugated regulatory nucleic acid agents are entirely within the capabilities of those skilled in the art (see, for example, Sorensen, DR, et al. (2003) J. Mol. Biol 327:761-766; Verma, UN, et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS, et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems that can be used for systemic delivery of ASO or iRNA agents include DOTAP (Sorensen, DR., et al. (2003), ibid.; Verma, UN, et al. (2003), ibid.), “solid nucleic acid lipid particles” (Zimmermann, TS, et al. (2006) Nature 441:111-114), cardiolipin (Chien, PY, et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME, et al. (2008) Pharm. Res. August 16 online advance; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), and Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487) and polyamide (Tomalia, DA, et al. (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al. (1999) Pharm. Res. 16:1799-1804).In some embodiments, the conjugated regulatory nucleic acid agent forms a complex with cyclodextrin for systemic administration. Methods of administration of the iRNA agent and cyclodextrin, and pharmaceutical compositions, are available in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0501] In some embodiments, the conjugated agent is characterized in that, when delivered to cells, tissues, or organisms, the payload portion is delivered to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target cells and / or expressed therein, compared to other similar cells, tissues, or organisms to which the unconjugated payload portion is delivered.

[0502] In some embodiments, the conjugated agent is characterized in that, when delivered to a tissue or organism, the payload portion is delivered to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target cells and / or expressed therein, compared to non-target cells.

[0503] In some embodiments, the conjugated agent is characterized in that, when delivered to cells, tissues, or organisms, the expression and / or activity of the target of the payload portion are modulated, for example, reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0504] In some embodiments, this disclosure provides a conjugated agent comprising: (i) a targeting portion specifically for internalized cell surface factors; and (ii) a payload portion comprising a nucleic acid agent, wherein the binding portion and the nucleic acid agent are conjugated to each other via a cleavable linker such that the conjugated agent is in a first associated state when outside of kidney cells and in a second dissociated state when inside cells in the presence of cell surface factors.

[0505] Dosing regimen Those skilled in the art will be able to determine the appropriate amount, dosage, or dose of the conjugate agent to be administered to a patient, based on known methods and taking into account factors such as age, weight, general health condition, route of administration, symptoms requiring treatment, nature of the disease or condition, and the presence of other drugs. For example, various antibody dosing regimens are disclosed in Hendrikx J et al. (2017). Oncologist The entire contents of 22(10): 1212-1221, PMID: 28754722 are hereby incorporated by reference.

[0506] In some embodiments, the conjugated regulatory nucleic acid agents of this disclosure (e.g., conjugated pharmaceutical agents comprising the target moiety disclosed herein and one or more regulatory nucleic acid compounds) are administered at a fixed dose, i.e., regardless of body weight. In some embodiments, the fixed dose reduces inter-patient variability, such as efficacy and / or PK / PD parameters.

[0507] In some embodiments, the regulatory nucleic acid agents of this disclosure (e.g., conjugates of the target portion and one or more regulatory nucleic acid compounds disclosed herein) are administered based on body weight, for example, at a dose of mg / kg.

[0508] In some embodiments, the conjugated regulatory nucleic acid agent of this disclosure (e.g., a conjugated pharmaceutical agent comprising the target moiety disclosed herein and one or more regulatory nucleic acid compounds) is administered at an initial dose. In some embodiments, one or more subsequent doses may follow the initial dose. In some embodiments, one or more subsequent doses may be administered daily, weekly, or monthly, or at other intervals in between. In some embodiments, the dosing regimen disclosed herein may be repeated once or multiple times.

[0509] Methods to suppress target gene expression This disclosure also provides a method for inhibiting the expression of target genes in cells. The method involves contacting cells with a regulatory nucleic acid agent (e.g., a target-conjugated double-stranded RNA agent) that effectively inhibits the expression of target genes in cells, thereby inhibiting the expression of target genes in cells.

[0510] Contacting cells with a conjugated regulatory nucleic acid agent (e.g., a double-stranded RNA agent conjugated to a target portion) can be performed in vitro or in vivo. Contacting cells in vivo with a conjugated regulatory nucleic acid agent includes contacting cells or cell populations within a subject (e.g., a human subject) with the conjugated regulatory nucleic acid agent. Combinations of in vitro and in vivo methods for cell contact are also possible. As described above, cell contact can be direct or indirect. Furthermore, cell contact can be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a conjugate of a cell surface factor (e.g., a conjugate of a macroprotein or cuboprotein), or any other ligand that directs the conjugated regulatory nucleic acid agent to a site of interest.

[0511] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0512] In some embodiments of the methods disclosed herein, the expression of the target gene is suppressed by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or suppressed to below the assayed detection level. In some embodiments, the expression of the target gene is suppressed by at least 70%. It should also be understood that it may be necessary to suppress the expression of the target gene in certain tissues (e.g., kidney) without significantly suppressing its expression in other tissues (e.g., brain).

[0513] Control cells, cell populations, or subject samples that can be used to evaluate the inhibition of target gene expression include cells, cell populations, or subject samples that have not yet been contacted with the conjugated regulatory nucleic acid agent of this disclosure. For example, control cells, cell populations, or subject samples may be derived from individual subjects (e.g., human or animal subjects) prior to treatment with the conjugated regulatory nucleic acid agent or an appropriately matched population control.

[0514] In some embodiments, the level of target mRNA expressed by cells or cell groups can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of target RNA in a sample is determined by detecting transcribed polynucleotides or portions thereof (e.g., mRNA of a target gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), the RNeasy™ RNA preparation kit (Qiagen®), or PAXgene™ (PreAnalytix™, Switzerland). Typical forms of assays utilizing ribonucleic acid hybridization include nuclear ligation assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis.

[0515] In some implementations, nucleic acid probes are used to determine the expression level of target RNA. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific target. Probes may be synthesized by those skilled in the art or derived from suitable biological agents. Probes may be specifically designed for labeling. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0516] Isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern blotting, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting isolated mRNA with a nucleic acid molecule (probe) capable of hybridizing with the target mRNA. In one embodiment, mRNA is immobilized on a solid surface and contacted with a probe, for example, by running isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane (such as nitrocellulose). In an alternative embodiment, one or more probes are immobilized on a solid surface and the mRNA is contacted with one or more probes, for example, in an Affymetrix® gene chip array. Those skilled in the art can readily employ known mRNA detection methods to determine the level of the target mRNA.

[0517] Alternative methods for determining the expression level of target RNA in a sample involve nucleic acid amplification of mRNA or reverse transcriptase (to prepare cDNA) in the sample, for example by RT-PCR (Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology) The amplified molecules can be detected using methods such as rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by detection using techniques well-known to those skilled in the art. These detection protocols are particularly useful for detecting nucleic acid molecules if the number of molecules is very small. In a particular aspect of this disclosure, the expression level of the target RNA is determined by quantitative fluorescent RT-PCR (i.e., the TaqMan™ system).

[0518] The expression level of target mRNA can be monitored using membrane blotting (e.g., for hybridization analysis, such as Northern, Southern, dot, etc.) or microwells, sample tubes, gels,...

Claims

1. A nucleic acid conjugate pharmaceutical agent comprising a nucleic acid conjugated to a polymyxin moiety or an analogue thereof, wherein the nucleic acid is conjugated to the polymyxin moiety or analogue via a linker.

2. The nucleic acid conjugate agent according to claim 1, wherein the linker comprises C 2-22 An alkylene or branched alkylene chain, wherein the carbon atoms of the alkylene chain are optionally interrupted by one or more -O-.

3. The nucleic acid conjugate agent according to any one of claims 1 to 2, wherein the linker comprises one or more -(OCH2CH2)- units.

4. The nucleic acid conjugate agent according to claim 3, wherein the linker comprises at least two -(OCH2CH2)- units.

5. The nucleic acid conjugate agent according to any one of the preceding claims, wherein the nucleic acid conjugate agent is capable of mediating enhanced delivery of the nucleic acid to a target tissue.

6. The nucleic acid conjugate agent according to any one of claims 1 to 5, wherein the nucleic acid conjugate agent is characterized in that, when delivered to cells, tissues or subjects, enhanced delivery of the nucleic acid to the cells, tissues or subjects is observed compared with a comparative agent.

7. The nucleic acid conjugate agent according to claim 6, wherein the comparative agent is other similar cells, tissues, or subjects that deliver unconjugated nucleic acids.

8. The nucleic acid conjugate agent according to claim 7 or 8, wherein the enhanced delivery of the nucleic acid is mediated by an aminoglycoside moiety or analogue.

9. The nucleic acid conjugate agent according to any one of claims 1 to 8, wherein the nucleic acid conjugate agent is characterized in that, when delivered to cells, tissues or subjects, the level of the target gene is reduced in the cells, tissues or subjects compared to a comparison agent.

10. The nucleic acid conjugate agent according to claim 9, wherein the comparative is other similar cells, tissues, or subjects delivering unconjugated nucleic acids.

11. The nucleic acid conjugate agent according to any one of the preceding claims, comprising an effective load portion, said effective load portion comprising a nucleic acid conjugated to the structure of Formula I: Wherein R1 is an optionally substituted C3-C7 alkyl or optionally substituted C3-C7 aryl. Where L is Or a branch link header; n is an integer from 1 to 16; and AA1, AA2, and AA3 are each independently chemical bonds or selected from the following amino acids: Where X is: Where L is an optional connector, and Where M is: Where Y = O, S, or NSO2Me, and Z = Nucleic acid.

12. The conjugate agent according to claim 11, wherein AA2 is or .

13. The conjugate pharmaceutical agent according to claim 11 or claim 12, wherein n is an integer from 5 to 12.

14. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein R1 is or .

15. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the structure of formula I is selected from the group consisting of: 、 、 、 ,as well as 。 16. The conjugate agent according to any one of the preceding claims, wherein the conjugate comprises a plurality of Formula I structures, optionally wherein the nucleic acid conjugate agent comprises at least two, at least three, at least four or at least five Formula I structures.

17. The conjugate formulation according to claim 16, wherein the plurality of Formula I structures are conjugated to the effective load portion via a branch connector (L), optionally wherein the branch connector L has a structure selected from the group consisting of: as well as Where Comp1, Comp2 and / or Comp3 independently contain compounds of formula I': 。 18. The conjugate agent according to claim 16 or claim 17, wherein the nucleic acid conjugate comprises two structures of formula I'.

19. The conjugate agent according to claim 16 or claim 17, wherein the nucleic acid conjugate consists of two, three, four or five structures of formula I'.

20. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid is conjugated with a ligand selected from the group consisting of: , 、 、 ,as well as 。 21. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid is or comprises an antisense sequence element, optionally wherein the antisense sequence element is complementary to at least a portion of one or more of the following in the target sequence: exon, intron, untranslated region, splice site, promoter region, enhancer region or non-coding region.

22. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid comprises a sequence element that is at least 80% complementary to a target sequence in the sense strand.

23. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid comprises a sequence element that is at least 80% complementary to the target sequence in the antisense strand.

24. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid comprises at least one sequence element having at least three consecutive nucleotides having at least 80% complementarity to a portion of the target sequence.

25. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid is single-stranded.

26. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid is double-stranded.

27. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid is or comprises RNA.

28. The conjugate agent according to claim 26 or claim 27, wherein the nucleic acid is an inhibitory RNA agent (RNAi), optionally wherein the RNAi is or comprises short interfering RNA (siRNA).

29. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid comprises an oligonucleotide chain of about 15-25 nucleotides in length.

30. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid comprises one or more modified nucleotides.

31. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid is or comprises DNA.

32. The conjugate agent of claim 31, wherein the DNA is or comprises a DNA analog, optionally wherein the DNA analog comprises one or more morpholino subunits linked together by phosphorus-containing bonds.

33. The conjugate agent according to claim 32, wherein the DNA analog is or comprises phosphoryldiamine morpholinonucleotide (PMO).

34. The conjugate pharmaceutical preparation of claim 33, wherein the PMO comprises about 12-40 nucleotides.

35. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid is or comprises an antisense oligonucleotide (ASO).

36. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid is or comprises peptide nucleic acid (PNA).

37. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid comprises a modification, the modification comprising: a modified backbone, a modified nucleobase, a modified ribose, a modified deoxyribose, or a combination thereof.

38. The conjugate agent according to any one of the preceding claims, wherein the nucleic acid comprises one or more modifications to the 5' end of the nucleic acid.

39. The conjugate formulation according to any one of the preceding claims, wherein the effective load portion is conjugated with the remainder of the compound of formula I at the 5' end of the effective load portion or at the 3' end of the effective load portion.

40. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid comprises one or more extended nucleic acid ("exNA") modifications, optionally wherein the one or more exNA modifications are located at or near the 3' end of the nucleic acid.

41. The conjugate pharmaceutical agent according to any one of the preceding claims, wherein the nucleic acid comprises one or more phosphoryl guanidine-containing backbones ("PN backbones") and / or methanesulfonyl aminophosphate modifications.

42. A pharmaceutical composition comprising a conjugate pharmaceutical agent according to any one of the preceding claims, and a pharmaceutically acceptable carrier.

43. A cell comprising a conjugate agent bound thereto according to any one of claims 1 to 41.

44. The cell of claim 43, wherein the cell is selected from kidney cells, including kidney cells of the subject, mammalian cells, human cells expressing macroprotein, and human cells expressing cuboprotein.

45. A method of delivering a conjugated pharmaceutical agent to cells, tissues, or a subject, the method comprising administering to the cells, tissues, or subject the conjugated pharmaceutical agent according to any one of claims 1 to 41, the pharmaceutical composition according to claim 42, or the cells according to claim 43 or 44.

46. ​​A method for regulating the expression of a target gene in a cell, the method comprising providing the cell with a conjugate agent according to any one of claims 1 to 41, a pharmaceutical composition according to claim 42, or a cell according to claim 43 or claim 44.

47. A method for treating a disease or condition in a subject who has or is susceptible to such a disease or condition, the method comprising administering to the subject a conjugate agent according to any one of claims 1 to 41, a pharmaceutical composition according to claim 42, or a cell according to claim 43 or 44.

48. The method of claim 47, wherein the disease is a disease associated with the expression of a cell surface receptor, optionally wherein the disease is a disease of a cell containing both the cell surface receptor and a target recognized by the payload portion.

49. A method for improving the delivery of a drug to cells, the method comprising contacting a system or subject containing at least one cell with a conjugated drug according to any one of claims 1 to 41, a pharmaceutical composition according to claim 42, or a cell according to claim 43 or 44.

50. The method according to any one of claims 45, 46, 48 or 49, wherein the cells are selected from: kidney cells, thyroid cells, parathyroid cells, inner ear cells or nervous system cells or combinations thereof.

51. The method of claim 50, wherein the kidney cells are selected from proximal tubular epithelial cells and / or podocytes.

52. The method according to any one of claims 45 to 51, wherein administering / providing the conjugate agent to the cells, tissues, or subject delivers the payload portion to at least 5% more target cells than: (a) Delivery of other similar cells, tissues, or subjects with unattached payload portions; (b) Non-target cells; or (c) Both (a) and (b).

53. The method of claim 52, wherein the target cell is or comprises a kidney cell.

54. The method of claim 52 or 53, wherein the target cell is or comprises a cell expressing a renal cell surface factor, optionally wherein the renal cell surface factor is a macroprotein or a cuboprotein.

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