Polymyxin for drug delivery to the kidneys

Abstract

This specification discloses conjugating agents comprising a payload portion and a targeting portion (in certain embodiments, a polymyxin or related compound) directly or indirectly conjugated thereto, compositions containing the same, and methods for preparing and using the same.

Description

TECHNICAL FIELD

[0001] Cross-Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 585,806 filed on September 27, 2023, and U.S. Provisional Patent Application No. 63 / 655,321 filed on June 3, 2024, the contents of which are hereby incorporated by reference in their entirety into the present specification.

[0002] Field of the Invention The present disclosure relates generally to molecular targeting of compositions to kidney cells, and related methods.

BACKGROUND ART

[0003] Background of the Invention Regulatory nucleic acid therapeutics have achieved significant clinical and commercial success in the treatment of liver diseases, at least in part due to the fenestrations in liver sinusoidal epithelial cells that make such cells particularly accessible to injected nucleic acids, especially those with N-acetylgalactosamine (GalNAc) modifications. Non-hepatic tissues, including the kidney, have tended to be relatively difficult to approach with regulatory nucleic acid therapeutics. However, recent findings have described the successful use of moieties targeting kidney cell surface factors to facilitate efficient delivery of partially conjugated regulatory nucleic acids to kidney cells (see, for example, PCT / US23 / 16319 (Patent Document 1)).

[0004] There is a need for a wide range of kidney cell targeting agents capable of delivering payload moieties (e.g., regulatory nucleic acids) to kidney cells.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0005]

PATENT DOCUMENT 1

SUMMARY OF THE INVENTION

[0006] Brief Summary of the Invention This disclosure provides techniques for achieving targeted delivery of therapeutic agents and / or nucleic acid agents, particularly to kidney cells, by including, in particular, polymyxin compounds and other compounds thought 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 when bound by a targeting moiety (e.g., a megalin targeting moiety). In some embodiments, targeted delivery by this disclosure (e.g., megalin targeted delivery) may be targeted delivery to kidney cells. In some embodiments, this disclosure provides techniques particularly useful for delivery to, for example, proximal tubular epithelial cells, podocytes, and / or renal cysts (e.g., in polycystic kidney disease).

[0007] In particular, this disclosure recognizes that some of the challenges that often arise with targeted delivery (e.g., megalin targeted delivery) include inefficient and / or insufficiently targeted delivery, undesirable off-target effects, or effects in cells or tissues that do not represent the intended site of action, which can be particularly problematic.

[0008] This disclosure provides, in particular, a conjugate comprising a polymyxin (e.g., polymyxin B, polymyxin E (colistin), etc.) or a polymyxin-related compound as a targeting moiety (e.g., a megalin targeting moiety) and directly or indirectly conjugated with a payload moiety, wherein the payload moiety comprises a nucleic acid. While not intended to be theoretically bound, the targeting moieties described herein specifically bind to factors present on the surface of target cells(s) of interest, e.g., kidney-related cells. In some embodiments, the techniques provided achieve, for example, targeted delivery of the payload nucleic acid moiety to target cells, tissues, organs, or organisms of interest with minimal off-target effects. In some embodiments, the targeting moieties described herein (e.g., a megalin targeting moiety) specifically bind to factors preferentially present on the surface of target cells(s) or tissues(s) of interest compared to, for example, one or more non-target cells(s) or tissues(s). In some embodiments, the targeting moiety described herein (for example, polymyxin or a polymyxin-related compound as the targeting moiety) specifically binds to a factor specific to the target cell(s) or tissue(s)(s) of interest.

[0009] In particular, this disclosure provides insight that targeting megalin with polymyxin (or polymyxin-related compound) presentation conjugates is a particularly useful strategy for delivering specific nucleic acid agents to cells. This disclosure provides specific insight that targeting megalin represents a particularly useful strategy for delivering specific drugs to kidney-related cells (e.g., kidney cells), specifically for the delivery of nucleic acid agents.

[0010] Furthermore, this specification particularly teaches that conjugates, such as those described herein, comprising a megalin-binding moiety conjugated with a nucleic acid agent (optionally by a linker), are particularly useful for delivering such nucleic acid agents to megalin-expressing cells. This specification establishes, in particular, the usefulness of such conjugates for delivering nucleic acid agents to kidney cells.

[0011] In some embodiments, the conjugates disclosed herein, when provided to a relevant system (e.g., including one or more cells, tissues, organs, or organisms), are characterized in that they affect the expression and / or activity of one or more targets or morphologies compared to when they come into contact with an unconjugated nucleic acid payload, all other conditions being equal.

[0012] In one embodiment, the disclosure provides a drug comprising a nucleic acid conjugated to a polymyxin moiety or analogue, wherein the nucleic acid is conjugated to the polymyxin moiety or analogue by a linker.

[0013] In a particular embodiment, the linker is C 2~22 The material comprises an alkylene or a branched alkylene chain, and the carbon atoms of the alkylene chain may be interposed by one or more -O- groups.

[0014] In certain embodiments, the linker includes one or more -(OCH2CH2)- units. Optionally, the linker includes at least two -(OCH2CH2)- units.

[0015] In some embodiments, the drug is characterized by the observation of enhanced delivery of nucleic acids to cells, tissues, or targets when delivered to cells, tissues, or targets, compared to a comparator. Optionally, the comparator is a similar cell, tissue, or target, except that it is delivered with unconjugated nucleic acids. In relevant embodiments, the enhanced delivery of nucleic acids is mediated by a polymyxin moiety or analogue.

[0016] In one embodiment, the drug, upon delivery to cells, tissues, or subjects, is characterized by a decrease in the level of a target gene in those cells, tissues, or subjects compared to a comparator. In related embodiments, the comparator is a similar cell, tissue, or subject, except that it is delivered with an unconjugated nucleic acid. In some embodiments, the decrease in the level of the target gene is mediated by the nucleic acid.

[0017] In another aspect, this disclosure includes (ii) Formula I: (i) Payload portion containing nucleic acids conjugated to the structure of TIFF2026534515000001.tif64165 The present invention provides a conjugate agent for regulating the expression of a target gene, including, In the formula, R1 is an optionally substituted C3-C7 alkyl or optionally substituted C3-C7 aryl. L is TIFF2026534515000002.tif22165 or branch linker, n is an integer between 1 and 16, and AA1, AA2, and AA3 are each independently of each other, and are chemically bonded or as follows: The amino acids are selected from TIFF2026534515000003.tif75165. Here, X is The file is TIFF2026534515000004.tif32165, where L is an optional linker, and M is The filename is TIFF2026534515000005.tif32165. Here, Y = O, S, or NSO2Me, and Z = nucleic acid.

[0018] In one embodiment, AA2 is The filename is TIFF2026534515000006.tif32165.

[0019] In another embodiment, n is an integer between 5 and 12.

[0020] In a particular embodiment, R1 is The filename is TIFF2026534515000007.tif27165.

[0021] In some embodiments, the structure of formula I is as follows: Selected from TIFF2026534515000008.tif197165 and TIFF2026534515000009.tif135165.

[0022] In one embodiment, the conjugate comprises multiple structures of formula I. Optionally, the nucleic acid conjugate comprises at least two, at least three, at least four, or at least five structures of formula I. In one embodiment, the multiple structures of formula I are conjugated to the payload portion via a branched linker (L). Optionally, the branched linker L is as follows: It has a structure selected from TIFF2026534515000010.tif94165, In the formula, compound 1, compound 2, and / or compound 3 are independently of formula I': Contains the compound TIFF2026534515000011.tif74165.

[0023] In one embodiment, the nucleic acid conjugate comprises two structures of formula I'.

[0024] In another embodiment, the nucleic acid conjugate comprises three structures of formula I'.

[0025] In certain embodiments, the nucleic acid conjugate consists of two, three, four, or five formula I' structures conjugated to a nucleic acid payload via one or more linkers (optionally branched).

[0026] In one embodiment, nucleic acids are It is conjugated to a ligand selected from TIFF2026534515000012.tif197165 and TIFF2026534515000013.tif135165.

[0027] In a particular embodiment, 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 exons, introns, untranslated regions, splice junctions, promoter regions, enhancer regions, or non-coding regions within the target sequence.

[0028] In certain embodiments, the nucleic acid is a sense sequence element or includes a sense sequence element. Optionally, the sense sequence element is substantially similar in sequence (e.g., at least 80% identical) to at least a portion of one or more of the exons, introns, untranslated regions, splice junctions, promoter regions, enhancer regions, or non-coding regions within the target sequence.

[0029] In one embodiment, the nucleic acid includes sequence elements that are at least 80% complementary to the target sequence in the sense strand.

[0030] In another embodiment, the nucleic acid includes sequence elements that are at least 80% complementary to the target sequence in the antisense strand.

[0031] In certain embodiments, the nucleic acid comprises at least one sequence element having at least three consecutive nucleotides that have at least 80% complementarity to a portion of the target sequence.

[0032] In one embodiment, the nucleic acid is single-stranded.

[0033] In another embodiment, the nucleic acid is double-stranded.

[0034] In certain embodiments, the nucleic acid is RNA or contains RNA.

[0035] In some embodiments, the nucleic acid is an inhibitory RNA agent (RNAi). Optionally, the RNAi is or contains small interfering RNA (siRNA).

[0036] In one embodiment, the nucleic acid includes an oligonucleotide chain approximately 15 to 25 nucleotides long.

[0037] In one embodiment, the nucleic acid comprises one or more modified nucleotides.

[0038] In another embodiment, the nucleic acid is DNA or contains DNA. Optionally, the DNA is a DNA analogue or contains a DNA analogue. Optionally, the DNA analogue contains one or more morpholino subunits linked together by a phosphorus-containing bond. In certain embodiments, the DNA analogue is a phosphorodiamidate morpholino nucleic acid (PMO) or contains a phosphorodiamidate morpholino nucleic acid. In some embodiments, the PMO contains about 12 to 40 nucleotides.

[0039] In certain embodiments, the nucleic acid is an antisense oligonucleotide (ASO) or contains an antisense oligonucleotide.

[0040] In another embodiment, the nucleic acid is a peptide nucleic acid (PNA) or comprises a peptide nucleic acid.

[0041] In some embodiments, the nucleic acid includes one or more of the following modifications: a modified skeleton, a modified nucleic acid base, a modified ribose, a modified deoxyribose, or a combination thereof.

[0042] In certain embodiments, the nucleic acid includes one or more modifications to the 5' end of the nucleic acid.

[0043] In one embodiment, the payload portion is conjugated to the rest of the compound of formula I at either its 5' or 3' end.

[0044] In another embodiment, the payload portion of nucleic acid includes one or more extended nucleic acid ("exNA") modifications. Optionally, one or more exNA modifications are located at or near the 3' end of the payload portion of nucleic acid.

[0045] In some embodiments, the nucleic acid of the payload portion comprises one or more phosphorylguanidine-containing skeletons ("PN skeletons") and / or mesylphosphoamidate modifications.

[0046] Another aspect of the present disclosure provides a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable carrier of the present disclosure.

[0047] A further aspect of the present disclosure provides a cell comprising a conjugate agent of the present disclosure bound thereto.

[0048] In one embodiment, the cell is a kidney cell. Optionally, the cell is a kidney cell of interest. Optionally, the cell is a kidney cell present in a living organism. In a particular embodiment, the cell may be a mammalian cell, such as a human cell. In a particular embodiment, the cell expresses megalin. In another embodiment, the cell expresses cubilin. In yet another embodiment, the cell expresses both megalin and cubilin. In a particular embodiment, the cell is a human cell expressing megalin and / or cubilin.

[0049] Further aspects of the present disclosure provide a method for delivering a conjugate agent to cells, tissues, or subjects, the method comprising delivering a conjugate agent to cells, tissues, or subjects by administering the conjugate agent, pharmaceutical composition, and / or cells of the present disclosure to the cells, tissues, or subjects.

[0050] Further aspects of this disclosure provide methods for modulating the expression of a target gene in cells, the methods including providing the conjugate agents, pharmaceutical compositions, and / or cells of this disclosure to cells.

[0051] Another aspect of the present disclosure provides a method for treating a disease or disorder in a subject that is suffering from or susceptible to the disease or disorder, the method comprising administering the conjugate agents, pharmaceutical compositions, and / or cells of the present disclosure to a subject.

[0052] In one embodiment, the disease is a disease associated with the expression of cell surface receptors. Optionally, the disease is a disease involving cells that contain both cell surface receptors and targets recognized by the payload portion.

[0053] Further aspects of the present disclosure provide a method for improving drug delivery to cells, the method comprising contacting a system or subject having at least one cell with a conjugate agent, pharmaceutical composition, and / or cell of the present disclosure.

[0054] In one embodiment, the cells are selected from kidney cells, thyroid cells, parathyroid cells, inner ear cells, or nervous system cells, or a combination thereof. Optionally, kidney cells are selected from proximal tubular epithelial cells and / or podocytes.

[0055] In certain embodiments, the Disclosure provides a method for administering the conjugate of the Disclosure to cells, tissues, or subjects to thereby deliver the payload portion to (a) similar cells, tissues, or subjects except that the unconjugated payload portion is delivered; (b) non-target cells; or (c) at least 5% more target cells compared to both (a) and (b).

[0056] In certain embodiments, the target cells are kidney cells or include kidney cells.

[0057] In another embodiment, the target cells are cells expressing a renal cell surface factor, or include cells expressing a renal cell surface factor. Optionally, the renal cell surface factor is megalin or cubilin.

[0058] In one embodiment, the nucleic acid payload is an antisense compound having an antisense chain as the first chain of the antisense compound. In one embodiment, the antisense compound includes a second chain complementary to the first chain, having a length of 15 to 60 nucleic acid bases.

[0059] In some embodiments, the compound is an antisense oligonucleotide (ASO).

[0060] In one embodiment, the payload-modulating nucleic acid compound of the present disclosure (e.g., an antisense compound, dsRNA, etc.) has at least one modified nucleoside bond, sugar moiety, or nucleic acid base. Optionally, the modified nucleoside bond is or contains a phosphorothioate bond.

[0061] In certain embodiments, at least one modified nucleoside bond, sugar moiety, or nucleic acid base is modified in the following ways: deoxy-nucleoside, 3'-terminal deoxythymine (dT) nucleoside, 2'-O-methyl (2'-OMe) modified nucleoside, 2'-fluoro (2'-F) modified nucleoside, 2'-deoxy modified nucleoside, locked nucleotide (LNA), unlocked nucleoside, conformationally restricted nucleoside, and restricted nucleotide. Tyl nucleosides, debasic nucleosides, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), 2'-amino-modified nucleosides, 2'-O-allyl-modified nucleosides, 2'-C-alkyl-modified nucleosides, 2'-hydroxy-modified nucleosides, 2'-methoxyethyl (2'-MOE)-modified nucleosides, 2'-O-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates, unnatural bases containing nucleosides, The material comprises one or more of the following: tetrahydropyran-modified nucleosides, 1,5-anhydrohexitol-modified nucleosides, cyclohexenyl-modified nucleosides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleosides containing adenosine-glycol nucleic acid (GNA), nucleosides containing thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and cholesteryl derivatives and terminal nucleosides bonded to a dodecanoate bisdecylamide group, as well as combinations thereof.

[0062] In certain embodiments, the modified nucleic acid base is 5-methylcytosine or contains 5-methylcytosine.

[0063] In certain embodiments, the double-stranded regions of the double-stranded nucleic acid payload of the present disclosure are 19 to 30 nucleotide pairs long; the double-stranded regions are 19 to 25 nucleotide pairs long; the double-stranded regions are 19 to 23 nucleotide pairs long; each strand is independently 30 nucleotides or less in length; the sense strand is 21 nucleotides long, the antisense strand is 23 nucleotides long; and / or the complementary region is at least 17 nucleotides long. Optionally, the complementary region is 19 to 23 nucleotides long. Optionally, the complementary region is 19 nucleotides long, and / or at least one strand includes a 3' overhang of at least 2 nucleotides.

[0064] In one embodiment, the payload nucleic acid of the Disclosure is or comprises an exon-skipping antisense oligonucleotide.

[0065] In one embodiment, the nucleic acid payload of the Disclosure (e.g., antisense compounds, RNAi compounds, exon-skipping antisense oligonucleotides, etc.) has at least one modified nucleoside linkage, sugar moiety, or nucleic acid base. Optionally, the modified nucleoside linkage includes a phosphorothioate linkage. In certain embodiments, the at least one modified nucleoside linkage, sugar moiety, or nucleic acid base is one of the following: deoxy-nucleoside, 3'-terminal deoxythymine (dT) nucleoside, 2'-O-methyl (2'-OMe) modified nucleoside, 2'-fluoro (2'-F) modified nucleoside, 2'-deoxy modified nucleoside, locked nucleotide (LNA), unlocked nucleoside, conformationally restricted nucleoside, or restricted ethyl Nucleosides, debasic nucleosides, 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), 2'-amino-modified nucleosides, 2'-O-allyl-modified nucleosides, 2'-C-alkyl-modified nucleosides, 2'-hydroxy-modified nucleosides, 2'-methoxyethyl (2'-MOE)-modified nucleosides, 2'-O-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates, unnatural bases containing nucleosides, tetra The modifications include rahydropyran-modified nucleosides, 1,5-anhydrohexitol-modified nucleosides, cyclohexenyl-modified nucleosides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleosides containing adenosine-glycol nucleic acid (GNA), nucleosides containing thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and cholesteryl derivatives and terminal nucleosides bonded to a dodecanoate bisdecylamide group, as well as modifications selected from combinations thereof.Optionally, the nucleic acid payload (e.g., antisense compound, dsRNA agent, exon-skipped antisense oligonucleotide, etc.) comprises one or more 2'-methoxyethyl (2'-MOE) modified nucleosides. In certain embodiments, all nucleosides in the nucleic acid payload are 2'-methoxyethyl (2'-MOE) modified nucleosides.

[0066] In some embodiments, the nucleic acid payload includes at least one modified nucleotide. Optionally, substantially all of the nucleotides in the sense strand of the nucleic acid payload, or substantially all of the nucleotides in the antisense strand of the antisense compound of the nucleic acid payload, include modifications, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand of the nucleic acid payload include modifications.

[0067] In certain embodiments, all nucleotides of the sense strand of the dsRNA agent nucleic acid payload are modified, or all nucleotides of the antisense strand of the antisense compound or other nucleic acid payload of the present disclosure are modified, or all nucleotides of the sense strand and all nucleotides of the antisense strand of the nucleic acid payload are modified.

[0068] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-restricted nucleotide, a restricted ethyl nucleotide, a debasalized 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 nucleotide (e.g., phospho These include logiamidate morpholino nucleic acids (PMOs), phosphoramidates, non-natural bases containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing phosphorylguanidine skeletons, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermally destabilized nucleotides, glycol-modified nucleotides (GNAs), and / or 2-O-(N-methylacetamide)-modified nucleotides, as well as combinations thereof.

[0069] In some embodiments, modifications to nucleotides include: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol, as well as combinations thereof; C7-modified deaza-adenine, C7-modified deaza-guanosine, C5-modified cytosine, C5-modified uridine, N1-methyl-pseudridine (m1ψ), 1-ethyl-pseudridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudridine, 5-methylcytidine, 5-methoxycytidine, or combinations thereof; phosphorothioate (PS) modification The following are selected from modifications, phosphorylguanidine (PN) modifications, boranophosphate modifications, alkylphosphonate nucleic acids (phNA), peptide nucleic acids (PNA), or combinations thereof; deoxyribonucleic acid (DNA) (optionally, DNA is or contains a DNA analog, optionally containing one or more morpholino subunits linked to each other by phosphorus-containing bonds, optionally containing a phosphorodiamidate morpholino nucleic acid (PMO), optionally containing a PMO containing about 12 to 40 nucleotides); peptide nucleic acid (PNA) modifications; and / or one or more modifications to the 5' end of an antisense compound or dsRNA agent (optionally, the modification to the 5' end is a 5' amino modification).

[0070] In certain embodiments, at least one modified nucleotide is selected from the following: deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxyribonucleotides, glycol modified nucleotides (GNAs), and / or vinyl phosphonate nucleotides; and / or combinations thereof.

[0071] In some embodiments, at least one modified nucleotide is selected from the following: deoxyribonucleotides, 2'-O-methyl modified nucleotides, and / or 2'-fluoro modified nucleotides; and / or combinations thereof.

[0072] In one embodiment, the nucleic acid payload of the Disclosure comprises one or more deoxynucleotides. Optionally, the nucleic acid payload is a component of a heterodouble-stranded oligonucleotide (HDO) and / or is a heterodouble-stranded oligonucleotide (HDO).

[0073] In one embodiment, the linker of the Disclosure is a cleavable linker. Optionally, the linker is cleaved when exposed to the intracellular environment.

[0074] The pharmaceutical compositions of this disclosure may be formulated for intravenous, subcutaneous, intramuscular, parenteral, or oral delivery, as optional.

[0075] Further aspects of this disclosure provide a kit for performing the methods described herein, the kit comprising a conjugate agent containing a modulated payload nucleic acid as described herein, and instructions for use thereof. Optionally, the kit also comprises means for administering the conjugate agent to a target.

[0076] definition To make this disclosure easier to understand, certain terms are first defined. Furthermore, whenever parameter values ​​or ranges of values ​​are enumerated, it should be noted that intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of this disclosure.

[0077] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects. For example, “an element” means one element or more than one element, e.g., multiple elements.

[0078] The term "including" is used herein to mean the phrase "including but not limited to," and is used interchangeably with it.

[0079] The term "or" is used herein to mean the term "and / or" unless the context otherwise explicitly indicates, and is used interchangeably with it.

[0080] The term “approximately” is used herein to mean a typical tolerance in the art. For example, “approximately” can be understood as approximately two standard deviations from the mean. In certain embodiments, approximately means ±10%. In certain embodiments, approximately means ±5%. When “approximately” precedes a set of numbers or ranges, it is understood that “approximately” can modify each number within that set of numbers or ranges.

[0081] The term "at least" preceding a number or a set of numbers is understood, as is clear from the context, to include the number following the term "at least," and all subsequent numbers or integers that can logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides out of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the stated characteristic. When "at least" precedes a set of numbers or a range, it is understood that each number within the set of numbers or range can be modified.

[0082] When used herein, “less than” or “less than” is understood as a set of consecutive values, and a logically low value or integer is zero, as logical from the context. For example, a double strand with an overhang of “less than or equal to 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. When “less than” precedes a set of numbers or a range, it is understood that “less than” can modify each number in the set of numbers or range. When used herein, a range includes both an upper and lower limit.

[0083] As used herein, the detection method may include a determination that the amount of analyte present is below the detection level of the method.

[0084] If the indicated target site is inconsistent with the nucleotide sequence of the sense or antisense strand, the indicated sequence takes precedence.

[0085] If there is a discrepancy between the chemical structure and the chemical name, the chemical structure takes precedence.

[0086] As used herein, the term “linker” means an organic part that connects two parts of a compound, for example, by covalent bonding. Linkers are typically directly bonded or to atoms such as oxygen or sulfur, NH, C(O), C(O)NH, SO, SO2, SO2NH, or chains of atoms, for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenyl The units include heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, etc., and one or more methylene units may be intercepted or terminated by O, S, S(O), SO2, NH, C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic compounds. The terms "linker" and "spacer" are used interchangeably herein.

[0087] In certain embodiments, the linker or bond may be a covalent bond linking two groups, or a chain of 1 to 100 atoms, e.g., a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, where the linker may be linear, branched, cyclic, or single-atom. In some cases, the linker is a branched linker referring to a linking portion linking three or more groups. In certain cases, one, two, three, four, or five or more carbon atoms in the linker skeleton may be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker skeleton may contain linking functional groups such as ethers, thioethers, aminos, amides, sulfonamides, carbamates, thiocarbamates, urea, thiourea, esters, thioesters, or imines. The bonds between the skeleton atoms may be saturated or unsaturated, and in some cases, one or fewer, two or fewer, or three or fewer unsaturated bonds may be present in the linker skeleton. The linker may contain one or more substituents, for example, with an alkyl, aryl, or alkenyl group. The linker may, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, and alkyl groups that may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker skeleton may contain a cyclic group, for example, an aryl, heterocyclic, or cycloalkyl group, and two or more atoms of the cyclic group, for example, two, three, or four atoms, are included in the skeleton. The linker may be cleavable or incleavable. In some cases, the linker is a branched linker, for example, a branched linker as described herein (e.g., a linker that branches to allow a multivalent targeting portion, a multivalent payload portion, or both within a single conjugate).

[0088] Certain compositions in this disclosure provide compounds having aliphatic hydrocarbons. The aliphatic chains include the classes alkyl, alkenyl, and alkynyl as defined below.

[0089] A linear aliphatic chain is limited to the unbranched carbon chain portion. As used herein, the term "aliphatic group" refers to a linear, branched, or cyclic aliphatic hydrocarbon group, and includes saturated and unsaturated aliphatic groups such as alkyl, alkenyl, or alkynyl groups.

[0090] "Alkyl" refers to a fully saturated cyclic or acyclic branched or unbranched carbon chain portion having a specified number of carbon atoms, or up to 30 carbon atoms if no number is specified. For example, alkyls with 1 to 8 carbon atoms refer to portions such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, as well as positional isomers of these portions. Alkyls with 10 to 30 carbon atoms include decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, and tetracosyl. In certain embodiments, linear or branched alkyls have 30 or fewer carbon atoms in their backbone (for example, C1 to C1 in the case of a linear chain). 30 In the case of branched chains, C3~C 30 ), optionally having 20 or fewer carbon atoms. The alkyl group may be substituted or unsubstituted.

[0091] As used herein, the term “heteroalkyl” refers to a previously defined alkyl moiety that contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms instead of carbon atoms.

[0092] As used herein, the term "alkylene" refers to an alkyl group having a specified number of carbon atoms, for example, 2 to 12 carbon atoms, and containing two bonding sites 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. The alkylene group may be a cyclic or acyclic, branched or unbranched carbon chain portion and may be optionally substituted with one or more substituents.

[0093] "Cycloalkyl" refers to a monocyclic, bicyclic, bridging, spirocyclic, or polycyclic saturated carbocyclic ring, each having 3 to 12 carbon atoms. Certain cycloalkyls may have 3 to 10 carbon atoms in their ring structure, and optionally 3 to 6 carbon atoms. Cycloalkyls may be substituted or unsubstituted.

[0094] Unless otherwise specified, the term "lower alkyl" as used herein means alkyl groups having 1 to 10 carbon atoms, and optionally 1 to 6 carbon atoms, in their skeletal structure, as defined above, 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 this disclosure, any particular alkyl group is a lower alkyl group. In certain embodiments, any substituent designated as alkyl herein is a lower alkyl group.

[0095] An "alkenyl" refers to a cyclic or acyclic branched or unbranched unsaturated carbon chain segment having a specified number of carbon atoms, or up to 26 carbon atoms if no limit on the number of carbon atoms is specified, and having one or more double bonds within the segment. Examples of alkenyls with 6 to 26 carbon atoms include hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicocenyl, heneicosoenyl, dococenyl, tricocenyl, and tetracocenyl, in their various isomers, where the unsaturated bond(s) can be located anywhere within the segment, and the double bond(s) can have either a (Z) or (E) configuration.

[0096] "Alkinyl" refers to a hydrocarbon portion within the alkenyl group that contains one or more triple bonds.

[0097] As used herein, the term “aryl” includes substituted or unsubstituted 3- to 12-membered monocyclic aromatic groups, where each atom of the ring is carbon (i.e., a carbocyclic aryl), or where one or more atoms are heteroatoms (i.e., a 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 in which two or more carbons are common to two adjacent rings, where at least one of these rings is aromatic, and for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. Heteroaryl groups include substituted or unsubstituted aromatic 3- to 12-membered ring structures, optionally 5- to 12-membered rings, and optionally 5- to 10-membered rings, where the ring structure contains 1 to 4 heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Aryls and heteroaryls can be monocyclic, bicyclic, or polycyclic.

[0098] The term "heterocyclyl" or "heterocyclic group" refers to a ring structure containing 1 to 4 heteroatoms in its ring structure, a 3 to 12-membered ring structure, optionally a 5 to 12-membered ring, or optionally a 5 to 10-membered ring. Heterocyclic rings can be monocyclic, bicyclic, spirocyclic, or polycyclic. Examples of heterocyclic groups include thiophene, thiantrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxatiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indidine, isoindole, indole, indazole, purine, quinoridine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carborin, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenalsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolan, o Heterocycles such as xazoles, piperidines, piperazines, morpholines, lactones, lactams such as azetidinone and pyrrolidinone, sultams, sultones, etc., can be substituted at one or more positions with substituents such as halogens, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, aminos, nitros, sulfhydryls, iminos, amides, phosphates, phosphonates, phosphinates, carbonyls, carboxyls, silyls, sulfamoyls, sulfinyls, ethers, alkylthios, sulfonyls, ketones, aldehydes, esters, heterocyclyls, aromatic or heteroaromatic moieties, -CF3, -CN, and the like.

[0099] The term "substituted" refers to a portion of a skeleton in which a substituent replaces one or more hydrogen atoms. It will be understood that "substituted" or "substituted with" implicitly includes the conditions that such substitution conforms to the permissible valencies of the substituted atom and substituent, and that the substitution results in a stable compound, such as one that does not undergo spontaneous transformation through rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In broad embodiments, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound.

[0100] The permissible substituents may be one or more for a given organic compound, and may be the same or different. For the purposes of this disclosure, heteroatoms such as nitrogen may have any permissible substituents of the organic compounds described herein that satisfy the valence of the hydrogen substituent and / or the heteroatom. Substituents may include any substituents described herein, e.g., halogens, hydroxyls, carbonyls (carboxyls, alkoxycarbonyls, formyls, or acyls, etc.), thiocarbonyls (thioesters, thioacetates, or thioformates, etc.), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. In certain embodiments, substituents on the substituted alkyl are C 1~6 Alkyl, C 3~6The substituents are selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In certain embodiments, the substituents on the substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will understand that, where appropriate, the substituents themselves may be substituted. Unless otherwise specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include substituted variants. For example, references to “aryl” groups or moieties implicitly include both substituted and unsubstituted variants.

[0101] As used herein, the term “agent” may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by certain properties and / or effects. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class, including, for example, small molecules, polypeptides, nucleic acids, sugars, lipids, metals, or combinations or complexes thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity containing a polymer. In some embodiments, the term may refer to a compound or entity containing one or more polymer portions. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that substantially does not contain a particular polymer or polymer portion. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or substantially does not contain a polymer or polymer portion.

[0102] 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 the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A “standard amino acid” refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A “non-standard amino acid” refers to any amino acid other than a standard amino acid, whether synthesized or obtained from a natural source. In some embodiments, amino acids in a polypeptide, including carboxyl and / or amino-terminal amino acids, may contain structural modifications compared to the general structure described above. For example, in some embodiments, an amino acid may be modified compared to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications can alter the cyclic half-life of a polypeptide containing modified amino acids compared to, for example, an identical polypeptide except for the presence of unmodified amino acids. In some embodiments, such modifications do not significantly alter the related activity of a polypeptide containing modified amino acids compared to an identical polypeptide except for the presence of unmodified amino acids. As will be apparent from the context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid. In some embodiments, amino acid may be used to refer to an amino acid residue of a polypeptide.

[0103] In certain embodiments, antisense compounds comprising antisense oligonucleotides, as well as other antisense compounds for use in modulating the expression and / or activity of target nucleic acid molecules, including, for example, the regulation of the expression of a target mRNA molecule, are disclosed. While not wishing to be constrained by theory, in certain embodiments, the regulation of target mRNA expression may be achieved by providing antisense compounds that hybridize with one or more target nucleic acid molecules, including hybridizing with one or more target mRNAs.

[0104] In some embodiments, double-stranded RNA (dsRNA) agents (e.g., siRNA) have been disclosed for use in regulating the expression of nucleic acid molecules encoding target mRNA. While we do not wish to be bound by theory, in certain embodiments, the regulation of target gene expression is achieved by providing dsRNA agents that involve an RNA interference mechanism (specifically, RNA-induced silencing complex (RISC)) to direct sequence-specific cleavage and degradation of one or more target nucleic acid molecules. In certain other embodiments, dsRNA compositions of the present disclosure (e.g., hetero-double-stranded oligonucleotides (HDOs), known in the art and comprising an antisense oligonucleotide chain that forms a double helix with a complementary RNA oligonucleotide chain (typically a “gapmer” type antisense oligonucleotide structure)) can achieve the regulation of target nucleic acid expression via an antisense oligonucleotide (ASO) mechanism that does not involve RISC.

[0105] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant,” as used interchangeably herein, refer to agents containing RNA, as defined herein, that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, for example, inhibits the expression of target genes in cells, such as cells, in subjects such as mammalian subjects.

[0106] In one embodiment, the RNAi agent of this disclosure comprises a single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. While we do not wish to be constrained by theory, it is thought that long double-stranded RNA introduced into a cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing a complementary antisense strand to induce target recognition (Nycanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC complex cleave the target, inducing silencing (Elbasil, et al., (2001) Genes Dev. 15:188). Therefore, in one embodiment, this disclosure relates to single-stranded RNA (siRNA) generated in cells that promote the formation of the RISC complex to achieve silencing of a target gene. Accordingly, the term “siRNA” is also used herein to refer to such siRNA.

[0107] In certain embodiments, the modulating 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 inhibit a target mRNA. While not wishing to be bound by theory, single-stranded RNAi agents bind to the RISC endonuclease Argonaut 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and are chemically modified. 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 incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0108] In certain embodiments, the “iRNA” for use in the compositions, uses, and methods of this disclosure is double-stranded RNA, and is referred herein to as “double-stranded RNA agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” means a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are referred to as having “sense” and “antisense” orientations with respect to target RNA (optionally, in one, more, or all of such ribonucleic acid strands of one or both strands of such dsRNA agent, including modified nucleotides as defined herein by substitution). In some embodiments of this disclosure, double-stranded RNA (dsRNA) induces degradation of target RNA via a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0109] As used herein, the term “antisense oligonucleotide (ASO)” refers to an oligonucleotide that can interact with and / or hybridize to pre-mRNA or mRNA having a complementary nucleotide sequence, thereby modifying gene expression.

[0110] As used herein, the term “exon skipping” refers to the modification of pre-mRNA splicing by targeting splice donors and / or acceptors and branching sites with one or more complementary antisense oligonucleotides (ASOs). By blocking spliceosome access to one or more splice donors, acceptors, or branching sites, ASOs can prevent the splicing reaction, thereby excluding one or more exons from fully processed mRNA. Exon skipping is achieved in the nucleus during the pre-mRNA maturation process. This involves masking key sequences involved in the splicing of the targeted exon by using antisense oligonucleotides (ASOs) complementary to splice donor / acceptor and branching point sequences, and / or by overlapping with ESEs (intra-exon) and ISEs (intra-intron) within the pre-mRNA.

[0111] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, "iRNA" or ASO may contain chemically modified ribonucleotides, and iRNA or ASO may contain substantial modifications to multiple nucleotides.

[0112] As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleotide-nucleotide bond, or a modified nucleic acid base, or any combination thereof. Therefore, the term “modified nucleotide” encompasses, for example, the substitution, addition, or removal of functional groups or atoms to the nucleoside bond, sugar moiety, or nucleic acid base. Modifications suitable for use in the agents of this disclosure include all types of modifications disclosed herein or known in the art. For the purposes of this specification and the claims, any such modification, when used in an siRNA-type molecule, is encompassed by “iRNA” or “RNAi agent,” or when used in an ASO-type molecule, is encompassed by “antisense oligonucleotide” or “ASO.”

[0113] In certain embodiments of this disclosure, the inclusion of a deoxynucleotide may be considered to constitute a modified nucleotide when present in an RNAi agent or in an ASO or other modulating nucleic acid agent.

[0114] As used herein, the term “targeting moiety” refers to a moiety that, upon contact with a system containing one or more target cells of interest (e.g., in culture, tissue, and / or in an organism), specifically binds to such target cells. In many embodiments, the targeting moiety binds to cell surface factors (e.g., factors preferentially or specifically found on the surface(s) of such target cells of interest). In some embodiments, the binding of the targeting moiety to the cell surface factors results in the internalization of the targeting moiety. Typically, useful targeting moieties according to this disclosure retain their specific binding properties when contained in a conjugate agent as described herein. In some embodiments, the binding of such a conjugate agent to the relevant cell surface factors results in the internalization of the conjugate agent. In some embodiments, the targeting moiety specifically binds to factors on the surface of kidney cells. In some embodiments, the targeting moiety specifically binds to cubilin. In some embodiments, the targeting moiety specifically binds to megalin. In some embodiments, the targeting moiety of this disclosure includes compounds of formula I or formula I', or those otherwise disclosed herein.

[0115] As used herein, the term “cell surface factor” refers to a factor (e.g., a polypeptide or containing a polypeptide) present on the surface of the cell(s) of interest (e.g., the target cells(s) described herein, which may be kidney cells in many embodiments). In some embodiments, the cell surface factor is preferentially present on the surface of the target cell(s) (e.g., kidney cells) compared to cells of one or more other tissues. In some embodiments, the cell surface factor is present on certain non-target cells in addition to the target cells. In some embodiments, the cell surface factor is preferentially or specifically absent on the relevant target cells of interest. In some embodiments, the cell surface factor is a receptor or contains a receptor. In some embodiments, the cell surface factor is internalized when bound by one or more specific ligands (e.g., having a targeting moiety as described herein). In some embodiments, the cell surface factor may interact (e.g., bind, form a complex, etc.) with one or more other components of the cell (e.g., one or more cell membrane components and / or one or more cell surface components and / or one or more intracellular components) on the surface of the cell on which it resides. 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 particular cellular state or condition (e.g., developmental stage, disease state, etc.).

[0116] In some embodiments, the “conjugate” has a structure represented by the following formula:(Xn1-Yn2-Zn3), where X is a targeting moiety (e.g., a polymyxin or other targeting substructure disclosed herein), n1 is an integer (i.e., 1 or greater, typically less than 5), Y is a linker (optionally, e.g., a branched linker to enable a conjugate structure comprising multiple targeting moieties associated with one or more payload moieties in a single structure), n2 is 0 or an integer (i.e., 1 or greater, typically 5), Z is a regulatory nucleic acid of the Disclosure, n3 is an integer (i.e., 1 or greater, typically less than 5), and in many embodiments, n2=n1 and / or n2=n3. In many embodiments, n1 and / or n3 is 1. In many embodiments, the conjugate has a structure represented by the formula (XYZ). In some embodiments, the conjugate has a structure represented by the formula (XY)nZ, where n is an integer greater than 1, and the conjugate includes two or more targeting moieties. In some embodiments, the conjugate has a structure represented by the formula X-(YZ)n, where n is an integer greater than 1, and the conjugate includes two or more regulatory nucleic acids.

[0117] The term "megalin," as used herein, refers to a receptor that is a member of the low-density lipoprotein receptor (LDLR) family. Megalin is encoded by the LRP2 gene. The amino acid sequence of full-length megalin, and / or the amino acid sequence of the nucleic acid encoding full-length megalin, can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human megalin (SEQ ID NO: 1, residues 27-4411 represent the extracellular domain containing the LDL receptor class A domain, the LDL receptor class B domain, and the EGF-like domain; residues 4589-4602 represent the DAB2 interaction domain; and residues 4453-4622 represent the cytoplasmic domain containing the NPXY motif, the SH2-binding domain, the SH3-binding domain, and the proline-rich domain) can be found under UniProt / Swiss-Prot accession number P98164, and the nucleic acid sequence encoding human megalin can be found under accession number NM_004525.3. Megalin is also known, for example, as a low-density lipoprotein receptor-associated protein 2 (LRP2), glycoprotein 330 (Gp330), calcium sensor protein, or Hayman nephritis antigen homolog. Those skilled in the art will understand that the sequence of SEQ ID NO: 1 is exemplary and that certain variations (including, for example, conserved substitutions of SEQ ID NO: 1 and codon-optimized variants of the associated nucleic acid sequence) are also or encode human megalin. Furthermore, those skilled in the art will understand that homologs and orthologues of human megalin are known and / or can be known by the exercise of the ordinary art, for example, based on the degree of sequence identity, the presence of one or more characteristic sequence elements, and / or one or more common activities. In some embodiments, megalin includes full-length megalin or a variant or fragment thereof. In some embodiments, the megalin targeted by this disclosure is megalin expressed by certain target cells and / or tissues (for example, in the organism of interest). In some embodiments, the megalin targeted by this disclosure is an engineered megalin.In many embodiments, the megalin targeted by this disclosure is present on the surface of the target cell(s) of interest (e.g., the target tissue(s), e.g., the kidney) and is internalized by such cells upon binding of the megalin binding moiety described herein. Megalin has been reported to be expressed in one or more of the following tissues and / or cells: immune cells (e.g., myeloid cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells]), 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; 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 gland; adrenal gland; pituitary gland; breast; skin; ovary; uterus; placenta; prostate; and testes. Megalin expression has been reported to be particularly abundant (e.g., higher than one or more other tissues) in the following tissues and / or cells: renal tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissue. Megalin has been particularly reported to be expressed (e.g., at relatively high levels) on the surface of renal cells, such as proximal tubular epithelial cells and podocytes. See Nielsen R. et al. (2016), Kidney Int. 89(1):58-67.

[0118] As used herein, the term “megalin-binding moiety” refers to a moiety that binds to megalin upon contact with megalin. Typically, the useful megalin-binding moieties provided herein bind specifically to megalin under contact conditions. In some embodiments, the megalin-binding moiety is or comprises a polymyxin or related structure, peptide, aminoglycoside, endogenous substance, xenobiotic, antibody or fragment thereof, or a combination thereof, as disclosed herein. In some embodiments, the megalin-binding moiety is internalized upon binding to megalin on the cell surface.

[0119] As used herein, the term “cubirin” refers to the receptor encoded by the CUBN gene. The amino acid sequences of full-length cubirin and / or the nucleic acid encoding full-length megalin can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human cubirin (SEQ ID NO: 2, residues 1-23 represent the signal peptide, residues 24-35 represent the propeptide which can be removed in the mature form, and residues 36-3623 represent the mature cubirin polypeptide) can be found under UniProt / Swiss-Prot accession number O60494, and the nucleic acid sequence encoding human cubirin can be found under accession number NM_001081.3. Cubilin is also known, for example, as IFCR, Gp280, endogenous factor-vitamin B12 receptor, MGA1, or IGS1. Those skilled in the art will understand that the cubirin sequence of SEQ ID NO: 2 is exemplary, and that certain variations (including, for example, conservative substitutions of SEQ ID NO: 2, codon-optimized variants of the nucleic acid sequence encoding cubirin, etc.) are also or also encode human cubirin. Furthermore, those skilled in the art will understand that homologs and orthologues of human cubirin are known and / or can be known by the exercise of the ordinary art, for example, based on the degree of sequence identity, the presence of one or more characteristic sequence elements, and / or one or more common activities. In some embodiments, cubirin includes full-length cubirin or a variant or fragment thereof. In some embodiments, the cubirin targeted by this disclosure is cubirin expressed by a specific target cell(s) and / or tissue(s) (for example, in the organism of interest). In some embodiments, the cubirin targeted by this disclosure is engineered cubirin. In many embodiments, the cubilin targeted by this disclosure is present on the surface of the target cell(s) of interest (e.g., the target tissue(s) of interest) and is internalized by such cells upon binding of the cubilin binding moiety described herein.Cubilin is found in the following tissues and / or cells: immune cells (e.g., bone marrow cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells]), red blood cells, eosinophils, neutrophils, and / or platelets); 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; kidneys; liver; lungs; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; and lower extremities. It has been reported that cubilin is expressed in one or more of the following: pituitary gland; breast; skin; ovary; uterus; placenta; prostate; and testes. Cubilin expression has been reported to be particularly abundant (e.g., higher than one or more other tissues) in the following tissues and / or cells: renal tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissue. Cubilin has been particularly reported to be expressed (e.g., at relatively high levels) on the surface of renal cells, such as proximal tubular epithelial cells and podocytes. See Nielsen R. et al. (2016), Kidney Int. 89(1):58-67.

[0120] As used herein, the term “cubillin-binding moiety” refers to a portion that binds to cubillin upon contact with cubillin. Typically, the useful cubillin-binding moieties provided herein bind specifically to cubillin under contact conditions. In some embodiments, the cubillin-binding moiety is internalized upon binding to cubillin on the cell surface.

[0121] As used herein, the expression “cells associated with the kidney” refers to cells that are present in or may be found in the kidney (e.g., during development, tissue homeostasis, or in the course of disease or impairment). In some embodiments, cells associated with the kidney are also referred to herein as “kidney cells.” In some embodiments, cells associated with the kidney include one or all of the following cell types: proximal tubular epithelial cells, podocytes, renal cyst cells (e.g., polycystic kidney disease), parietal epithelial cells, graft cells, renal stem cells, epithelial progenitor cells, fibroblasts, myofibroblasts, pericytes, ascending loop of Henle cells, descending loop of Henle cells, distal tubular cells, connecting tubular cells, interstitial cells, and chief cells. An exemplary population of kidney cells is provided in Schumacher et al., (2021)npj Regen Med 6, 45, the entire contents of which are incorporated herein by reference. In some embodiments, kidney cells are cells derived from the kidney, e.g., renal tumor cells and / or metastatic renal tumor cells, or include them.

[0122] As used herein, the term “combination therapy” refers to a situation 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., all “doses” of the first regimen are administered before any dose of the second regimen is administered). In some embodiments, such regimens are administered in overlapping drug regimens. In some embodiments, “administration” of combination therapy may involve administering one or more drugs or modalities to a subject receiving other drugs or modalities in combination. For clarity, combination therapy does not require that individual drugs be administered in a single composition (or not necessarily simultaneously), however, in some embodiments, two or more drugs or their active portions may be administered together in a combined composition, or even a combined compound (e.g., as part of a single chemical complex or covalent entity).

[0123] A composition or method described herein as “comprising” one or more named elements or steps is open, meaning that the named elements or steps are essential, but other elements or steps may be added to the scope of the composition or method. To avoid redundancy, any composition or method described herein as “comprising” (or “comprises”) one or more named elements or steps is also construed to describe a corresponding, more restrictive composition or method that “consisting essentially of” (or “consists essentially of”) the same named elements or steps. This means that a composition or method may include an essential named element or step and may include additional elements or steps that do not substantially affect the basic and novel characteristics of the composition or method. Any composition or method described herein as “comprising” or “consisting essentially of” one or more named elements or steps is also consisting of (or consists essentially of) the named elements or steps. It is understood that this also describes a corresponding, more limited and closed composition or method that excludes any other unnamed element or step. In any composition or method disclosed herein, any known or disclosed equivalent of any named principal element or step may be substituted for such element or step.

[0124] As used herein, the term "peptide" typically refers to a polypeptide that is relatively short, for example, having a length of 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.

[0125] As used herein, “polypeptide” refers to a polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an engineered amino acid sequence in that it is designed and / or produced by human action. In some embodiments, the polypeptide may contain or consist of natural amino acids, non-natural amino acids, or both.

[0126] As used herein, the term “pharmaceutical composition” refers to a composition comprising an active agent (e.g., a modulated nucleic acid agent disclosed herein) with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose appropriate for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in a particular form (e.g., solid or liquid form), and / or, for example, oral administration (e.g., oral medication [aqueous or nonaqueous solution or suspension], e.g., tablets, capsules, boluses, powders, granules, pastes, etc., which may be specially formulated for absorption into the buccal mucosa, sublingually, or systemically; parenteral administration (e.g., as a sterile solution or suspension, or as a sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous or epidural injection); topical administration (e.g., as a cream, ointment, patch or spray, e.g., applied to the skin, lungs, or oral cavity); vaginal or rectal administration (e.g., as a pessary, suppository, cream, or foam); intraocular administration; nasal or pulmonary administration, etc.

[0127] As used herein, the term “subject” means an organism, e.g., 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 child subject. In some embodiments, a subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a susceptible subject has a predisposition to developing a disease, disorder, or condition and / or exhibits an increased risk of developing a disease, disorder, or condition (compared to the mean risk observed in a reference subject or population). In some embodiments, a subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any specific symptoms (e.g., clinical symptoms of a disease) or features of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, the subject is an individual who is being diagnosed and / or treated. In one embodiment, the subject is a human being, for example, a human being being treated or evaluated for a disease or disorder that would benefit from a reduction in the target mRNA expression described herein; a human being at risk of a disease or disorder that would benefit from a reduction in the target mRNA expression; a human being having a disease or disorder that would benefit from a reduction in the target mRNA expression; or a human being being treated for a disease or disorder that would benefit from a reduction in the target mRNA expression. In some embodiments, the subject is a human female. In other embodiments, the subject is a human male. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0128] Where used herein, “reference” refers to the standard or control on which the comparison is made. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control drug, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or measured substantially concurrently with the test or measurement of interest. In some embodiments, the reference or control is a historical reference or control, sometimes embodied in a more distinct medium. Typically, as will be understood by those skilled in the art, the reference or control is measured or characterized under equivalent conditions or circumstances during these evaluations. Those skilled in the art will understand that there are similarities sufficient to justify reliance on and / or comparison to a particular possible reference or control.

[0129] As used herein, the term “specific binding” refers to the ability to distinguish between possible binding partners in the environment in which binding occurs. A binder that interacts with one particular target in the presence of other potential targets is said to “specifically bind” to the target it interacts with. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binder and its partner. In some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binder-partner complex. In some embodiments, specific binding is assessed by detecting or measuring 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 measurement over a range of concentrations.

[0130] When the term “specific” is used herein in reference to an active drug, it will be understood by those skilled in the art to mean that the drug distinguishes a potential target entity or state. For example, in some embodiments, a drug is said to bind “specifically” to a target if it preferentially binds to that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction depends on the presence of specific structural features of the target entity (e.g., epitopes, fissures, binding sites). It should be understood that specificity does not have to be absolute. In some embodiments, specificity may be evaluated in comparison to the specificity of the binder with respect to one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated in comparison to the specificity of a reference specific binder. In some embodiments, specificity is evaluated in comparison to the specificity of a reference nonspecific binder. In some embodiments, a drug or entity does not detectably bind to a competing alternative target under conditions that it binds to its target entity. In some embodiments, a binder binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to a competing alternative target(s).

[0131] As is well known in the art, "specificity" is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

[0132] As used herein, “complementary” means a nucleic acid molecule that can form hydrogen bonds with another nucleic acid molecule by either conventional Watson-Crick base pairing or other unconventional types of pairing (e.g., Hougsteen or inverse Hougsteen hydrogen bonds) between complementary nucleosides or nucleotides. With respect to the oligonucleotides of this disclosure, the binding free energy of the antisense oligonucleotide / antisense chain and its complementary sequence is sufficient to allow the relevant function of the oligonucleotide agent to proceed, and there is sufficient complementarity to avoid nonspecific binding of the antisense oligonucleotide / antisense chain to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo therapeutic procedures. The measurement of 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") is a term that indicates a sufficient degree of complementarity or precise pairing so that stable and specific binding occurs between the antisense oligonucleotide / antisense strand and the pre-mRNA or mRNA target. It is well known in the art that nucleic acid molecules do not need to be 100% complementary to the target nucleic acid sequence in order to be specifically hybridizable. That is, two or more nucleic acid molecules may not be perfectly complementary.

[0133] Complementarity is indicated by the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if the first nucleic acid molecule has 10 nucleotides and the second nucleic acid molecule also has 10 nucleotides, then 5, 6, 7, 8, 9, or 10 nucleotide base pairings between the first and second nucleic acid molecules represent 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. A "perfectly" or "sufficiently" complementary nucleic acid molecule means that all consecutive residues in the first nucleic acid molecule form hydrogen bonds with the same number of consecutive residues in the second nucleic acid molecule, where both nucleic acid molecules have the same number of nucleotides (i.e., the same length) or the two molecules have different lengths.

[0134] The term “variant,” as used herein, refers to a molecule or entity that exhibits significant structural identity with a reference molecule or entity, but is structurally different from the reference molecule or entity, for example, in the presence or absence of one or more chemical moieties, or at a different level thereof (e.g., nucleic acids, proteins, or small molecules, or those containing them). In some embodiments, “variant” may be referred to as “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 appropriately considered a “variant” of a reference depends on the degree of structural identity with the reference molecule. As will be 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 different molecule or entity that shares one or more such characteristic structural elements but differs from the reference molecule or entity in at least one aspect. To give some examples, polypeptides may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to specific structural motifs and / or biological functions. Similarly, nucleic acids may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequences and / or one or more differences in chemical parts (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., those attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid indicates that the overall sequence identity with the reference polypeptide or nucleic acid is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, the polypeptide or nucleic acid of interest is considered a “variant” of the reference polypeptide or nucleic acid if it has the same amino acid sequence or nucleotide sequence as the reference polypeptide or nucleic acid, but differs only in a few sequence modifications at specific positions. Typically, about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of residues in the variant are substituted, inserted, or deleted compared to the reference. 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. Often, the variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, less than 4, less than 3, less than 2, or less than 1) substituted, inserted, or deleted functional residues (i.e., residues involved in specific biological activity) compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains about 5 or fewer, about 4 or fewer, about 3 or fewer, about 2 or fewer, or about 1 or fewer additions or deletions compared to the reference, and in some embodiments, it contains no additions or deletions compared to the reference.In some embodiments, the variant polypeptide or nucleic acid includes additions or deletions of about 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, 6, and generally less than 5, 4, 3, or 2 compared to the reference polypeptide. In some embodiments, the reference polypeptide or nucleic acid is naturally occurring. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0135] In some embodiments, regulatory RNA compounds (e.g., inhibitory RNA compounds such as siRNA, inhibitory antisense oligonucleotides, and antisense compounds designed to modulate splicing) are provided for the prevention, improvement, and / or treatment of diseases or conditions such that the disease(s) or condition(s) in a subject can be prevented, improved, and / or treated by delivering nucleic acid molecules capable of specifically and selectively modulating the expression of a target mRNA to cells of a target kidney or other organ.

[0136] As used herein, the terms “to treat” or “treatment” refer to a beneficial or desired outcome, such as reducing at least one sign or symptom of a target RNA-associated disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with the expression of an undesirable target RNA and / or target polypeptide; reducing the degree of activation or stabilization of an undesirable target RNA and / or target polypeptide; and improving or mitigating the activation or stabilization of an undesirable target RNA and / or target polypeptide. “Treatment” may also mean extending survival compared to the survival expected without treatment. The term “lower” in the context of levels of target RNA and / or target polypeptide in a subject or disease marker or symptom refers to a statistically significant reduction in such levels. The reduction may 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 certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction is at least 50% of the expression level of a disease marker, e.g., a protein or gene. In the context of the level of target RNA and / or target polypeptide in the subject, “lower” preferably means reduced to a level that is acceptable within the normal range for an individual without such disorder. In certain embodiments, “lower” means a reduction in the difference between the level of a marker or symptom for a subject suffering from the disease and a level that is acceptable within the normal range for the individual, e.g., the level of weight loss between an obese individual and an individual with a weight that is acceptable within the normal range.

[0137] As used herein, “prevention” or “prevention” means, when used in relation to a disease, disorder, or condition that can be treated or improved by reducing the expression of a target gene, a reduction in the likelihood of the subject developing symptoms associated with the disease, disorder, or condition, such as symptoms of undesirable or excessive target RNA and / or target polypeptide expression, for example, metabolic disorders and / or kidney diseases or conditions (e.g., glomerular disorders, renal tubular disorders, other kidney disorders), congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, viral infections, etc. For example, if an individual with one or more risk factors for kidney disease or condition does not develop the kidney disease or condition, or develops the disease or condition with less severe severity compared to a population with the same risk factors but who has not received the treatment described herein, then the likelihood of developing kidney disease or condition is reduced. The absence of the onset of a disease, disorder, or condition, or a reduction or delay in the onset of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically acceptable scale for that disease or disorder) (e.g., by days, weeks, months, or years), is considered effective prevention.

[0138] As used herein, the term “Target RNA-related disease” refers to a disease, disorder, or condition caused by or associated with undesirable or excessive levels and / or expression of target RNA. The term “Target RNA-related disease” includes diseases, disorders, or conditions that can be treated or improved by reducing target RNA expression. The term “Target RNA-related disease” includes nephropathy and various other kidney diseases or conditions, such as glomerular disorders, renal tubular disorders, other kidney disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, viral infections, phenylketonuria (PKU) and related amino acid metabolic disorders.

[0139] An "effective dose" is a quantity sufficient to produce a beneficial or desired result.

[0140] When used herein, “therapeutic dose” is intended to include the amount of a modified 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 of its symptoms). “Therapeutic dose” may vary depending on the modified nucleic acid agent, the method of drug administration, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other individual characteristics of the subject being treated.

[0141] As used herein, “prophylactic effective dose” is intended to include an amount of a modulated nucleic acid agent sufficient to prevent or improve a disease or one or more symptoms of a disease when administered to a subject with a target RNA-associated disorder. Improving a disease includes slowing the progression of the disease or reducing the severity of a disease that develops later. The “prophylactic effective dose” may vary depending on the modulated nucleic acid agent, the method of drug administration, the degree of the disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other individual characteristics.

[0142] The “therapeutic effective dose” or “prophylactic effective dose” also includes the amount of a modulated nucleic acid agent that produces some desired effect with a reasonable benefit / risk ratio applicable to either treatment. The conjugated modulated nucleic acid (e.g., conjugated iRNA) used in the method of this disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0143] The term "pharmaceutically acceptable" is used herein to mean a compound, substance, composition, or dosage form that is suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic response, or other problems or complications, and that is commensurate with a reasonable benefit-to-risk ratio, within the bounds of sound medical judgment.

[0144] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium, or zinc, or steric acid), or a solvent encapsulation material used to support or transport a compound of interest from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Such carriers are known in the art. Examples of pharmaceutically acceptable carriers include carriers for administration by injection.

[0145] The term “sample,” as used herein, includes similar fluids, cells, or tissues isolated from a subject, as well as aggregates of fluids, cells, or tissues present within the subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may originate from a specific organ, a part of an organ, or fluids or cells within those organs. In certain embodiments, a sample may originate from a kidney (e.g., the entire kidney or a specific segment of the kidney, or a specific type of cell in the kidney, such as proximal tubular epithelial cells or podocytes). In some embodiments, “sample derived from subject” refers to urine obtained from the subject. “Sample derived from subject” may refer to blood from the subject or serum or plasma derived from blood.

[0146] 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” are used for convenience to encompass RNA (including pre-mRNA and mRNA, or parts 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 certain embodiments, the target nucleic acid is mRNA encoding a human target polypeptide.

[0147] The 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” means a perturbation of function, e.g., an increase (stimulation or induction) or decrease (inhibition or reduction) in the expression of a target mRNA. Another example of regulation of expression may include disrupting splice site selection in pre-mRNA processing. “Expression” includes all functions that translate the information encoded by a gene into structures that exist and function within the cell. These structures include the products of transcription and translation. The functions of RNA that can be regulated may include, but are not limited to, transposition functions, which include, the transposition of RNA to protein translation sites, the transposition of RNA to intracellular sites away from RNA synthesis sites, and the translation of RNA into proteins. RNA processing functions that can be regulated include, but are not limited to, RNA splicing to yield one or more RNA species, RNA capping, RNA 3' maturation, and catalytic activity or complex formation in which RNA is involved or facilitated. Expression regulation can result in an increase or decrease in the levels of one or more nucleic acid species, either transiently or at a net steady-state level. One consequence of such interference with target nucleic acid function is the regulation of the expression of target RNA (e.g., target mRNA) and / or target polypeptide. Thus, in one embodiment, expression regulation may mean an increase or decrease in the levels of target RNA or protein. In another embodiment, expression regulation may mean an increase or decrease in one or more RNA splice products, or a change in the ratio of two or more splice products.

[0148] The effect of regulatory RNA compounds on target nucleic acid expression can be tested in any of various cell types, provided that the target nucleic acid is present at measurable levels and can be routinely determined, for example, by PCR or Northern blot analysis. Cell lines are derived from both normal tissues and cell types, as well as from cells associated with various disorders (e.g., hyperproliferative disorders). Cell lines derived from multiple tissues and species can be obtained from the American Type Culture Collection (ATCC, Manassas, Va.) and other public sources. Primary cells, or cells isolated from animals and not subjected to serial culture, may be prepared according to methods known in the art or may be obtained from various commercial suppliers. Furthermore, primary cells may be obtained from donor human subjects (i.e., blood donors, surgical patients) in a clinical setting. These techniques are well known to those skilled in the art.

[0149] When used herein, the definitions of each expression, such as alkyl, m, η, etc., are intended to be independent of their definitions elsewhere in any given structure if they occur more than once in that structure.

[0150] This disclosure is further illustrated by the following detailed description. [Modes for carrying out the invention]

[0151] Detailed description of the invention This specification discloses conjugate agents comprising a payload portion and a targeting portion (e.g., a polymyxin compound, a variant or derivative thereof, or other targeting compounds disclosed herein) directly or indirectly conjugated thereto. In some embodiments, the targeting portion specifically binds to surface factors on target cells of interest (e.g., kidney cells). In some embodiments, the payload portion is or comprises a nucleic acid agent. In some embodiments, the payload portion is or comprises a therapeutic agent (e.g., a therapeutic oligonucleotide).

[0152] In particular, this disclosure provides insights into the possibility that conjugate agents as described herein may be particularly useful or effective in delivering nucleic acid agents to kidney cells and / or other cells that express or otherwise possess surface factors (e.g., megalin) that are specifically bound by the targeting moieties described herein.

[0153] Polymyxin and polymyxin-related targeting portion In certain embodiments, the conjugates of the Disclosure include a polybasic structure, e.g., polymyxin, polymyxin-type, or other related compound, as a targeting moiety. The targeting moieties for use disclosed herein can bind, for example, selectively, to surface factors (e.g., part or portion thereof, and / or specific forms, e.g., disease-related forms) present on the surface of the target cell(s) of interest disclosed herein (e.g., kidney cells).

[0154] Polymyxin B (PMB) has the following structure. TIFF2026534515000014.tif78165

[0155] Polymyxin E (colistin) has the following structure. TIFF2026534515000015.tif73165

[0156] It has been identified in the art that both PMB and colistin bind to megalin with high affinity, thereby resulting in the internalization of such compounds into the kidney (Moestrup et al. J Clin Invest. 96:1404-1413). Specific aspects of this disclosure relate to identifying and using genera of polybasic compounds related to PMB and colistin as targeting moieties for conjugation with molecular payloads (e.g., nucleic acid agents) via linker sites (binding to such linkers optionally comprising one or more amino acid structures).

[0157] Therefore, a particular targeting portion of the conjugate composition of this disclosure is Formula I: The compound comprises TIFF2026534515000016.tif64165, wherein R1 is an optionally substituted C3-C7 alkyl or optionally substituted C3-C7 aryl, and L is a linker as disclosed herein.

[0158] While we do not wish to be bound by theory, this disclosure proposes that internalization of a cell surface factor present on the surface of a relevant cell (e.g., a tissue cell) can be achieved by binding a targeting moiety (polymyxin, polymyxin-type, or other related compound) to the cell surface factor, together with the bound targeting moiety (which may, for example, be part of a conjugate described herein). In some embodiments, such internalization may mean that the relevant cell surface factor becomes unavailable (at least for a certain period of time) on the surface of the cell (e.g., a tissue cell) for purposes such as signal transduction and / or ligand binding.

[0159] In particular, this disclosure provides the finding that inducing the internalization of surface factors can be usefully achieved for the delivery of targeting portions (and / or conjugate agents, such as those described herein, including the targeting portions) to intracellular compartments, such as vesicles and / or organelles, and / or to the cytoplasm of cells. This disclosure further provides the finding that such internalization may be particularly useful for the delivery of conjugate agents and / or parts thereof (e.g., their payload portions) into cells. This disclosure provides specific finding that such internalization may be particularly useful for the delivery of nucleic acid agents described herein, particularly in the context of conjugate agents described herein (e.g., as their payload portions).

[0160] 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%) are internalized upon binding to the targeting moiety. In some embodiments, substantially all or all of the cell surface factors are internalized upon binding to the targeting moiety.

[0161] In some embodiments, the binding of a targeting portion to a cell surface factor on the surface of a cell, for example a tissue cell, does not internalize the cell surface factor.

[0162] In some embodiments, the conjugate described herein comprises one or more payload portions and / or one or more targeting portions.

[0163] In some embodiments, the conjugate described herein comprises one payload portion and one or more targeting portions.

[0164] In some embodiments, the conjugate described herein comprises one or more payload portions and one targeting portion.

[0165] In some embodiments, the cell surface factor is a polypeptide present on the surface of cells, such as tissue cells (e.g., detectable), or comprises such polypeptides. In some embodiments, the cell surface factor is present on the surface of cells expressing, for example, megalin as described herein (e.g., detectable). In some embodiments, the cell surface factor comprises a receptor.

[0166] In some embodiments, the cell surface factor is or comprises renal cell surface factor. In some embodiments, the renal cell surface factor is present on (e.g., detectable on) the surface of kidney-associated cells, such as kidney cells or cells that may be found within the kidney during development, maintenance of tissue homeostasis, or in the course of disease or impairment. In some embodiments, the renal cell surface factor is present on proximal tubular epithelial cells, podocytes and / or renal cysts and can be detected, for example.

[0167] In some embodiments, kidney cell surface factors may be present on the surface of kidney-associated tissues, for example, tissues that are part of the kidney or may be found within the kidney during development, maintenance of tissue homeostasis, and / or during disease or impairment, and may be detected.

[0168] In some embodiments, the kidney cell surface factor is, for example, a detectable receptor present on the surface of a cell, e.g., a kidney-associated cell as described herein, or a kidney-associated tissue as described herein. In some embodiments, the kidney cell surface factor may bind to one or more co-receptors on the surface of a cell, e.g., a tissue cell. In some embodiments, the kidney cell surface factor may be internalized when a kidney-specific binding portion in the conjugate agent binds to the kidney cell surface factor. In some embodiments, the internalization of the kidney cell surface factor as a result of binding to the kidney-specific binding portion in the conjugate agent also internalizes the conjugate agent (e.g., a portion thereof, e.g., the payload portion) into the cell. In some embodiments, the internalized conjugate agent (e.g., a portion thereof, e.g., the payload portion) is delivered to intracellular vesicles (e.g., lysosomes, endosomes, cratrin-coated pits, or intracellular membranous organelles, or a combination thereof). In some embodiments, the internalized conjugate (e.g., a portion thereof, e.g., the payload portion) is delivered to intracellular compartments, such as the cytoplasm, mitochondria, ribosomes, nucleus, nucleolus, or other intracellular compartments, or a combination thereof.

[0169] In some embodiments, an internalized conjugate agent (e.g., a portion thereof, e.g., a payload portion) in a cell (e.g., a vesicle or intracellular compartment) can reduce the expression and / or activity of the target of the payload portion.

[0170] In some embodiments, the internalization of a conjugate agent (e.g., a portion thereof, e.g., a payload portion) into a cell (e.g., a vesicle or intracellular compartment) detaches the targeting portion from the payload portion, e.g., separates it. In some embodiments, the targeting portion is detached from the payload portion, e.g., separated it, by chemical reaction and / or mechanical separation. In some embodiments, the chemical reaction includes an enzymatic reaction to cleave the linker connecting the targeting portion to the payload portion.

[0171] In some embodiments, internalization of a conjugate agent (e.g., a portion thereof, e.g., a payload portion) into a cell (e.g., a vesicle or intracellular compartment) detaches the targeting portion from the payload portion.

[0172] In some embodiments, the conjugates disclosed herein can be filtered by glomerular capillaries, for example, into Bowman capsules. In some embodiments, the conjugates disclosed herein have size, charge, conformation, and / or other properties that enable filtration by glomerular capillaries. In some embodiments, the threshold for glomerular filtration is in the range of 30 to 50 kDa.

[0173] In some embodiments, the cell surface factor (e.g., kidney cell surface factor) is a receptor selected from megalin, cubilin, or both, or includes one such receptor.

[0174] Linker Certain aspects of this disclosure feature a linker that conjugates a targeting portion (e.g., a polybasic portion as described herein) to a payload portion (e.g., a nucleic acid agent). In some embodiments, the bioconjugate or conjugate agent comprises a regulatory nucleic acid chemically conjugated or covalently bound to the targeting portion.

[0175] In some embodiments, the conjugate is prepared by conjugating or covalently bonding a modulating nucleic acid agent to the targeting moiety. In some embodiments, the modulating nucleic acid may be linked to the targeting moiety, for example, by a reaction between the modulating nucleic acid and the targeting moiety in solution. The conjugate may also be prepared in a single synthesis using, for example, known synthetic methods for the preparation of GalNAc conjugate nucleic acids by solid-phase means. (See, for example, U.S. Patents 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 whole.)

[0176] Regardless of the method of production, and depending on the desired properties of the conjugate, it may or may not be advantageous to include a spacer or linker between the regulatory nucleic acid and the binding site. When including a linker is advantageous, the linker can be of many different types and chemical compositions.

[0177] Generally, linkers are designated as "cleavable" or "non-cleavable." Cleavable linkers are typically used when it is desirable for either or both of the payload and the conjugated binding site to be released so that they can perform their functions better (see, for example, U.S. Patent No. 10,808,039 and U.S. Patent No. 9,463,252, each of which is incorporated by reference in whole). Non-cleavable linkers are typically used to maintain the desired activity, performance, and stability of a conjugated agent, such as an enzyme linked to a probe or (m)Ab to facilitate an ELISA assay and improve affinity, bispecificity, etc. Some linkers that can be cleaved are chemically cleaved by, for example, hydrolysis, pH changes, reduction, or oxidation, and others are enzymatically cleaved by, for example, proteases, esterases, glucosidases, glucuronidases, galactosidases, phosphatases, phosphodiesterases, nucleases, lipases, or any enzyme capable of cleaving linkers and releasing biomolecules from other compounds.

[0178] In some embodiments, the cleavable linker is a disulfide bond, an ester, a phosphodiester, a sugar, or a lipid, or comprises these.

[0179] In some embodiments, the uncleavable linker is chemically, enzymatically, biochemically, and physiologically stable. Therefore, the uncleavable linker does not contain any bonds that are chemically, biochemically, or enzymatically cleavable, or otherwise physiologically unstable.

[0180] In some embodiments, the linker, whether cleavable or not, may be introduced by a chemical coupling reaction between the regulatory nucleic acid and the binding site to which it is conjugated. The regulatory nucleic acid and binding site may or may not be modified beforehand to increase or enhance their reactivity with respect to each other. Such modifications may also increase or improve the specificity and degree of conjugation of the conjugation reaction, if desired. The linker may be introduced in a single reaction or in a stepwise reaction until the desired linker and regulatory nucleic acid are prepared.

[0181] Non-limiting examples of chemical coupling reactions for forming conjugates include the reactions of various thiols to form disulfides, the reactions of thiols with alkyl halides or maleimides to form thioethers, the reactions of alkynes with azides to form triazoles ("click reactions"), the reactions of aldehydes with hydrazides or amines or aminooxy compounds to form hydrazones, imines and oxyimines, and the reactions of carboxylic acids with amines, thiols or alcohols (i.e., nucleophiles) to form amides, thioesters and esters. Carboxylic acids can be activated in situ to make them reactive in the presence of amines, thiols or alcohols, or pre-activated before the addition of nucleophiles by, for example, conversion to activated esters of N-hydroxysuccinimide (NHS) or sulfonated NHS. Numerous reviews exist on chemical coupling reactions; see, for example, Spier et al. (2018) Chem. Rev. 2018, 118, 16, 7702-7743.

[0182] The reaction of thiols with maleimides is very widely used, see, for example, Revasco et al (2018) Chem.Eur.J.10.1002 / chem.201803174, as well as click reactions (e.g., Fantoni et al., (2021) Chem.Rev., 121, 12, 7122-7154) and hydrazide formation (e.g., HyNic Peptide Conjugation Protocol, Dirksen et al (2006) J.Am.Chem.Soc., 128, 49, 15602-15603, Kozlov et al. (2004) Biopolymers 73(5):621-30.doi:10.1002 / bip.20009). Many companies sell chemical compounds and kits with protocols that allow for the simple preparation of conjugates containing various linkers.

[0183] As generally defined above and herein, a linker is a divalent group that connects or ligates a binding site to a regulatory nucleic acid site.

[0184] In some embodiments, the linker is a divalent linear or branched C 1~40 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=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)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocycle having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a group selected from sulfur, wherein, R 1 is an amino acid side chain, R is hydrogen or optionally substituted C 1~6 aliphatic, 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, 3- to 6-membered saturated or partially unsaturated heterocycle having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0185] In some embodiments, the linker is or comprises a divalent linear or branched C 1~35 aliphatic chain, wherein one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0186] In some embodiments, the linker is a divalent linear or branched C 1~30 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0187] In some embodiments, the linker is a divalent linear or branched C 1~25 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0188] In some embodiments, the linker is a divalent linear or branched C 1~20 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0189] In some embodiments, the linker is a divalent linear or branched C 1~15 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0190] In some embodiments, the linker is a divalent linear or branched C 1~10 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0191] In some embodiments, the linker is a divalent linear or branched C 1~5 It is an aliphatic chain or contains an aliphatic chain, where one or more methylene units of the aliphatic chain are -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-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)- It is substituted with a group selected from -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3-6 member saturated or partially unsaturated carbocyclic ring, a 3-6 member saturated or partially unsaturated heterocycle having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and sulfur.

[0192] In some embodiments, the linker is The structure is selected from TIFF2026534515000017.tif89165, or contains it. In the formula, X is either NH or O.

[0193] In some embodiments, the cleavable linker is a cathepsin-cleavable linker. In some such embodiments, the linker is a valine-citrulline (Val-Cit) motif: TIFF2026534515000018.tif48165, or containing it, where R is hydrogen or C 1~6 It is an aliphatic.

[0194] In some embodiments, the valine-citrulline linker is The filename is TIFF2026534515000019.tif48165, or contains it.

[0195] In some embodiments, the valine-citrulline linker is TIFF2026534515000020.tif53165, or containing it, where R is hydrogen or C 1~6 It is an aliphatic.

[0196] In some embodiments, the linker includes a disulfide bond. In some embodiments, the linker includes a poly(ethylene glycol) moiety (e.g., -(CH2CH2O) b -) is included, and b is between 1 and 50.

[0197] In some embodiments, the linker is A base selected from TIFF2026534515000021.tif203165TIFF2026534515000022.tif212165TIFF2026534515000023.tif168165, or containing such a base, where each of k, m, n, p, q, r, s, t, u, v, w, x, y, and z is between 1 and 20. R is hydrogen or C 1~10 It is an aliphatic.

[0198] In some embodiments, k is 3.

[0199] In some embodiments, m is 3.

[0200] In some embodiments, n is 2. In some embodiments, n is 12.

[0201] In some embodiments, p is 3.

[0202] In some embodiments, m and p are each 3.

[0203] In some embodiments, q is 1.

[0204] In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 6.

[0205] In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 6.

[0206] In some embodiments, r and s are each 3. In some embodiments, r and s are each 4. In some embodiments, r and s are each 6.

[0207] In some embodiments, t is 3. In some embodiments, t is 5.

[0208] In some embodiments, u is 3. In some embodiments, u is 5.

[0209] In some embodiments, t and u are each 3. In some embodiments, t and u are each 5.

[0210] In some embodiments, v is 3.

[0211] In some embodiments, w is 4.

[0212] In some embodiments, x is 8.

[0213] In some embodiments, y is 2.

[0214] In some embodiments, z is 1.

[0215] In embodiments, as described in Formula I, certain polybasic structures of the present disclosure may be linked to one or more amino acids, which are also linked in exemplary structures having an L-linker group as defined herein. In such embodiments, the amino acid groups AA1, AA2, and AA3 are each independently intended to be either a chemical bond (if an amino acid is not present at that position) or an amino acid selected from the following: TIFF2026534515000024.tif74165

[0216] In certain embodiments, the particular L group of the present disclosure is L, Includes TIFF2026534515000025.tif22165 or branch linker, In the formula, X is The filename is TIFF2026534515000026.tif181165. Alternatively, X is The file is TIFF2026534515000027.tif32165, where L is an optional linker. M is The filename is TIFF2026534515000028.tif32165, where Y=O, S, or NSO2Me, and Z is the nucleic acid payload.

[0217] nucleic acid payload In many embodiments, the payload portion for use in this disclosure is or includes an entity whose presence in the relevant cells, e.g., tissue cells, achieves (e.g., correlates) a particular effect (e.g., a particular detectable effect). In some embodiments, the relevant effect is or includes a particular biological and / or physiological effect. In some embodiments, the relevant effect is or includes an increase or decrease in the level or activity of a particular nucleic acid (or form thereof) in the cell.

[0218] 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 some embodiments, the payload portion is or contains double-stranded nucleic acids. In some embodiments, the payload portion is or contains oligonucleotides.

[0219] In some embodiments, the regulatory nucleic acids of this disclosure are 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, approximately 10-35 nucleotides Otides have lengths and / or chain lengths (in the case of multistranded regulatory nucleic acids, e.g., dsRNA) in the range of 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 acids of this disclosure are 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, approximately 35-60 nucleotides, and approximately 36 They have lengths and / or chain lengths (in the case of multi-stranded regulatory nucleic acids, e.g., dsRNA) in the range of ~60 nucleotides, approximately 37~60 nucleotides, approximately 38~60 nucleotides, approximately 39~60 nucleotides, approximately 40~60 nucleotides, approximately 41~60 nucleotides, approximately 42~60 nucleotides, approximately 43~60 nucleotides, approximately 44~60 nucleotides, approximately 45~60 nucleotides, approximately 46~60 nucleotides, approximately 47~60 nucleotides, approximately 48~60 nucleotides, approximately 49~60 nucleotides, approximately 50~60 nucleotides, approximately 51~60 nucleotides, approximately 52~60 nucleotides, approximately 53~60 nucleotides, approximately 54~60 nucleotides, approximately 55~60 nucleotides, approximately 56~60 nucleotides, approximately 57~60 nucleotides, approximately 58~60 nucleotides, and approximately 59~60 nucleotides.

[0220] For some purposes, the regulatory nucleic acids of this disclosure have lengths of approximately 10 nucleotides, approximately 11 nucleotides, approximately 12 nucleotides, approximately 13 nucleotides, approximately 14 nucleotides, approximately 15 nucleotides, approximately 16 nucleotides, approximately 17 nucleotides, approximately 18 nucleotides, approximately 19 nucleotides, approximately 20 nucleotides, approximately 12 nucleotides, approximately 22 nucleotides, approximately 23 nucleotides, approximately 24 nucleotides, approximately 25 nucleotides, approximately 26 nucleotides, approximately 27 nucleotides, approximately 28 nucleotides, approximately 29 nucleotides, approximately 30 nucleotides, approximately 31 nucleotides, approximately 32 nucleotides, approximately 33 nucleotides, and approximately 34 nucleotides. The creotides consist of approximately 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, and 60 nucleotides.

[0221] In some embodiments, the nucleic acid agent used by the present disclosure, for example, the oligonucleotide agent, may include a single strand. In some embodiments, the nucleic acid may include two or more strands. In some embodiments, the nucleic acid may include one or more double-stranded portions. In some such embodiments, some or all of such portions may be formed by self-hybridization of sequences on a single strand. In some embodiments, some or all of such portions may be formed by hybridization of separate strands. In some embodiments, a nucleic acid including one or more double-stranded portions may include one or more nicks or gaps and / or one or more bulges or loops.

[0222] In some embodiments, the nucleic acid agents used by the present disclosure, such as oligonucleotide agents, may include one or more structural features or properties related to their mechanism of action. For example, those skilled in the art will recognize that there is extensive literature on the structural features of oligonucleotides that, for example, cause the degradation of their targets (e.g., by recruiting RNase H) (such oligonucleotides are often called “antisense” agents or “ASOs”) and / or cause the degradation of Dicer and / or other elements of the RNA-induced silencing complex (RISC) (such oligonucleotides are often called “siRNA” agents), as well as oligonucleotides that regulate the splicing of target transcripts (e.g., make the production of one splice form more favorable than the production of another splice form), and / or function as guide RNAs for recruiting other mechanisms (e.g., nucleases such as CRISPR / Cas or dsRNA-binding proteins or their conjugates) to specific nucleic acid sequences, or as aptamers that bind to specific targets.

[0223] In some embodiments, the nucleic acid is or contains an interfering RNA (RNAi) agent. In some embodiments, the RNA is or contains a small 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.

[0224] In some embodiments, the nucleic acid is or contains a small interfering RNA (siRNA) agent. In some embodiments, the nucleic acid containing the siRNA agent can be ligated to the targeting moiety (e.g., directly or indirectly) with a sense strand. In some embodiments, the nucleic acid containing the siRNA agent can be ligated to the targeting moiety (e.g., directly or indirectly) with an antisense strand. In some embodiments, the nucleic acid containing the siRNA agent can be ligated to the targeting moiety (e.g., directly or indirectly) at the 5' end of the siRNA agent. In some embodiments, the nucleic acid containing the siRNA agent can be ligated to the targeting moiety (e.g., directly or indirectly) at the 3' end of the siRNA agent.

[0225] In some embodiments, the nucleic acid is or includes an exon skipping agent, an exon inclusion agent, or another splicing modulator.

[0226] In some embodiments, the nucleic acid is an aptamer agent or comprises one.

[0227] In some embodiments, the nucleic acid is or comprises an antisense oligonucleotide (ASO). In some embodiments, the ASO regulates gene expression via an RNase H-mediated mechanism. In some embodiments, the ASO regulates gene expression via steric hindrance.

[0228] In some embodiments, the nucleic acid agent is or comprises a phosphorodiamidate morpholino oligonucleotide (PMO).

[0229] In some embodiments, the nucleic acid agent is or comprises peptide nucleic acid (PNA).

[0230] In some embodiments, the nucleic acid agent is a nucleic acid analog, such as an RNA analog or a DNA analog, or a combination thereof, or includes such analogs.

[0231] In some embodiments, the nucleic acid can be ligated to the targeting region (e.g., directly or indirectly) with a sense strand. In some embodiments, the nucleic acid can be ligated to the targeting region (e.g., directly or indirectly) with an antisense strand. In some embodiments, the nucleic acid can be ligated to the targeting region (e.g., directly or indirectly) at the 5' end of the nucleic acid. In some embodiments, the nucleic acid can be ligated to the targeting region (e.g., directly or indirectly) at the 3' end of the nucleic acid.

[0232] For example, in some embodiments, the nucleic acid analog comprises one or more modified nucleotides (compared to canonical DNA and / or RNA). In some embodiments, the modified nucleotide comprises one or more of the following: a modified backbone, a modified nucleic acid base, a modified sugar (e.g., modified ribose or modified deoxyribose), or a combination thereof. In some embodiments, the modified nucleotide may be or comprise one or more naturally occurring modifications. In some embodiments, the modified nucleotide may be or comprise one or more non-naturally occurring modifications.

[0233] In some embodiments, the nucleic acid analog comprises one or more bonds other than phosphodiester bonds (e.g., phosphorothioate bonds or phosphorodiamidate bonds, or including them).

[0234] In some embodiments, the nucleic acid analog comprises one or more morpholino subunits linked together by a phosphorus-containing bond. In some embodiments, one or more morpholino subunits in the oligonucleotide analog are linked by a phosphorodiamidate bond. The synthesis, structure, and binding properties of the morpholino oligomers are described in detail in U.S. Patents 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 Patent Application No. PCT / US07 / 11435 (Cationic Bonding) and U.S. Patent Application No. 08 / 012,804 (Improved Synthesis), all of which are incorporated herein by reference. Morpholino subunits linked by phosphorodiamidate 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 PMO. In some embodiments, the PMO is substantially uncharged, for example, has a neutral charge.

[0235] In some embodiments, the nucleic acid agent has a negative charge.

[0236] In some embodiments, the nucleic acid agent is substantially uncharged, for example, has a neutral charge.

[0237] Those skilled in the art will understand, upon reading this disclosure, that in some embodiments, the nucleic acid agent for use according to this disclosure may comprise one or more DNA residues or analogues, one or more RNA residues or analogues, and / or combinations thereof. Furthermore, those skilled in the art will understand that in some embodiments, the nucleic acid agent may comprise one or more or entirely phosphodiester bonds, phosphorothioate bonds, or other preferred bonds.

[0238] In some embodiments, the nucleic acid agent includes native residues, such as DNA residues and / or RNA residues.

[0239] In some embodiments, the nucleic acid agent comprises one or more analogues, such as a DNA analogue and / or an RNA analogue.

[0240] In some embodiments, the nucleic acid agent comprises DNA residues and / or RNA residues, such as native residues or analogs.

[0241] In some embodiments, nucleic acids contain one or more chiral centers (for example, as they may be present in phosphorothioate bonds). In some embodiments, preparations of nucleic acids having a chiral center are stereochemically pure with respect to that center, in that they contain only one stereoisomer of that center. In some embodiments, both stereoisomers are present. In some embodiments, the preparation represents a racemic mixture of stereoisomers at that position. In some embodiments, preparations of nucleic acids having multiple chiral bonds may be stereochemically pure with respect to one or more centers and a mixture (e.g., racemic) with respect to one or more other centers. In some embodiments, the preparation may be stereochemically pure with respect to all chiral centers. In some embodiments, the preparation may be a racemic mixture (e.g., with respect to all chiral centers or as a whole).

[0242] In some embodiments, the nucleic acid comprises a structure comprising a first wing sequence, a gap sequence, and a second wing sequence. Nucleic acids comprising such a wing-gap-wing sequence are typically referred to as gapmers. In some embodiments, the gap sequence is flanked by the first and second wing sequences. In some embodiments, the gap sequence comprises about 6 to 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, as disclosed herein, for example. In some embodiments, the gapmer acts by recruiting RNaseH. In certain embodiments, the gapmer is a chimeric antisense oligonucleotide (ASO) comprising a central sequence of phosphorothioate DNA nucleotides (forming a "DNA gap") flanked at both ends by sequences of modified RNA residues. While we do not wish to be bound by theory, it is thought that such wing sequence modified RNA residues protect the DNA gap region from nuclease degradation, while the central DNA gap region allows for RNase-H mediated cleavage of target RNA.

[0243] In some embodiments, the nucleic acid includes an overhang. In some embodiments, the overhang is a 3' overhang or a 5' overhang. In some embodiments, the overhang is a 3' overhang. In some embodiments, the overhang contains 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 an overhang.

[0244] In some embodiments, the nucleic acid includes at least one stem-loop structure.

[0245] The oligonucleotides disclosed herein typically comprise at least one sequence element that hybridizes to a target sequence. In some embodiments, a nucleic acid agent, for example, an oligonucleotide, is or comprises an antisense sequence element. In some embodiments, the antisense sequence element is complementary to at least a portion of one or more of an exon, an intron, an untranslated region, a splice junction, a promoter region, an enhancer region, or a non-coding region (e.g., those in a gene transcript) within the target sequence. In some embodiments, the antisense sequence element is complementary to a portion of the target sequence within the sense strand.

[0246] In some embodiments, the nucleic acid comprises a sequence element that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target sequence within the sense strand. In some embodiments, the nucleic acid comprises a sequence element that is complementary (i.e., 100% complementary) to the target sequence within the sense strand.

[0247] In some embodiments, the nucleic acid comprises a sequence element that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target sequence within the antisense strand. In some embodiments, the nucleic acid comprises a sequence element that is complementary (i.e., 100% complementary) to the target sequence within the antisense strand.

[0248] In some embodiments, the nucleic acid includes 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 have at least 80% complementarity to a portion of the target sequence. In some embodiments, the nucleic acid includes 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 have at least 85% complementarity to a portion of the target sequence. In some embodiments, the nucleic acid includes 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 have at least 90% complementarity to a portion of the target sequence. In some embodiments, the nucleic acid includes 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 have at least 95% complementarity to a portion of the target sequence. In some embodiments, the nucleic acid includes 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 have at least 96% complementarity to a portion of the target sequence. In some embodiments, the nucleic acid includes 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 have at least 97% complementarity to a portion of the target sequence. In some embodiments, the nucleic acid includes 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 have at least 98% complementarity 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 contiguous nucleotides that have at least 99% complementarity 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 contiguous nucleotides that have 100% complementarity to a portion of the target sequence.

[0249] In some embodiments, the nucleic acid comprises two or more sequence elements 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 contiguous nucleotides that have at least 80% complementarity to a portion of the target sequence.

[0250] 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.

[0251] In some embodiments, the nucleic acid, e.g., an oligonucleotide, is characterized in that when the oligonucleotide, a composition comprising the oligonucleotide, or a conjugate agent comprising the oligonucleotide is delivered to a cell, tissue, or organism that expresses the target, a decrease in the expression and / or activity of the target is observed as compared to a cell, tissue, or organism to which the oligonucleotide, the composition comprising the oligonucleotide, or the conjugate agent comprising the oligonucleotide has not been delivered.

[0252] In some embodiments, nucleic acids, such as oligonucleotides, are characterized in that when an oligonucleotide, a composition containing an oligonucleotide, or a conjugate containing an oligonucleotide is delivered to cells, tissues, or organisms expressing the target, a decrease in the expression and / or activity of the target is observed compared to cells, tissues, or organisms that do not express the target (e.g., do not have detectable expression of the target).

[0253] In some embodiments, nucleic acids, such as oligonucleotides, are characterized in that when an oligonucleotide, a composition containing an oligonucleotide, or a conjugate containing an oligonucleotide is delivered to a cell, tissue, or organism expressing a target, a modification of the target's expression and / or activity is observed compared to that observed with a suitable reference agent known to have a specific effect on the target. In some embodiments, the target's expression and / or activity is modified in a manner and / or to a degree that is reasonably comparable to, or determined to be, 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.

[0254] In some embodiments, when nucleic acids, such as oligonucleotides, are delivered to cells, tissues, or organisms expressing a target, the expression and / or activity of the target is regulated, for example, reduced, compared to cells, tissues, or organisms to which the oligonucleotide has not been delivered.

[0255] The modulated nucleic acid agents of this disclosure may be designed to target human target genes, including portions of such genes that are conserved in target orthologues of other mammalian species. While not intended to be theoretical, the aforementioned properties of these modulated nucleic acid agents and specific target sites or specific modification combinations or partial combinations are thought to confer improved efficacy, stability, potency, persistence, and safety to the modulated nucleic acid agents of this disclosure.

[0256] Accordingly, this disclosure provides a method for treating and / or preventing target gene-related disorders and / or target RNA-related disorders, such as glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, or viral infections, or combinations thereof, using a conjugate comprising a modulating nucleic acid composition that results in RNase H-mediated degradation and / or RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of a target gene.

[0257] In some embodiments, the conjugated regulatory nucleic acid agent of the present disclosure comprises an RNA chain (antisense chain) having a region of up to about 30 nucleotides in length, 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, 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, wherein the region is substantially complementary to at least a portion of the mRNA transcript of the target gene.

[0258] In certain embodiments, one or both strands of the antisense oligonucleotide (ASO) and / or double-stranded RNAi agent of the present disclosure are up to 66 nucleotides long, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides long, and have 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 having longer antisense strands may preferably include a second RNA strand (sense strand) of 20-60 nucleotides long, where the sense strand and antisense strand form a double helix of 18-30 consecutive nucleotides.

[0259] The use of ASOs and / or iRNAs of this disclosure enables targeted inhibition and / or degradation of mRNA of corresponding genes (target genes) in mammals. In vitro assays can demonstrate that ASOs and / or iRNAs targeting target genes potently mediate RNAi, resulting in significant inhibition of target gene expression. Therefore, methods and compositions comprising these ASOs and / or iRNAs are useful in treating subjects with target gene-related disorders and / or target RNA-related disorders, such as glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, and viral infections.

[0260] Accordingly, this disclosure provides methods and combination therapies for treating subjects having disorders that would benefit from the regulation (e.g., inhibition or reduction) of the expression of a target gene, such as target gene-related disorders and / or target RNA-related disorders, such as glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, or viral infections, or combinations thereof, using conjugated regulatory nucleic acid agents, such as conjugated ASOs and / or iRNA compositions that result in RNase H-mediated degradation and / or RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a target gene.

[0261] This disclosure also provides a method for preventing at least one symptom in subjects having disorders that would benefit from inhibition or reduction of the expression of a target gene, such as glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, viral infections, or combinations thereof.

[0262] This specification discloses methods for preparing and using compositions containing modulating nucleic acid agents, such as ASOs and / or iRNAs, to regulate (e.g., inhibit) the expression of target genes, as well as compositions, uses, and methods for treating subjects who would benefit from the regulation (e.g., inhibition and / or reduction) of the expression of target genes, such as subjects who are susceptible to or have already been diagnosed with target gene-related disorders and / or target RNA-related disorders.

[0263] In certain embodiments, the Disclosure provides ASOs and / or iRNAs that inhibit the expression of target genes. In certain embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of target genes in cells of a mammal, such as a human, that is susceptible to developing metabolic disorders, including, for example, nephropathy and various other kidney diseases or conditions, such as target gene-related disorders and / or target RNA-related disorders.

[0264] dsRNA agents The double-stranded region of the dsRNA disclosed herein may be of any length that enables the specific degradation of a desired target RNA via the RISC pathway, and is approximately 19 to 36 base pairs long, for example, approximately 19 to 30 base pairs long, for example, approximately 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 long, for example, approximately 19 to 3 The lengths may range from 0, 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 certain embodiments, the double-stranded region is 19-21 base pairs long, for example, 21 base pairs long. Intermediate ranges and lengths between the above ranges and lengths are also intended to be part of this disclosure.

[0265] The two strands forming a double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore connected by an unbroken nucleotide chain between the 3' end of one strand and the 5' end of the other, forming a double-stranded structure, the connecting 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 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0266] If two substantially complementary strands of dsRNA are contained within separate RNA molecules, those molecules do not need to be covalently linked, but can be. If the two strands are covalently linked by means other than a continuous nucleotide chain between the 3' end of one strand and the 5' end of the other, forming a double-stranded structure, that linkage structure is called a “dsRNA linker.” RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA, minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs. In one embodiment of an 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 includes a 5' overhang of at least one nucleotide. In specific embodiments, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0267] In certain embodiments, the iRNA agent of the present disclosure is a dsRNA, each strand comprising 19 to 23 nucleotides, which interacts with a target RNA sequence, such as a target gene, to direct the cleavage of the target RNA.

[0268] In some embodiments, the iRNA of this disclosure is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a target mRNA gene, to direct the cleavage of the target RNA.

[0269] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded iRNA. For example, when the 3'-end of one strand of the dsRNA extends beyond the 5'-end of the other strand, or vice versa, a nucleotide overhang is present. A dsRNA can comprise an overhang of at least one nucleotide. Alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. The overhang(s) can be present on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang can be present at the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0270] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end or 5'-end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end or 5'-end. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside phosphorothioate.

[0271] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0272] In certain embodiments, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at its 3' or 5' end. In certain embodiments, the overhang on the sense strand, the overhang on the antisense strand, or both may include an extended length longer than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is on the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 5' end of the antisense strand of the double helix. In certain embodiments, one or more nucleotides in the extended overhang are substituted with nucleoside thiophosphates. In certain embodiments, the overhang includes a self-complementary moiety such that the overhang can form a stable hairpin structure under physiological conditions.

[0273] "Blunt-ended" or "blunt-ended" means that a double-stranded RNA agent has no unpaired nucleotides at its ends, i.e., no nucleotide overhangs. A "blunt-ended" double-stranded RNA agent is double-stranded along its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of this disclosure include RNAi agents that have no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end), or RNAi agents that have no nucleotide overhangs at either end. In most cases, such molecules are double-stranded along their entire length.

[0274] The terms "antisense strand" or "guide strand" refer to a strand of iRNA, such as dsRNA, that contains a region substantially complementary to the target sequence, such as the target mRNA.

[0275] 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, e.g., a target nucleotide sequence, as defined herein. If the complementary region is not entirely complementary to the target sequence, the mismatch may be located within the molecule or in a terminal region. Generally, the most acceptable mismatches are in terminal regions, e.g., within 5, 4, or 3 nucleotides at the 5' or 3' end of an iRNA. In some embodiments, the double-stranded RNA agent of the Disclosure includes nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the Disclosure includes four or fewer mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the Disclosure includes four or fewer mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the Disclosure contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the Disclosure contains four or fewer mismatches with the antisense strand, for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, located in the 5, 4, or 3 nucleotides at the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, located in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) are not present in the seed region.

[0276] Therefore, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agent described herein contains three or fewer mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agent described herein contains two or fewer mismatches. In one embodiment, the RNAi agent described herein contains one or fewer mismatches. In one embodiment, the RNAi agent described herein contains zero mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch may be limited to the last 5 nucleotides from either the 5' or 3' end of the complementarity region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the region of the target gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the target gene. Investigating the efficacy of RNAi agents with mismatches in inhibiting target gene expression is particularly important when specific complementary regions of the target gene are known to have polymorphic sequence variations within the population.

[0277] As used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA that contains a region substantially complementary to the antisense strand region, as defined herein.

[0278] As used herein, “substantially all nucleotides are modified” means that most of the nucleotides are modified, but not completely, and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0279] As used herein, the term “cleavage region” refers to a region located immediately adjacent to a cleavage site. A cleavage site is a site on the target where cleavage occurs. In some embodiments, the cleavage region includes one end of the cleavage site and three bases immediately adjacent to the cleavage site. In some embodiments, the cleavage region includes one end of the cleavage site and two bases immediately adjacent to the cleavage site. In some embodiments, the cleavage site occurs specifically at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0280] When used herein, unless otherwise specified, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double structure, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be, for example, stringent conditions, which may include washing at 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C, or 70°C for 12–16 hours (see, for example, “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered within living organisms, may also be applied. Those skilled in the art will be able to determine the set of conditions most appropriate for testing the complementarity of the two sequences, depending on the final use of the hybridized nucleotides.

[0281] When described herein, complementary sequences within iRNA, for example, within dsRNA, involve base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence spanning the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred herein as “substantially complementary,” the two sequences may be fully complementary, or they may form one or more mismatched base pairs during hybridization of a double helix up to 30 base pairs, but not generally more than 5, 4, 3, or 2, while retaining their ability to hybridize under conditions most appropriate for their final use, e.g., inhibition of gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be described as "perfectly complementary" for the purposes described herein.

[0282] When used herein, “complementary” sequences also include, or may be entirely formed from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, provided that the above requirements regarding the ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogsteen-type base pairs.

[0283] In this specification, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used in reference to base pairing between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of a double-stranded RNA agent, as can be understood from the context in which they are used.

[0284] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding the target polypeptide). For example, a polynucleotide is complementary to at least a portion of the target mRNA if its sequence is substantially complementary to a continuous portion of the mRNA encoding the target polypeptide.

[0285] Those skilled in the art are well aware that dsRNAs having double-stranded structures of approximately 20–23 base pairs, for example, 21 base pairs, are highly valued for their particular effectiveness in inducing RNA interference (Elbasil et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA14:1714-1719, Kim et al. (2005) Nat Biotech 23:222-226).

[0286] Generally, “iRNA” includes ribonucleotides having chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. When used in a dsRNA molecule, any such modification is included in “iRNA” for the purposes of this specification and the claims.

[0287] In certain embodiments of this disclosure, the inclusion of a deoxyribonucleotide, when present in an RNAi agent, may be considered to constitute a modified nucleotide.

[0288] The dsRNA agents of this disclosure include those having an antisense strand containing a complementary region that is complementary to at least a portion of the mRNA formed in the expression of a target gene. In some embodiments, the complementary region is about 19 to 30 nucleotides long (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides). When the iRNA comes into contact with cells expressing the target gene, it inhibits the expression of the target gene (e.g., a human, primate, non-primate, or rat target gene) by at least about 50% when assayed, for example, by PCR or branched DNA (bDNA) based methods, or by protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry techniques. In certain embodiments, the expression inhibition is determined in a suitable biological cell line, for example, by a qPCR method known in the art using siRNA at a concentration of 10 nM. In certain embodiments, in vivo inhibition of expression is determined, for example, by knockdown of the human gene in rodents expressing the human gene, such as mice expressing the human target gene or AAV-infected mice, when the human target gene is administered as a single dose (e.g., 3 mg / kg) at the lowest point of RNA expression (NADI).

[0289] A dsRNA comprises two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementarity region that is substantially complementary to the target sequence and is generally fully complementary. The target sequence may be derived from the sequence of mRNA formed during the expression of a target gene. The other strand (the sense strand) contains a region complementary to the antisense strand, and as a result, the two strands hybridize to form a double-stranded structure when combined under appropriate conditions. As described elsewhere in this specification and as known in the art, the complementary sequence of the dsRNA may be included as a self-complementary region of a single nucleic acid molecule, as opposed to being located on a separate oligonucleotide.

[0290] Generally, double-stranded structures are 15-30 base pairs long, 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, 19-2 The lengths are 9, 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 certain embodiments, the double-stranded structure is 18–25 base pairs long, 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 long, for example, 19–21 base pairs long. Intermediate ranges and lengths between the above ranges and lengths are also intended to be part of this disclosure.

[0291] Similarly, regions complementary to the target sequence are 15-30 nucleotides in length, e.g., 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, Lengths of 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, lengths of 19-23 nucleotides or lengths of 21-23 nucleotides. Intermediate ranges and lengths between the above ranges and lengths are also intended to be part of this disclosure.

[0292] In some embodiments, the double-stranded structure is 19–30 base pairs long. Similarly, the complementary region to the target sequence is 19–30 nucleotides long.

[0293] In some embodiments, dsRNAs are about 19–23 nucleotides long, or about 25–30 nucleotides long. Generally, dsRNAs are long enough to function as substrates for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21–23 nucleotides can function as substrates for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is most often a larger RNA molecule, often a part of an mRNA molecule. Where applicable, the “part” of the mRNA target is a sequence of mRNA targets long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0294] Those skilled in the art will also recognize that the double-stranded region is the primary functional portion of dsRNA, for example, a double-stranded region of about 19 to about 30 base pairs, for example, about 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Therefore, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region greater than 30 base pairs to the extent that it is processed into a functional double-stranded functional double-stranded RNA of 15-30 base pairs that targets the desired RNA for cleavage is a dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting gene expression is not generated in the target cell by cleaving a larger dsRNA.

[0295] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may include, or consist of, nucleotide / nucleoside analogs containing deoxynucleotides / nucleosides. Overhangs may reside on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhangs may reside at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0296] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi compounds of this disclosure can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using liquid-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.

[0297] Modified Nucleic Acids of the Disclosure In some embodiments, the conjugated modulating nucleic acid agents of the present disclosure comprise one or more modified (compared 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 nucleic acid base, a modified sugar (e.g., modified ribose or modified deoxyribose), or a combination thereof. In some embodiments, the modified nucleotide may be or comprise one or more naturally occurring modifications. In some embodiments, the modified nucleotide may be or comprise one or more non-naturally occurring modifications.

[0298] In some embodiments, the modulating nucleic acid agent comprises one or more bonds other than phosphodiester bonds (e.g., phosphorothioate bonds or phosphorodiamidate bonds, or including them).

[0299] In some embodiments, the modulating nucleic acid agent comprises one or more morpholino subunits linked together by a phosphorus-containing bond. In some embodiments, one or more morpholino subunits in the oligonucleotide agent are linked by a phosphorodiamidate bond. The synthesis, structure, and binding properties of the morpholino oligomers are described in detail in U.S. Patents 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 Patent Application No. PCT / US07 / 11435 (cationic binding) and U.S. Patent Application No. 08 / 012,804 (improved synthesis), all of which are incorporated herein by reference. Morpholino subunits linked by phosphorodiamidate 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 modulating nucleic acid agent is or includes PMO. In some embodiments, the PMO is substantially uncharged, for example, has a neutral charge.

[0300] In some embodiments, the regulatory nucleic acid agent has a negative charge.

[0301] In some embodiments, the modulating nucleic acid agent is substantially uncharged, for example, has a neutral charge.

[0302] Those skilled in the art will understand, upon reading this disclosure, that in some embodiments, the modulating nucleic acid agents of this disclosure may comprise one or more DNA residues or analogues, one or more RNA residues or analogues, and / or combinations thereof. Furthermore, those skilled in the art will understand that in some embodiments, the modulating nucleic acid agents of this disclosure may comprise one or more or entirely phosphodiester bonds, phosphorothioate bonds, or other preferred bonds.

[0303] In some embodiments, the regulatory nucleic acid agent includes native residues, such as DNA residues and / or RNA residues.

[0304] In some embodiments, the regulatory nucleic acid agent comprises one or more analogues, such as a DNA analogue and / or an RNA analogue.

[0305] In some embodiments, the regulatory nucleic acid agent comprises DNA residues and / or RNA residues, such as native residues or analogs.

[0306] In some embodiments, the regulatory nucleic acids of this disclosure contain one or more chiral centers (for example, as they may be present in phosphorothioate bonds). In some embodiments, preparations of regulatory nucleic acids having a chiral center are stereochemically pure with respect to that center, in that they contain only one stereoisomer of that center. In some embodiments, both stereoisomers are present. In some embodiments, the preparation represents a racemic mixture of stereoisomers at that position. In some embodiments, preparations of regulatory nucleic acids having multiple chiral bonds may be stereochemically pure with respect to one or more centers and a mixture (e.g., racemic) with respect to one or more other centers. In some embodiments, the preparation may be stereochemically pure with respect to all chiral centers. In some embodiments, the preparation may be a racemic mixture (e.g., with respect to all chiral centers or as a whole).

[0307] In some embodiments, the modulated nucleic acids of the Disclosure comprise one or more modified nucleotides. In some embodiments, the modified nucleotide comprises one or more of a modified skeleton, a modified nucleic acid base, a modified ribose, a modified deoxyribose, or a combination thereof.

[0308] In some embodiments, the modified nucleotide is selected from 2'-O-methyl-modified nucleotides, 5-methylcytidine, 5-methyluridine, nucleotides containing a 5'-phosphorothioate group, morpholinonucleotides (e.g., PMO), cholesteryl derivatives or terminal nucleotides bonded to a dodecanoate bisdecylamide group, 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholinonucleotides (e.g., PMO), phosphoramidates, phosphorylguanidine (PN)-based skeletons, or non-natural bases containing nucleotides, or combinations thereof.

[0309] In some embodiments, the modified nucleic acid bases include C7-modified deaza-adenine, C7-modified deaza-guanosine, C5-modified cytosine, C5-modified uridine, N1-methyl-pseudridine (m1ψ), 1-ethyl-pseudridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudridine, 5-methylcytidine, 5-methoxycytidine, or combinations thereof.

[0310] In some embodiments, the modified sugar (e.g., modified ribose or modified deoxyribose) includes 2'-fluoro 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 a combination thereof.

[0311] In some embodiments, the modified skeleton includes phosphorothioate (PS) modification, phosphorylguanidine (PN) modification, boranophosphate modification, alkylphosphonate nucleic acid (phNA), peptide nucleic acid (PNA), or a combination thereof.

[0312] In some embodiments, the modulating nucleic acid agent of the Disclosure comprises, for example, one or more modified nucleotides to the 5' end of an oligonucleotide. In some embodiments, the modulating nucleic acid agent of the Disclosure comprises a 5' amino modification.

[0313] In some embodiments, the modulating nucleic acid agents of the present disclosure are partially modified (e.g., at least 5%) for a specific modification, for example, over the entire length of the sequence.

[0314] In some embodiments, the modulating nucleic acid agents of the present disclosure are fully modified for a specific modification over the entire length of the sequence.

[0315] In some embodiments, at least 5% of a specific nucleotide (e.g., A, G, C, T, or U) is modified in the oligonucleotide.

[0316] In some embodiments, all (e.g., 100%) of a particular nucleotide (e.g., A, G, C, T, or U) are modified in the oligonucleotide.

[0317] In certain embodiments, the nucleotide sequences of the regulatory nucleic acid agents of the Disclosure, e.g., ASO and / or dsRNA, are unmodified and do not contain, for example, chemical modifications or conjugations known in the Art and described herein. In other embodiments, the nucleotide sequences of the regulatory nucleic acid agents of the Disclosure, e.g., ASO and / or dsRNA, are chemically modified to enhance stability or other beneficial properties. In certain embodiments of the Disclosure, substantially all of the nucleotides of the conjugate regulatory nucleic acid (e.g., ASO, dsRNA, etc.) of the Disclosure are modified. In other embodiments of the Disclosure, all of the nucleotides of the conjugate regulatory nucleic acid (e.g., ASO, dsRNA, etc.), or substantially all of the nucleotides of the conjugate regulatory nucleic acid (e.g., ASO, dsRNA, etc.) are modified, i.e., there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer unmodified nucleotides in the chain of the conjugate regulatory nucleic acid (e.g., ASO, dsRNA, etc.).

[0318] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, for example, those described by reference herein, “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, reverse bond) or 3'-terminal modifications (conjugation, DNA nucleotide, reverse bond, etc.); base modifications, e.g., stabilizing bases, destabilizing bases, or bases that form base pairs with the extended repertoire of a partner, base removal (debasing nucleotide), or substitution with a conjugate base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; or skeletal modifications including modification or substitution of phosphodiester bonds. Specific examples of conjugate-modulating nucleic acids useful in the embodiments described herein (e.g., ASOs, dsRNAs, etc.) include, but are not limited to, RNA that does not contain a modified skeleton or any natural internucleoside bonds. RNAs with modified skeletons include, among other things, those that do not have a phosphorus atom in their skeleton. For the purposes of this specification, and as is often referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides. In some embodiments, modified regulatory nucleic acids (e.g., ASOs, dsRNAs, etc.) will have a phosphorus atom in their internucleoside skeleton.

[0319] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylenephosphonates and chiralphosphonates, phosphinates, phosphoramidoates including 3'-aminophosphoramidoates and aminoalkylphosphoramidoates, thionophosphoramidoates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' and 2'-5' linkage analogs thereof, as well as those having inverted polarity where adjacent nucleoside unit pairs are linked from 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 the present disclosure, the conjugate modifying nucleic acid (e.g., ASO, dsRNA, etc.) agents of the present disclosure are in free acid form. In other embodiments of the Disclosure, the conjugate-modulating nucleic acid (e.g., ASO, dsRNA, etc.) agents of the Disclosure are in salt form. In one embodiment, the conjugate-modulating nucleic acid (e.g., ASO, dsRNA, etc.) agents of the Disclosure are in sodium salt form. In certain embodiments, when the conjugate-modulating nucleic acid (e.g., ASO, dsRNA, etc.) agent of the Disclosure is in sodium salt form, the agent contains sodium ions as counterions to substantially all phosphodiester groups and / or phosphorothioate groups present in the agent. Agents in which substantially all phosphodiester and / or phosphorothioate bonds have sodium counterions contain 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer phosphodiester and / or phosphorothioate bonds that do not have sodium counterions. In some embodiments, when the conjugate-modulating nucleic acid (e.g., ASO, dsRNA, etc.) agent of the Disclosure is in sodium salt form, the agent contains sodium ions as counterions to all phosphodiester groups and / or phosphorothioate groups present in the agent.

[0320] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above are incorporated herein by reference in their entirety: U.S. Patents Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, and 5,278,302. No. 5,286,717, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,46 6,677, 5,476,925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, No. 5,571,799, No. 5,587,361, No. 5,625,050, No. 6,028,188, No. 6,124,445, No. 6,160,109, No. 6,169,1 No. 70, No. 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6, This includes, but is not limited to, U.S. patent nos. 531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. patent no. RE 39464.

[0321] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar moiety of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons; amide skeletons; and others having mixed N, O, S, and CH2 constituent parts.

[0322] Representative U.S. patents teaching the preparation of the oligonucleosides described above are U.S. Patents Nos. 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, and 5,466,677, the entire contents of which are incorporated herein by reference. This includes, but is not limited to, the following publications: No. 5,470,967, No. 5,489,677, No. 5,541,307, No. 5,561,225, No. 5,596,086, No. 5,602,240, No. 5,608,046, No. 5,610,289, No. 5,618,704, No. 5,623,070, No. 5,663,312, No. 5,633,360, No. 5,677,437, and No. 5,677,439.

[0323] Suitable RNA mimetics are intended for use in conjugate regulatory nucleic acid (e.g., ASO, dsRNA, etc.) agents provided herein, in which both the sugar and nucleoside bonds of the nucleotide units, i.e., the backbone, are replaced with novel bases. The base units can be maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, including an RNA mime that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleic acid bases can be retained and directly or indirectly bonded 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. Patents 5,539,082, 5,714,331, and 5,719,262, the entire contents of which are incorporated herein by reference. Additional PNA compounds suitable for use with 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.

[0324] In certain embodiments, the regulatory nucleic acids of this disclosure include RNAs having a phosphorothioate backbone and oligonucleotides having a heteroatom backbone, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as the methylene(methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [the natural phosphodiester backbone is represented as -OPO-CH2-], as well as the amide backbone of U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of U.S. Patent No. 5,034,506.

[0325] Modified RNA may also contain one or more substituted sugar moieties. The regulatory nucleic acids characterized herein, for example, conjugated regulatory nucleic acid agents (e.g., ASO, dsRNA, etc.) contain at the 2' position one of the following: OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 Alkenyls and alkynyls may also be used. An example of appropriate modification is 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 is one example, where n and m are approximately 1 to 10. In other embodiments, dsRNA has C1-C at the 2' position. 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of ASO and / or iRNA, or groups for improving the pharmacodynamic properties of ASO and / or iRNA, as well as one of other substituents having similar properties. In some embodiments, the modifications include 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. Other exemplary modifications include 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the -O(CH2)2ON(CH3)2 group, as described in the examples herein, and 2'-dimethylaminoethoxyethoxy (also known 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 R and S isomers of these three families); 2'-alkoxyalkyls; and 2'-NMA (N-methylacetamide).

[0326] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the RNA of the ASO and / or iRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide (or the 3' position of the sugar on the 2'-5' linked dsRNA) and the 5' position of the 5' terminal nucleotide. The ASO and / or iRNA may also have sugar mimics such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are incorporated herein by reference in their entirety, U.S. Patents 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, and 5,514,785. This includes, but is not limited to, Nos. 9,134, 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. The entirety of each of the aforementioned contents is incorporated herein by reference.

[0327] ASOs and / or iRNAs may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, 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-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine This includes thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, STand Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleic acid bases are particularly useful for improving the binding affinity of the oligomeric compounds featured in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, as well as 2-aminopropyladenine, 5-propyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid double helix by 0.6-1.2°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, especially when combined with 2'-O-methoxyethyl sugar modification.

[0328] Representative U.S. patents teaching the preparation of the specific modified nucleic acid bases and other modified nucleic acid bases described above are incorporated herein by reference in their entirety: U.S. Patents No. 3,687,808, No. 4,845,205, No. 5,130,30, No. 5,134,066, No. 5,175,273, No. 5,367,066, No. 5,432,272, No. 5,457,187, No. 5,459,255, No. 5,484,908, No. 5,502,177, No. 5,525,711, No. 5,552,540, and No. 5,58 This includes, but is not limited to, Nos. 7,469, 5,594,121, 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.

[0329] The RNA in ASO or iRNA agents can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety that includes an additional crosslink connecting the 2' and 4' carbon atoms of the ribose moiety. This structure effectively "locks" the ribose in the higher-order structure of the 3'-end structure. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA 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).

[0330] The RNA of the ASO or iRNA agent may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S-higher-order structure referred to herein as “S-cEt”.

[0331] The ASO or iRNA agents of this disclosure may also comprise one or more “higher-order structure-restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of ribose, or the C3 and -C5' carbons of ribose. CRNs lock the ribose ring into a stable higher-order structure, increasing hybridization affinity to mRNA. The linker is long enough to position oxygen optimally for stability and affinity, reducing ribose ring puckering.

[0332] Representative publications teaching the preparation of the specific CRNs described above include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication WO2013 / 036868, the entire contents of which are incorporated herein by reference.

[0333] In some embodiments, the ASO or iRNA agent of this disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which a sugar bond has been removed to form an unlocked "sugar" residue. In one example, UNA also comprises monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, 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, incorporated herein by reference). Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0334] Potentially stabilizing modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3”-phosphate, and inverted base dT (idT). Disclosure of these modifications can be found in PCT Publication WO2011 / 005861.

[0335] Other nucleotide modifications of the ASO or iRNA agents of this disclosure include 5' phosphates or 5' phosphate mimics, for example, a 5' terminal phosphate or phosphate mimic on the antisense strand of the ASO or iRNA agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication 2012 / 0157511, the entirety of which is incorporated herein by reference.

[0336] In one aspect of this disclosure, the agent for use 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 in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by forming base pairs with the mRNA and physically interfering with the translation mechanism (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may contain a sequence that is at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0337] When used herein, the phrase “bring an iRNA, such as dsRNA, into contact with a cell” includes contacting the cell by any possible means. Bringing an iRNA into contact with a cell includes bringing the iRNA into contact with a cell in vitro or in vivo. Contact can be carried out directly or indirectly. For example, the iRNA can be brought into physical contact with a cell by the person performing the method, or alternatively, the iRNA can be placed in a situation that allows or causes subsequent contact with a cell.

[0338] In vitro contact with cells can be achieved, for example, by incubating cells with iRNA. In vivo contact with cells can be achieved, for example, by injecting iRNA into or near the tissue in which the cells are located, or by injecting iRNA into another region, such as the bloodstream or subcutaneous space, so that the drug can then reach the tissue in which the cells to be contacted are located. For example, the iRNA may contain or be bound to a ligand that induces the iRNA to a site of interest, such as kidney cells, such as a megalin-binding, cubelin-binding, or other cell surface factor binding moiety. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may also be contacted with iRNA in vitro and then transplanted into a target.

[0339] In certain embodiments, contacting an ASO or iRNA agent with cells includes “introducing the ASO or iRNA agent into cells” or “delivering the ASO or iRNA agent into cells” by promoting or achieving uptake or absorption into cells. Absorption or uptake of the ASO or iRNA agent may occur through unassisted diffusion or active cellular processes, or by adjuvants or devices. Introduction of an ASO or iRNA agent into cells may be in vitro or in vivo. For example, in vivo introduction may involve injecting the ASO or iRNA agent into a tissue site or administering it systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below or known in the art.

[0340] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, iRNA, or a plasmid on which iRNA is transcribed. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0341] Regulatory nucleic acids conjugated to ligands Another modification of the RNA of the conjugate-modulating nucleic acid (e.g., ASO, dsRNA, etc.) agents of this disclosure includes chemically linking one or more ligands, moieties, or conjugates to the conjugate-modulating nucleic acid agent that enhance the activity, intracellular distribution, or cellular uptake (e.g., introduction into cells) of the conjugate-modulating nucleic acid agent. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556).In other embodiments, the ligand may be cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NYAcad. Sci., 660:306-309, Manoharan et al., (1993) Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), or aliphatic chains, such as dodecanediol 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, such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-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 adamantane acetate (Manoharan et al., Tetrahedron These are the palmityl moiety (Lett., 1995, 36:3651-3654), the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or the octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0342] In certain embodiments, a ligand alters the distribution, targeting, or lifespan of the conjugate-modulating nucleic acid agent into which it is incorporated. In certain embodiments, a ligand enhances the affinity for selected targets, such as molecules, cells or cell types, compartments, such as cellular or organ compartments, tissues, organs, or regions of the body, compared to, for example, species in which such a ligand is absent. Certain ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0343] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid), or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers or oligomers, such as synthetic polyamino acids. Examples of polyamino acids, including polyamino acids, include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) 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 polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-mimicking polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helix peptides.

[0344] The ligand may also include a targeting group that binds to a specific cell type, such as kidney cells, e.g., a cell or tissue targeting agent, e.g., lectins, glycoproteins, lipids or proteins, e.g., antibodies (the kidney cell targeting portion is discussed in more detail elsewhere in this specification). The targeting group may be tyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, RGD peptide, or RGD peptide mimetic. In certain embodiments, the ligand is a polyvalent galactose, e.g., N-acetyl-galactosamine.

[0345] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl) Examples include cholenic acid, dimethoxytrityl, or phenoxazine and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, or AP.

[0346] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules that have a specific affinity for a co-ligand, or antibodies, such as antibodies that bind to specific cell types, such as hepatocytes. Ligands also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, and polyvalent fucose. Ligands can be, for example, lipopolysaccharides, p38MAP kinase activators, or NF-κB activators.

[0347] A ligand can be a substance, such as a drug, that can increase the uptake of a conjugate-modulating nucleic acid agent into a cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfibrils, or intermediate filaments. A drug may be, for example, Taxol, Vincristine, Vinblastine, Cytochalasin, Nocodazole, Japlaquinolide, Latrantrin A, Phalloidin, Swinford A, Indanosine, or Myoserbine.

[0348] In some embodiments, ligands conjugated to the conjugate-modulating nucleic acid agents described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Since oligonucleotides containing several phosphorothioate bonds are also known to bind to serum proteins, short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases with multiple phosphorothioate bonds in their backbone, are also suitable as ligands (e.g., as PK-modulating ligands) in this disclosure. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0349] The ligand-conjugate nucleic acid agents of this disclosure can be synthesized by using oligonucleotides having pendant-reactive properties, such as those derived from the binding of linking molecules to oligonucleotides. These reactive oligonucleotides can be directly reacted with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands having a linking portion that binds to them.

[0350] The oligonucleotides used in the conjugates of this disclosure can be prepared simply and routinely via well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several manufacturers, including, for example, Applied Biosystems® (Foster City, Calif.). Any other methods for such synthesis known in the art can be used additionally or alternatively. It is also known to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives, using similar techniques.

[0351] In the ligand-conjugate nucleic acid agents and ligand molecules having sequence-specific linked nucleosides of the present disclosure, oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, nucleotide or nucleoside conjugate precursors already having a linking portion, ligand nucleotide or nucleoside conjugate precursors already having a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0352] When a nucleotide-conjugate precursor already having a linking portion is used, the synthesis of a sequence-specific linking nucleoside is typically completed, and then the ligand molecule reacts with the linking portion to form a ligand-conjugate oligonucleotide. In some embodiments, the oligonucleotides or linking nucleosides of this disclosure are synthesized by an automated synthesizer using standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis, as well as phosphoramidites derived from ligand-nucleoside conjugates.

[0353] Carbohydrate conjugate In some embodiments of the compositions and methods of this disclosure, the targeting partial conjugate nucleic acid agent (e.g., ASO or iRNA agent) further comprises a carbohydrate. Carbohydrate-conjugate nucleic acid agents are advantageous for compositions suitable for in vivo delivery of nucleic acids and in vivo therapeutic applications. As used herein, “carbohydrate” refers to a carbohydrate itself, which consists of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound which has as part a carbohydrate consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Certain monosaccharides contain sugars with C5 or more (e.g., C5, C6, C7, or C8). Disaccharides and trisaccharides contain sugars with two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0354] Longer carbohydrate modifications (e.g., C16 or C20 modifications) have also been reported to be effective for the delivery of modulated nucleic acid drugs to the central nervous system (CNS).

[0355] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is a monosaccharide.

[0356] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US8,106,022, the full contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand for targeting a regulatory nucleic acid agent to specific cells. In some embodiments, the GalNAc conjugate targets a regulatory nucleic acid agent to liver cells, for example, by functioning as a ligand for the asialocrycoprotein receptor in liver cells (e.g., hepatocytes).

[0357] kidney cell targeting portion and conjugate In certain embodiments, a targeting moiety is used that specifically binds to factors present on the surface of target cells(s), such as kidney-related cells. In some embodiments, the provided technology achieves targeted delivery of the payload moiety to target cells, tissues, organs, or organisms with minimal off-target effects. In some embodiments, the targeting moiety described herein specifically binds to factors that preferentially exist on the surface of target cells(s) or tissues(s) compared to one or more non-target cells(s) or tissues(s). In some embodiments, the targeting moiety described herein specifically binds to factors specific to target cells(s) or tissues(s).

[0358] In certain embodiments, the targeting moieties disclosed herein associate with (e.g., conjugate or otherwise link) modulated nucleic acid agents of this disclosure that target megalin and / or cubilin (kidney cell surface factor receptors). Certain examples of such targeting moieties have been previously described as particularly useful for delivering nucleic acid agents to cells, in particular kidney-associated cells (e.g., kidney cells) (see PCT / US23 / 16319). Conjugated agents comprising a megalin-binding moiety conjugated with a nucleic acid agent (optionally by a linker) are particularly useful for delivering such nucleic acid agents to megalin-expressing cells. Such conjugated agents are particularly useful for delivering nucleic acid agents to kidney cells.

[0359] The targeting portions for use disclosed herein can bind, for example, selectively, to surface factors (e.g., parts or portions thereof, and / or specific forms, e.g., disease-related forms) present on the surface of the target cell(s) disclosed herein (e.g., kidney cells).

[0360] While we do not wish to be bound by theory, certain embodiments of this disclosure provide for the conjugation of a targeting moiety that associates with a regulatory nucleic acid agent of this disclosure to a cell surface factor present on the surface of a relevant (e.g., kidney) cell (e.g., tissue cell) to achieve internalization of the cell surface factor together with the conjugated targeting moiety (as part of a conjugate agent comprising the targeting moiety disclosed herein and one or more regulatory nucleic acid compounds). In some embodiments, such internalization may mean that the relevant cell surface factor is unavailable (for at least a certain period of time) on the surface of the cell (e.g., tissue cell) for purposes such as signal transduction and / or binding to ligands.

[0361] In some embodiments, renal cell surface factors are present on the surface of kidney-associated tissues, e.g., tissues that are part of the kidney or may be found within the kidney during development, maintenance of tissue homeostasis, and / or during disease or impairment, and can be detected, for example. In some embodiments, renal cell surface factors are present on proximal tubular epithelial cells, podocytes, and / or renal cysts (e.g., in polycystic kidney disease), and can be detected, for example. In some embodiments, the internalized conjugate (e.g., a portion thereof, e.g., the payload portion) is delivered to intracellular vesicles (e.g., lysosomes, endosomes, cratrin-coated pits, or intracellular membranous organelles, or a combination thereof). In some embodiments, the internalized conjugate (e.g., a portion thereof, e.g., the payload portion) is delivered to intracellular compartments, e.g., cytoplasm, mitochondria, ribosomes, nucleus, nucleolus, or other intracellular compartments, or a combination thereof. In some embodiments, an internalized conjugate agent in a cell (e.g., a vesicle or intracellular compartment) (e.g., a part thereof, e.g., a regulatory nucleic acid agent associated with a targeting moiety) can reduce the expression and / or activity of the target of the regulatory nucleic acid agent.

[0362] In some embodiments, internalization of a conjugate agent (e.g., a portion thereof, e.g., a regulatory nucleic acid agent associated with a targeting moiety) into a cell (e.g., a vesicle or intracellular compartment) involves cleaving the targeting moiety from the regulatory nucleic acid agent, e.g., separation. In some embodiments, the targeting moiety is cleaved from the regulatory nucleic acid agent, e.g., separated, by chemical reaction and / or mechanical separation. In some embodiments, the chemical reaction includes an enzymatic reaction to cleave the linker connecting the targeting moiety to the regulatory nucleic acid agent.

[0363] In some embodiments, internalization of a conjugate agent (e.g., a portion thereof, e.g., a regulatory nucleic acid agent associated with a targeting moiety) into a cell (e.g., a vesicle or intracellular compartment) involves cleaving the targeting moiety from the regulatory nucleic acid agent associated with the targeting moiety.

[0364] In some embodiments, the conjugates disclosed herein can be filtered by glomerular capillaries, for example, into Bowman capsules. In some embodiments, the conjugates disclosed herein have size, charge, conformation, and / or other properties that enable filtration by glomerular capillaries. In some embodiments, the threshold for glomerular filtration is in the range of 30 to 50 kDa.

[0365] In some embodiments, the cell surface factor (e.g., kidney cell surface factor) is a receptor selected from megalin, cubilin, or both, or includes one such receptor.

[0366] Megalin Megalin is a receptor with a length of approximately 600 kDa (approximately 4655 amino acids) and belongs to the low-density lipoprotein receptor family (disclosed in Nielsen R. et al. (2016), Kidney Int. 89(1):58-67). Megalin is also known as LDL receptor-related protein 2 (LRP2), glycoprotein 330 (Gp330), calcium sensor protein, or Hayman nephritis antigen homolog. Human megaphosphorus protein sequence:

[0367] The extracellular domain of megalin contains a cluster of cysteine-rich complement-type repeats. These repeats are separated by a β-propeller domain containing a YWTD motif and an EGF-type repeat. Megalin has a single transmembrane domain, which allows it to be positioned in a portion of the cell membrane containing cholesterol and / or sphingoglycolipids. Megalin also possesses an intracellular C-terminal cytoplasmic domain capable of regulating receptor transport and / or endocytosis. The cytoplasmic domain of megalin contains an NPXY motif and several other domains, including a proline-rich sequence and a PDZ motif. The cytoplasmic domain of megalin is associated with receptor internalization. A typical structure of megalin is disclosed in Figure 1 of Marzolo and Farfan (2011), Biol Res 44:89-105, its entirety incorporated herein by reference. The extracellular domain of megalin may also contain one or more posttranslational modifications, such as glycosylation.

[0368] Megalin has been described as interacting with its co-receptor, cubilin, at least in certain cases. Megalin has been identified on the surface of one or more of the following tissues and / or cells: immune cells (e.g., myeloid cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells]), erythrocytes, eosinophils, neutrophils, and / or platelets); 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; muscle (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 renal tissue, megalin has been reported to be found on the surface of proximal tubular epithelial cells and podocytes. In renal proximal tubular epithelial cells, megalin expression has been observed in brush margins, endocytic vesicles, and dense apical tubules and / or lysosomes. Several ligands for megalin 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 described in particular as high-affinity megalin ligands.

[0369] In some embodiments, the targeting kidney cell surface factor for delivery of the targeting partial association modulated nucleic acid compound of the present disclosure is megalin, a fragment thereof, or a variant thereof.

[0370] In some embodiments, the targeting moiety is or includes a megalin-binding moiety. In certain embodiments, the targeting moiety binds to the extracellular domain of megalin (e.g., a site on the extracellular domain, e.g., a site exposed when megalin is on the cell surface). In other specific embodiments, the conjugate includes a targeting moiety that binds to the extracellular domain of megalin (e.g., a site on the extracellular domain, e.g., a site exposed when megalin is on the cell surface) and causes internalization of megalin upon binding to megalin.

[0371] Cubilin Cubilin is a receptor with a size of approximately 460 kDa. Cubilin is also known as IFCR, Gp280, the endogenous factor-vitamin B12 receptor, MGA1, or IGS1. As an extracellular protein, cubilin can interact with other membrane proteins, such as megalin. One of cubilin's functions is as a receptor for the endogenous factor-vitamin B12 complex. Human cubillin protein sequence:

[0372] The extracellular domain of cubilin contains CUB domain repeats (complement C1r / C1s, Uegf [epidermal growth factor-related sea urchin protein], and osteomorphocyte 1) and EGF-type repeats. A typical structure of cubilin is disclosed in Figure 1 of Marzolo and Farfan (2011), Biol Res 44:89-105, and its entirety is incorporated herein by reference. The extracellular domain of cubilin may also contain one or more posttranslational modifications, such as glycosylation.

[0373] Cubilin has been reported to be found on the surface of one or more of the following tissues and / or cells: immune cells (e.g., myeloid cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells]), erythrocytes, eosinophils, neutrophils, and / or platelets); 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; 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 renal tissue, cubilin has been reported to be found on the surface of proximal tubular epithelial cells and podocytes. Several ligands for cubilin have been identified, some of which are disclosed in Nielsen et al. 2016.

[0374] Further exemplary cubillin-binding moieties or ligands are disclosed in U.S. Patent No. 10,065,993, International Patent Application WO2017 / 100700, International Patent Application WO2018 / 232122, and International Patent Application WO2015 / 027205, the entirety of which is incorporated herein by reference.

[0375] In some embodiments, the kidney cell surface factor is cubilin, a fragment thereof, or a variant thereof.

[0376] In some embodiments, the targeting portion is or includes a cubillin-binding portion.

[0377] In some embodiments, the targeting portion (e.g., the megalin-binding portion) is or includes a xenobiotic, such as those disclosed elsewhere in this specification. In some embodiments, the xenobiotic is selected from polymyxin, aprotinin, tricosanthin, or a combination thereof.

[0378] Other nucleic acid modifications In certain embodiments, the kidney cell targeting moieties disclosed herein are used for the delivery of nucleic acid payloads. Such kidney cell targeting moieties are expressly intended to be used in combination with one or more of the various other nucleic acid modifications known in the art. Specific nucleic acid modifications expressly intended for use in combination with the kidney cell targeting moieties of this disclosure include, but are not limited to, “extended nucleic acid” (“exNA”) modifications and various phosphorylguanidine-containing scaffoldings (“PN chemistry”).

[0379] "Extended nucleic acid" ("exNA") modification typically refers to a modification in which a methyl group is inserted between the 3'-phosphate group of the first nucleoside of an oligonucleotide chain and the 5'-carbon (progressing from 5' to 3') of the ribose of the next nucleoside. Therefore, such a canonical "exNA" modification has the following general structure: In certain embodiments, “exNA” modifications are also explicitly contemplated, which are optionally debased and / or have an extension longer than methyl, and / or may be substituted with one or more of a base, a 2'- substituent, and / or an extended carbon chain. Thus, in some embodiments of the present disclosure, the term “exNA” may refer to any part having the following structure: TIFF2026534515000030.tif41165, R 1is either a nucleic acid base or a debase structure (e.g., H, CH3, or other modified structures which may be substituted), and R 2 This includes, for example, but is not limited to, 2'-O-alkyl (e.g., 2'-O-methyl), 2'-F, and C 1~10 This refers to a carbon chain having 1 to 10 carbon atoms, which is optionally saturated or unsaturated and has 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, octylene, nonylene, or decyl), or alkynyl (e.g., ethynnyl, optionally substituted propynyl, butynyl, pentynyl, hexynyl, heptynyl, octinyl, noninyl, or decynyl) chains.

[0380] In related embodiments, C shown above 1~10 The branching of the domain is also intended for the “exNA” modifier. For example, in some embodiments, the “exNA” modifier of this disclosure is also an expression It can be represented by TIFF2026534515000031.tif37165, where R 1 is either a nucleic acid base or a debase structure (e.g., H, CH3, or other modified structures which may be substituted), and R 2 These are 2' substituents, for example, but not limited to, 2'-O-alkyl (e.g., 2'-O-methyl), 2'-F, etc., and the lateral substitution on the 5' of ribose can consist of 2 to 11 atoms and have varying amounts of substituents on X and Y (from X=Y=H to long n- or branched alkyl structures).

[0381] In certain embodiments, the "exNA" and / or other internucleoside modifications may be located at or near the 3' end of a therapeutic oligonucleotide (e.g., at or near the 3' end of the guide strand of an siRNA, or at or near the 3' end of an antisense agent), and we do not wish to be constrained by theory, but exNA modifications may confer an exNA-modified nucleic acid payload that is resistant to 3'-exonuclease-mediated digestion (Takeda, K. et al., Nature Biotechnology 2024). In some embodiments, the exNA-phosphorothioate internucleoside bond may be located at the last, or optionally second-to-last, nucleoside bond(s) at the 3' end of the nucleic acid payload (e.g., exNA-PS modifications(s) located in the 3' terminal region of the guide strand of an siRNA, and / or exNA-PS modifications(s) located in the 3' terminal region of an antisense oligonucleotide). As disclosed in WO2021 / 195533, exNA modifications may be used in combination with a variety of other commonly used oligonucleotide modifications, including, but not limited to, the following: In such embodiments, such exNA modifications also include, but are not limited to, the following representative natural and artificially modified "bases" or R known in the art, as described above. 1 It is also explicitly intended that it may be used with virtually any base recognized in the relevant art at any given position: TIFF2026534515000033.tif49165

[0382] Phosphorylguanidine-containing skeleton ("PN skeleton") binding is also expressly intended to be used in combination with the kidney cell targeting moiety of this disclosure. Such PN skeleton modifications were first identified in WO2023 / 201095 as members of the following genus: TIFF2026534515000034.tif42165PN Exemplary versions of skeletal modifications are, but are not limited to, The formula includes TIFF2026534515000035.tif92165, where W is O or S. In certain embodiments, the PN skeleton modification is The latter structure is selected from TIFF2026534515000036.tif37165 (the latter structure is shown as the “n001” modification of WO2023 / 201095). Exemplary PN skeletal bonds are also disclosed, for example, in U.S. Patent No. 11,208,430.

[0383] In addition to the express intent herein of using the above-described phosphorylguanidine structure for modifying nucleoside bonds of the nucleic acid agents disclosed herein, various other guanidine moieties are also expressly intended herein for bonding phosphorus groups. Such other guanidine moieties include, but are not limited to, the following: TIFF2026534515000037.tif214165TIFF2026534515000038.tif200165TIFF2026534515000039.tif197165TIFF2026534515000040.tif204165TI FF2026534515000041.tif168165TIFF2026534515000042.tif203165TIFF2026534515000043.tif196165TIFF2026534515000044.tif200165In the formula, R LS These are independently -Cl, -Br, -F, N(Me)2, or NHCOCO3.

[0384] Other skeletal modifications known in the art are also intended to be used with the targeting portion and payload of the Disclosure. In particular, the nucleic acid agents of the Disclosure are explicitly intended to include one or more mesylphosphoroamidate modifications and / or budylphosphoroamidate (and / or other structurally related modifications). Compared to phosphorothioate modifications, mesylphosphoroamidate and budylphosphoroamidate modifications have the following structures (see Segeeva et al. (2024) Front Chem., 12-2024 doi.org / 10.3389 / fchem.2024.1342178): In certain embodiments, the oligomeric compound (e.g., an antisense oligonucleotide, siRNA, etc., or a part thereof) comprises linked nucleosides and contains or comprises oligonucleotides having at least one internucleoside binding group represented by the following formula: In formula TIFF2026534515000046.tif42165, X is selected from O or S, and R is selected from aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, 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 conjugated group. In certain embodiments, X is O, R is methyl, and the nucleoside bond group in the above formula is the nucleoside bond group in the following formula.

[0385] In certain embodiments, the oligomeric compound (e.g., an antisense oligonucleotide, siRNA, or a portion thereof) contains or comprises oligonucleotides consisting of linked nucleosides and having at least one internucleoside linking group represented by the following formula. TIFF2026534515000047.tif37165 (Mesylphosphoamide nucleoside bond, see, e.g., WO2023 / 278589).

[0386] In certain embodiments, the oligomeric compound (e.g., an antisense oligonucleotide, siRNA, or a portion thereof) comprises linked nucleosides and contains or consists of oligonucleotides having at least one internucleoside linking group represented by the following formula. TIFF2026534515000048.tif37165

[0387] In certain embodiments, the oligomeric compound (e.g., an antisense oligonucleotide, siRNA, or a portion thereof) comprises linked nucleosides and contains or consists of oligonucleotides having at least one internucleoside linking group represented by the following formula. Modifications to the phosphate backbone can be positioned within the oligonucleotide agents of this disclosure at any position known in the art and / or disclosed herein.

[0388] The modified oligonucleotides of this disclosure include at least one modification compared to an unmodified oligonucleotide (i.e., at least one modified nucleoside (including a modified sugar moiety, a non-standard configurational nucleoside, and / or a modified nucleic acid base) and / or at least one modified internucleoside bond). In certain embodiments, the modified internucleoside bond is a modified internucleoside bond group having one of the above formulas. In certain embodiments, the compounds described herein are oligomeric compounds (including antisense agents, siRNA, etc., or oligomeric compounds that are part thereof) having at least one modified internucleoside bond group having one of the above formulas.

[0389] Expression regulation assay The regulation of target RNA and / or target polypeptide expression can be assayed using various methods known in the art. For example, the level of target mRNA can be quantified by, for example, Northern blotting, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed by methods known in the art to analyze total cellular RNA or poly(A) + This can be performed on mRNA. Methods for RNA isolation are taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.

[0390] Northern blot analysis is commonplace in the art and is taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative PCR can be conveniently achieved using the commercially available ABI PRISM® 7700 sequence detection system, available from PE-Applied Biosystems, Foster City, Calif., according to the manufacturer's instructions. Methods for analyzing RNA-level regulation are not limited to this disclosure.

[0391] The level of target protein encoded by target mRNA or DNA can be quantified by various methods well known in the art, such as immunoprecipitation, Western blotting, ELISA, or fluorescence-activated cell sorting (FACS). Antibodies against target protein encoded by target mRNA can be identified and obtained from various sources, such as the MSRS antibody catalog (Aerie Corporation, Birmingham, Mich.), or prepared by conventional antibody production methods. Methods for preparing polyclonal antiserum are taught, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. The method for preparing monoclonal antibodies is described, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0392] Immunoprecipitation is standard practice in this field and is described, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is also standard practice in this field and is described, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997.

[0393] Active target segment The location on a target nucleic acid defined by targeting one or more active regulatory RNA compounds is referred to as the “active target segment.” Substantial variation (e.g., defined by inhibition rate) may exist in the activity of regulatory RNA compounds within the active target segment. Active regulatory RNA compounds are those that have been shown to regulate the expression of their target RNAs. In some embodiments, active regulatory RNA compounds are inhibitory, optionally inhibiting the expression of their target RNAs by at least about 50%, optionally at least about 70%, and optionally at least about 80%, or more. In some embodiments, the level of inhibition required to define an active inhibitory RNA compound is defined based on results from screening used to define the active target segment. Those skilled in the art will understand that the inhibition rate by an inhibitory RNA compound against a target mRNA may vary between assays due to factors related to the assay conditions.

[0394] Hybridization As used herein, “hybridization” means the pairing of complementary strands of antisense compounds with their target sequences, and / or the pairing of complementary strands of double-stranded nucleic acid molecules with one another. While not limited to specific mechanisms, the most common pairing mechanisms include hydrogen bonding, which can be Watson-Crick hydrogen bonds, Hoogsteen hydrogen bonds, or reverse Hoogsteen hydrogen bonds between complementary nucleosides or nucleotide bases (nucleic acid bases). For example, the natural base adenine is complementary to the natural nucleic acid bases thymidine and uracil, which pair via the formation of hydrogen bonds. The natural base guanine is complementary to the natural base 5-methylcytosine and the artificial base known as G-clamp. Hybridization can occur under a variety of circumstances.

[0395] Regulatory RNA compounds can specifically hybridize under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic procedures, or under the conditions under which the assay is performed in the case of in vitro assays, provided that there is sufficient complementarity to avoid nonspecific binding of the regulatory RNA compound to non-target nucleic acid sequences.

[0396] As used herein, “stringent hybridization conditions” or “stringent conditions” refer to conditions under which a regulatory RNA compound hybridizes to its target sequence, but also to a minimum number of other sequences. Stringent conditions are sequence-dependent and vary depending on the context, and the “stringent conditions” under which a regulatory RNA compound hybridizes to its target sequence are determined by the properties and composition of the regulatory RNA compound, as well as the assay in which they are being investigated.

[0397] Complementarity As used herein, “complementarity” refers to the ability of two nucleic acid bases on either two oligomeric compound chains or an antisense compound having a target nucleic acid to precisely pair. For example, if a nucleic acid base at a particular position on an antisense compound can hydrogen bond with a nucleic acid base at a particular position on a target nucleic acid, the hydrogen bonding position between the oligonucleotide and the target nucleic acid is considered a complementary position. An antisense compound and further DNA or RNA are complementary if a sufficient number of complementary positions in each molecule are occupied by nucleic acid bases that can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms used to indicate that there is a sufficient degree of precise pairing or complementarity across a sufficient number of nucleic acid bases to result in a stable and specific bond between an antisense compound and a target nucleic acid, or between each sense strand and antisense strand (or a subsequence thereof) of a double-stranded nucleic acid molecule.

[0398] identity Double-stranded RNA compounds, antisense compounds, or parts thereof may have a defined percentage of identity with respect to a target sequence and / or its complement. For use herein, a sequence is identical to a sequence disclosed herein if it has the same nucleic acid base pairing ability. For example, RNAs containing uracil instead of thymidine in the disclosed sequences will both pair with adenine and are therefore considered identical. This identity may extend over the entire length of the oligomeric compound or to a portion of a given strand, such as a portion of an antisense compound (for example, nucleic acid bases 1-20 of a 27-mer can be compared to a 20-mer to determine the percentage of identity of the 27-mer over residues 1-20 to the comparator-20mer (or a defined sequence of 20 nucleotides in the target nucleic acid molecule)). It will be understood by those skilled in the art that regulatory RNA compounds (e.g., siRNA compounds or antisense compounds) do not need to have the same sequence as those described herein in order to function similarly to the regulatory RNA compounds described herein. Shortened or extended versions of regulatory RNA compounds taught herein, or, in the case of siRNA compounds, on one or both strands, or non-identical versions of regulatory RNA compounds taught herein, are within the scope of this disclosure. Non-identical versions are those in which each base does not have the same pairing activity as the regulatory RNA compound disclosed herein. The bases do not have the same pairing activity by being shorter or having at least one debase site. Alternatively, non-identical versions may include at least one base that has been replaced with a different base having different pairing activity (e.g., G may be replaced with C, A, or T). The identity percentage is calculated according to the number of bases that have identical base pairs corresponding to the reference sequence being compared (e.g., target sequence, complementary strand of dsRNA, antisense oligonucleotide and / or antisense strand sequence). Non-identical bases may be adjacent to each other, dispersed throughout the oligonucleotide, or both.

[0399] For example, a 16-mer having the same sequence as nucleic acid bases 2-17 of a 20-mer is 80% identical to the 20-mer throughout its entire length. Alternatively, a 20-mer containing four nucleic acid bases that are not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleic acid bases 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the capabilities of those skilled in the art.

[0400] The identity percentage is based on the proportion of nucleic acid bases in the original sequence that are present in a portion of the modified sequence. Therefore, a 30-nucleotide antisense compound containing the complete sequence of the complement of a 20-nucleotide active target segment has a portion that is 100% identical to the complement of the 20-nucleotide active target segment, while further containing an additional 10-nucleotide portion. The complement of the active target segment can constitute a single portion. In certain embodiments, the oligonucleotide is at least about 80%, optionally at least about 85%, more preferably at least about 90%, and most preferably at least 95% identical to at least a portion of the complement of the presented active target segment.

[0401] It is well known to those skilled in the art that it is possible to increase or decrease the length of a regulatory RNA compound, and / or introduce mismatched bases (target sequence, or complementary strand(s) of the double-stranded RNA, or both), without reducing its activity. For example, Wolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992, incorporated herein by reference) tested a series of ASOs ranging from 13 to 25 nucleic acid bases in length for their ability to induce cleavage of target RNA. A 25-nucleotide-length ASO containing eight or eleven mismatched bases near the end was able to induce specific cleavage of target mRNA, though not as effectively as ASOs without mismatches. Regulatory RNA compounds having shorter or longer consecutive nucleic acid base compositions (independent of either or both strands of the double-stranded nucleic acid compound), or regulatory RNA compounds containing mismatches, are contemplated in this disclosure, as long as regulatory RNA activity against the target sequence is maintained.

[0402] Treatment drugs The conjugated regulatory nucleic acid compounds of this disclosure may be used to modulate the expression of target RNA and / or target polypeptide in animals such as humans. In one non-limiting embodiment, the method comprises the step of administering an effective amount of the regulatory RNA compound that inhibits the expression of target RNA and / or target polypeptide to the animal requiring treatment for a disease or condition related to the target RNA and / or target polypeptide. Diseases or conditions related to the target RNA and / or target polypeptide include, but are not limited to, glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, or viral infections, or combinations thereof. In one embodiment, the conjugated regulatory RNA compound effectively inhibits the level or function of the target mRNA. A decrease in the target mRNA level may also result in a change in the level of the protein product encoding the mRNA expression, and such resulting changes can also be measured. A regulatory RNA compound that effectively inhibits the level or function of the expression of the target RNA or target protein product is considered an inhibitory RNA compound. In one embodiment, the regulatory RNA compound of the present disclosure inhibits the expression of a target RNA, reducing the 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%.

[0403] For example, a decrease in the expression of target RNA and / or target polypeptide can be measured in the body fluids, tissues, or organs of an animal. Methods for obtaining samples for analysis of body fluids (e.g., blood, plasma, urine, saliva, etc.), tissues (e.g., biopsy), or organs, and methods for preparing samples that enable analysis, are well known to those skilled in the art. Methods for analysis at the RNA and protein levels have been considered above and are well known to those skilled in the art. The efficacy of the treatment can be evaluated by measuring biomarkers associated with the expression of target RNA and / or target polypeptide in the aforementioned fluids, tissues, or organs collected from animals that have come into contact with one or more compounds, using routine clinical methods known in the art.

[0404] The modulated nucleic acid agents of this disclosure may 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 the diluent or carrier is based on several factors, including, but not limited to, the solubility of the compound and the desired route of administration. Such considerations will be well understood by those skilled in the art. In one embodiment, the compound inhibits the expression of target RNA and / or target polypeptide. Modulated nucleic acid agents may also be used in the manufacture of agents for the treatment of diseases and conditions related to the expression of target RNA and / or target polypeptide.

[0405] This also involves a method for contacting a body fluid, organ, or tissue with an effective amount of one or more regulatory RNA compounds or compositions. Contacting a body fluid, organ, or tissue with one or more compounds disclosed herein can result in the regulation of target RNA and / or target polypeptide expression in cells of the body fluid, organ, or tissue.

[0406] Accordingly, this specification provides for the use of isolated single-stranded or double-stranded regulatory nucleic acid agents targeting target RNA and / or target polypeptides in the manufacture of agents for the treatment of diseases or disorders by the methods described above. In certain embodiments, the regulatory nucleic acid agent is a single-stranded antisense compound. In other embodiments, the regulatory nucleic acid agent is a double-stranded inhibitory RNA compound.

[0407] In some embodiments, the modulating nucleic acid agents of the Disclosure, such as oligonucleotides, are characterized in that when the modulating nucleic acid agent of the Disclosure, a composition comprising the modulating nucleic acid agent of the Disclosure, or a conjugate comprising the modulating nucleic acid agent of the Disclosure is delivered to cells, tissues, or organisms 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 with the modulating nucleic acid agent of the Disclosure, a composition comprising the modulating nucleic acid agent of the Disclosure, or a conjugate comprising the modulating nucleic acid agent of the Disclosure.

[0408] In some embodiments, the modulating nucleic acid agents of the Disclosure, such as oligonucleotides, are characterized in that when the modulating nucleic acid agent of the Disclosure, a composition comprising the modulating nucleic acid agent of the Disclosure, or a conjugate comprising the modulating nucleic acid agent of the Disclosure is delivered to cells, tissues, or organisms expressing the target, a decrease in the expression and / or activity of the target is observed compared to cells, tissues, or organisms that do not express the target (e.g., do not have detectable expression of the target).

[0409] In some embodiments, the modulating nucleic acid agent of the Disclosure, for example, an oligonucleotide, is characterized in that, when the modulating nucleic acid agent of the Disclosure, a composition comprising the modulating nucleic acid agent of the Disclosure, or a conjugate comprising the modulating nucleic acid agent of the Disclosure is delivered to cells, tissues, or organisms expressing the target, a modification of the expression and / or activity of the target is observed compared to that observed with a suitable reference agent known to have a specific effect on the target. In some embodiments, the expression and / or activity of the target is modified in a manner and / or to a degree that is reasonably comparable to, or determined to be, 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.

[0410] In some embodiments, the modulating nucleic acid agents of the Disclosure, such as oligonucleotides, dsRNAs, etc., when delivered to cells, tissues, or organisms expressing a target, are characterized in that the expression and / or activity of the target is regulated, for example, reduced, compared to cells, tissues, or organisms to which the modulating nucleic acid agent of the Disclosure has not been delivered.

[0411] In some embodiments, the targeting portion, for example, the polymyxin or other compound disclosed herein, may be conjugated with the regulatory nucleic acid agent of this disclosure, such as oligonucleotides, dsRNA, etc.

[0412] This disclosure also provides pharmaceutical compositions comprising or delivering a conjugate agent disclosed herein. In some embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intramuscular, parenteral, or oral delivery. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains less than 5% impurities. In some embodiments, the impurities comprise one or more endotoxins, cellular components, or aggregates.

[0413] In some embodiments, what is provided herein are cells containing the conjugate agent disclosed herein. In some embodiments, the cells are present in tissues, organs, or organisms.

[0414] This disclosure provides a payload portion containing a target-recognizing nucleic acid agent, ligated to a first cleaved portion of a linker. In some embodiments, the payload portion is located within a cell where cell surface factors are present. In some embodiments, the cell further includes a targeting portion ligated to a second cleaved portion of the linker.

[0415] Provided herein are methods for delivering a conjugate agent to a target, comprising the step of administering to the target a conjugate agent, or a pharmaceutical composition containing a conjugate agent, which includes a targeting portion directly or indirectly linked to a payload portion.

[0416] Furthermore, this specification discloses a method for treating a disease or disorder, comprising the step of administering to a subject a conjugate agent or a pharmaceutical composition containing a targeting portion directly or indirectly linked to a payload portion.

[0417] Further disclosed herein are methods for treating a disease with a nucleic acid agent, the improvement comprising, for example, the step of administering the nucleic acid agent as a conjugate with a targeting moiety, as disclosed herein.

[0418] In some embodiments, the present disclosure provides a method for improving drug delivery to cells, the method comprising contacting a system or subject comprising at least one cell with a conjugate agent disclosed herein, or a pharmaceutical composition comprising such conjugate agent.

[0419] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered to cells expressing a cell surface factor. In some embodiments, the cell surface factor is a kidney cell surface factor. In some embodiments, the kidney cell surface factor is selected from megalin and / or cubilin.

[0420] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered to a tissue, organ, or fluid compartment.

[0421] In some embodiments of the methods, conjugates, compositions, or cells disclosed herein, the conjugate is internalized upon binding to a cell surface factor. In some embodiments, the internalization of the conjugate delivers the payload portion to an internal compartment or vesicle of the cell.

[0422] In some embodiments of the methods, conjugates, compositions, or cells disclosed herein, the payload reduces the expression and / or activity of targets provided in any one or a combination thereof in Tables 1-4.

[0423] In some embodiments of any of the methods disclosed herein, contact includes administering a conjugate agent to cells, tissues containing cells, or organisms containing cells.

[0424] In some embodiments of any of the methods disclosed herein, administering a conjugated agent to cells, tissues, or organisms delivers the payload portion to at least 5%, at least 10%, 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% more target cells compared to similar cells, tissues, or organisms to which the unconjugated payload portion was delivered.

[0425] In some embodiments of any of the methods disclosed herein, administering a conjugate agent to cells, tissues, or organisms delivers the payload portion to at least 5%, at least 10%, 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% more target cells compared to non-target cells.

[0426] In some embodiments of the methods, conjugates, compositions, or cells disclosed herein, the target cells are or include kidney cells.

[0427] In some embodiments of the methods, conjugates, compositions, or cells disclosed herein, the target cells are cells having (e.g., detectable) expression of a cell surface factor. In some embodiments, the cell surface factor is a kidney cell surface factor or comprises a kidney cell surface factor. In some embodiments, the kidney cell surface factor is megalin, or a variant or fragment thereof. In some embodiments, the kidney cell surface factor is cubilin, or a variant or fragment thereof.

[0428] In some embodiments of the methods, conjugates, compositions, or cells disclosed herein, the target cells are or comprise one or more expression entities of targets selected from any of the targets listed in Tables 1 to 4.

[0429] In some embodiments of any of the methods, conjugates, compositions, or cells disclosed herein, non-target cells are cells that do not express (e.g., detectable expression of) cell surface factors, or include such cells. In some embodiments, non-target cells are cells that do not express (e.g., do not have detectable expression of) kidney cell surface factors (e.g., megalin and / or cubilin), or include such cells.

[0430] In some embodiments of any of the methods disclosed herein, administering a conjugated agent to a cell, tissue, or organism reduces the expression and / or activity of the target of the unconjugated payload 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 a similar cell, tissue, or organism to which the unconjugated payload portion was delivered.

[0431] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered to cells expressing a cell surface factor, for example, as described herein. In some embodiments, the cell surface factor is selected from megalin and / or cubilin.

[0432] In some embodiments, the cells are selected from immune cells, nervous system cells, muscle cells, intestinal cells, colon cells, adipocytes, kidney cells, liver cells, 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 a combination thereof.

[0433] In some embodiments, the cells are selected from kidney cells, thyroid cells, parathyroid cells, inner ear cells, or nervous system cells, or a combination thereof.

[0434] In some embodiments, cells are selected from proximal tubular epithelial cells and / or podocytes.

[0435] In some embodiments of any of the methods disclosed herein, the disease is a disease related to the expression of cell surface receptors. In some embodiments, the disease is a disease comprising cells in which both cell surface receptors and targets recognized by the payload portion are present.

[0436] In some embodiments of any of the methods disclosed herein, the disease or disorder is selected from glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, viral infections, or a combination thereof.

[0437] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered intravenously, subcutaneously, intramuscularly, parenterally, or orally.

[0438] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered in one or more doses.

[0439] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered in combination with one or more additional conjugate agents. In some embodiments, the one or more additional conjugate agents include different payload portions, different linkers, different targeting portions, or a combination thereof.

[0440] In some embodiments of any of the methods disclosed herein, the conjugate agent is delivered in combination with one or more additional therapeutic modes.

[0441] Nucleic acid payload target Disclosed herein, in particular, are conjugate agents comprising a payload portion capable of acting on one or more targets, such as those disclosed herein.

[0442] In some embodiments, the targets are located in cells or tissues selected from immune cells (e.g., myeloid cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells]), 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; kidneys; liver; lungs; spleen; stomach; esophagus; bladder; pancreas; thyroid gland; salivary glands; adrenal glands; pituitary gland; breast; skin; ovaries; uterus; placenta; prostate; or testes, or combinations thereof).

[0443] In some embodiments, the target is located in a tissue or cell selected from renal tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissue.

[0444] In some embodiments, the targets are present (for example, at relatively high levels) on kidney cells such as proximal tubular epithelial cells and / or podocytes.

[0445] In some embodiments, the target is located in kidney-associated cells, for example, in kidney cells or cells that may be found within the kidney during development, maintenance of tissue homeostasis, or in the course of disease or injury. In some embodiments, the target is located in kidney-associated tissue, for example, in tissue that is part of the kidney during development, maintenance of tissue homeostasis, or in the course of disease or injury.

[0446] In some embodiments, cells of the tissue expressing the target also express the targeting moiety, as described herein, for example.

[0447] In some embodiments, cells of the tissue expressing the target also express a kidney-specific targeting moiety, as disclosed herein, for example.

[0448] In some embodiments, the expression and / or activity of the target may be dysregulated in disease or disorder. In some embodiments, delivery of a conjugate agent to cells in tissues expressing the target reduces the expression and / or activity of the target.

[0449] In some embodiments, delivery of a conjugate agent to an organism having abnormal expression and / or activity of a target within cells, for example, within tissue cells, treats a disease or disorder and / or alleviates the symptoms of the disease or disorder in the organism.

[0450] In some embodiments, the target is selected from any one of the targets provided in Tables 1-4, or a combination thereof.

[0451] In some embodiments, the target is or includes a gene product (e.g., a transcript) expressed in specific cells (e.g., cell types) and / or tissues described herein.

[0452] In some embodiments, the target is or includes non-coding RNA (or other regulatory RNA species) expressed in specific cells (e.g., cell types) and / or tissues described herein. In some embodiments, the target is or includes long non-coding RNA (lncRNA), microRNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snoRNA), or a combination thereof.

[0453] In some embodiments, the target is expressed in cells and / or tissues having an internalized receptor on its surface. In some embodiments, the target is expressed in cells and / or tissues having megalin on its surface. In some embodiments, the target is expressed in cells and / or tissues having cubilin 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., myeloid cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells], 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; liver cells; 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 embodiments, the target is expressed in renal proximal tubular epithelial cells (RPTECs), podocytes, and / or a combination thereof.

[0454] In some embodiments, the target is a gene expressed in renal proximal tubular epithelial cells (RPTECs), or includes such genes. 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 features and / or functions, or combinations thereof, provided in Table 2. In some embodiments, the target is A-kinase anchoring protein; acyl-CoA dehydrogenase family; acyl-CoA thioesterase; aldo-ketoreductase; ankyrin repeat domain-containing protein; apolipoprotein; basic helix-loop-helix protein; basic leucine zipper protein; beta-gamma crystallin; blood group antigens; BPI fold-containing protein; C-type lectin domain-containing protein; C1q and TNF-related proteins; C2 domain-containing protein; cadherin; CAP superfamily; CD molecule; chemokine ligand; claudin; collagen; complement system; CTAGE family; cytochrome P450; Dbl family Rho GEF; EF hand domain-containing proteins; erythrocyte membrane protein band 4.1; F-BAR domain-containing proteins; fatty acid binding protein family; fatty acid desaturase; fibronectin type III domain-containing proteins; G protein-coupled receptors; galectin; gelsolin / bilin; glycoside hydrolase family 31; GOLD domain-containing proteins; GRAM domain-containing proteins; GRAM domain-containing proteins; haloate dehalogenase-like hydrolase domain-containing proteins; heat shock proteins; histones; homeoboxes; I-BAR domain-containing proteins; immunoglobulin superfamily domain-containing proteins; interleukin receptors; intermediate filaments; ion channels; kinesin; late keratinization envelope proteins; ligand-gated ion channels; low-density lipoprotein receptors; M14 carboxypeptidase; Maestro heat-like repeat-containing proteins; 4-domain membranes; metallothionein methyltransferase family; mitochondrial respiratory chain complex builders; mitochondrial respiratory chain complex; mucin; myosin heavy chain;N-BAR domain-containing proteins; N-terminal EF hand calcium-binding proteins; Na+ / K+ transporting ATPase-interacting proteins; NLR family; non-coding RNA; regulatory RNA; oxysterol-binding proteins; paraneoplastic Ma antigen; PDZ domain-containing proteins; phospholipase; plextrin homology domain-containing proteins; protein phosphatase 1 regulatory subunit; PWWP domain-containing proteins; Ras-related domain family; Ras small GTPase superfamily; receptor accessory proteins; receptor kinases; receptor ligands; RNA-binding motif-containing proteins; serine proteases; serpin peptidase inhibitors; SH2 domain-containing proteins Proteins possessing one or more characteristics and / or functions selected from the following: proteins; short-chain dehydrogenase / reductase superfamily; sideroflexins; RNA metabolic signaling and activation families; solute carriers; sorting nexins; sterile alpha-motif domain-containing proteins; STRIPAK complexes; sulfatases; Sushi domain-containing proteins; synapsins; synaptotagmins; tetraspanins; tetratricopeptide repeat domain-containing proteins; trielement motif-containing proteins; tubulin tyrosine ligase family; tubulin; WD repeat domain-containing proteins; zinc fingers; ZYG11 cell cycle regulator family, or combinations thereof.

[0455] In some embodiments, the target is a gene expressed in podocytes, or includes such a gene. In some embodiments, the target is selected from the podocyte genes provided in Table 3, or combinations thereof. In some embodiments, the target has one or more features and / or functions, or combinations thereof, provided in Table 4.In some embodiments, the targets are abhydrolase domain-containing proteins; ADAM metalloptidase with a thrombospondin type 1 motif; ankyrin repeat domain-containing proteins; apolipoproteins; armadillo-like helix domain-containing proteins; basic leucine zipper proteins; blood group antigens; bone morphogenetic proteins; C-type lectin domain-containing proteins; C1q and TNF-related proteins; carbonic anhydrase; CD molecules; chitinase; fimbriae and flagella-related proteins; Crumbs complexes; Dbl family Rho GEF; EF hand domain-containing protein; F-BAR domain-containing protein; Fibronectin type III domain-containing protein; Forkhead box; Formin; G protein-coupled receptor; Gla domain-containing protein; Glycosyltransferase; Homeobox; Immunoglobulin superfamily domain-containing protein; Ion channel; Junctophilin; Kallikrein; Ligand-dependent ion channel; Lipocalin; Myosin light chain kinase family; Netrin; PDZ domain-containing protein; Phospholipase; Plexstrin homology domain-containing protein; Potassium voltage-dependent channel regulatory subunit; Protein phosphatase; Ras small GTPase superfamily; Receptor kinase; Receptor ligand; Rho Having one or more features and / or functions selected from GTPase-activating proteins; RNA-binding motif-containing proteins; semaphorins; serine proteases; serpin peptidase inhibitors; Shisa family members; solute carriers; sterile alpha motif domain-containing proteins; stomatin family; T cell receptors; tetraspan junction superfamily; transcription elongation factor A-like family; troponin complex subunits; tubulin polymerization-promoting proteins; WD repeat domain-containing proteins; Wnt family; zinc fingers, or combinations thereof.

[0456] [Table 1] TIFF2026534515000051.tif188165TIFF2026534515000052.tif192165TIFF2026534515000053.tif193165TIFF2026534515000054.tif191165TIFF2026534515000055.tif192165TIFF2026534515000056.tif197165TIFF2026534515000057.tif194165TIFF2026534515000058.tif192165TIFF2026534515000059.tif195165TIFF2026534515000060.tif193165TIFF2026534515000061.tif185165TIFF2026534515000062.tif195165TIFF2026534515000063.tif186165TIFF2026534515000064.tif145165

[0457] [Table 2] TIFF2026534515000066.tif233165TIFF2026534515000067.tif234165TIFF2026534515000068.tif229165TIFF2026534515000069.tif230165TIFF2026534515000070.tif235165TIFF2026534515000071.tif230165TIFF2026534515000072.tif230165TIFF2026534515000073.tif233165TIFF2026534515000074.tif123165

[0458] [Table 3] TIFF2026534515000076.tif201165TIFF2026534515000077.tif192165TIFF20265345150 00078.tif194165TIFF2026534515000079.tif203165TIFF2026534515000080.tif219165

[0459] [Table 4] TIFF2026534515000082.tif233165TIFF2026534515000083.tif232165TIFF2026534515000084.tif176165

[0460] Characterization of conjugates In some embodiments, the conjugate(s) provided and / or utilized herein are characterized in that, when provided to a relevant system (e.g., including one or more cells, tissues, organs, or organisms), they affect the expression and / or activity of one or more targets or their forms(s).

[0461] In some embodiments, the drug in question is characterized by its effect on RNA (e.g., mRNA) and / or protein (e.g., encoded by mRNA) targeted by its nucleic acid payload. In some such embodiments, such effect is evaluated in vivo (i.e., in an organism). Alternatively or additionally, in some such embodiments, the effect is evaluated in vitro (e.g., in a cell line).

[0462] In some embodiments, the conjugate(s) described and / or utilized pursuant to this disclosure are characterized in comparison to an unconjugated nucleic acid agent (as a payload). In some embodiments, when evaluated under equivalent conditions, a suitable in vivo or in vitro system exhibits a significantly greater effect when in contact with the conjugate(s) described herein than the effect otherwise observed when the system is in contact with an unconjugated nucleic acid agent under equivalent conditions.

[0463] Pharmaceutical composition This disclosure provides, in particular, pharmaceutical compositions comprising or otherwise delivering a conjugate modulated nucleic acid agent. Typically, such a pharmaceutical composition comprises an active agent (e.g., an ASO or iRNA agent or a composition comprising the same) conjugated to a targeting agent disclosed herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0464] Pharmaceutical compositions containing iRNA are useful for preventing or treating metabolic disorders, including target RNA-related disorders such as nephropathy and various other kidney diseases or conditions. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery, for example, by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of this disclosure may be administered in doses sufficient to inhibit the expression of target genes.

[0465] In some embodiments, the pharmaceutical compositions of the Disclosure are sterile. In other embodiments, the pharmaceutical compositions of the Disclosure do not contain pyrogens.

[0466] The pharmaceutical compositions of this disclosure may be administered in doses sufficient to inhibit the expression of a target gene. Generally, appropriate doses of the ASO or iRNA agents of this disclosure range from about 0.001 to about 200.0 milligrams per kilogram of body weight per day of the recipient, generally ranging from about 1 to 50 mg per kilogram of body weight per day. Typically, appropriate doses of the ASO or iRNA agents of this disclosure range from about 0.1 mg / kg to about 5.0 mg / kg, optionally about 0.3 mg / kg and about 3.0 mg / kg. Repeated-dose regimens may involve administering a therapeutic dose of the ASO or iRNA agent regularly, for example, monthly, every 3 to 6 months, or once a year. In certain embodiments, the ASO or iRNA agent is administered about once a month to about once every 6 months.

[0467] After the initial treatment regimen, less frequent treatments may be administered. The duration of treatment can be determined based on the severity of the disease.

[0468] In other embodiments, a single dose of the pharmaceutical composition is highly sustained, allowing administration at intervals of 1, 2, 3, or 4 months or less. In some embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered approximately once a month. In other embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered quarterly (i.e., approximately every 3 months). In other embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered twice a year (i.e., once every 6 months).

[0469] Those skilled in the art will understand that certain factors, including but not limited to mutations present in the subject, past treatments, the subject's overall health or age, and other pre-existing diseases, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of the subject with a prophylactic or therapeutically effective dose of the composition, as needed, may consist of a single treatment or a series of treatments.

[0470] iRNAs can be delivered in a manner that targets specific tissues (e.g., kidney cells).

[0471] The pharmaceutical compositions of this disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components, including, but are not limited to, pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Formulations include those targeting the kidneys.

[0472] The pharmaceutical formulations of this disclosure, which can be conveniently provided as single dosage forms, can be prepared according to the prior art well known in the pharmaceutical industry. Such arts include the step of associating an active ingredient with a pharmaceutical carrier(s) or excipient(s). Generally, formulations are prepared by homogeneously and closely associating an active ingredient with a liquid carrier.

[0473] In some embodiments, the pharmaceutical compositions described herein may include a buffer containing neutral buffered saline or phosphate-buffered saline (PBS); carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol; proteins, polypeptides, 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 (e.g., no detectable levels of contaminants (e.g., endotoxins)).

[0474] In some embodiments, the pharmaceutical compositions described herein may be administered in a manner suitable for the disease, disorder, or condition to be treated or prevented. In some embodiments, the dosage 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, disorder, or condition, although the appropriate dosage may be determined by clinical trials.

[0475] In some embodiments, the pharmaceutical compositions provided by this disclosure may be in the form of, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., solutions for injection and infusion), dispersions or suspensions, liposomes, and suppositories. Typically, pharmaceutical compositions containing or delivering antibody agents are injectable or infusion preparations. In some such embodiments, such compositions can be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, transarterial, sublingual, intranasal, topical, or intraperitoneal administration. In some embodiments, the pharmaceutical compositions provided are formulated for intravenous administration. In some embodiments, the pharmaceutical compositions provided are formulated for subcutaneous administration.

[0476] The pharmaceutical compositions described herein may be formulated for administration by means of injection techniques commonly known in the art (for example, see Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, which is incorporated herein in whole by reference).

[0477] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously with, or after) additional therapies for symptoms, diseases, or disorders, such as SOC therapy for symptoms, diseases, or disorders. In some embodiments, the pharmaceutical compositions described herein may be administered before or after surgery.

[0478] In some embodiments, the dose of any of the aforementioned therapies administered to a subject varies with the disease, disorder, or condition being treated and is determined on a particular basis. Scaling of human dosing can be carried out in accordance with established practices in the art.

[0479] Use of conjugate modulated nucleic acid compounds Cell type for delivery In some embodiments, the regulatory nucleic acid agent of the present disclosure (e.g., a conjugate comprising a targeting moiety and one or more regulatory nucleic acid compounds disclosed herein) is delivered to cells, for example, tissue cells, and optionally to cells in which cell surface factors are present.

[0480] In some embodiments, the cells are or include cells (e.g., from tissue) selected from kidney cells; immune cells (e.g., myeloid cells, lymph node cells, thymocytes, peripheral blood mononuclear cells [e.g., myeloid cells and / or lymphoid cells]), 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; liver cells; 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.

[0481] In some embodiments, the cells are or include cells (e.g., tissue cells) selected from kidney cells, thyroid cells, parathyroid cells, inner ear cells, or nervous system cells.

[0482] In some embodiments, the cells are, for example, kidney cells as described herein, or include them. In some embodiments, the cells are renal cyst cells (for example, those in polycystic kidney disease (PKD)). In some embodiments, the cells are, or include proximal tubular epithelial cells, podocytes, or both.

[0483] In some embodiments, the cells to which the conjugates disclosed herein are delivered express both cell surface factors (e.g., megalin and / or cubilin) ​​and the targets of the payload portion.

[0484] In some embodiments, the modulating nucleic acid agent of this disclosure (e.g., a conjugate comprising a targeting moiety and one or more modulating nucleic acid compounds as disclosed herein) is administered to a subject having a disease or disorder, for example, as disclosed herein. In some embodiments, the disease or disorder includes cells in which surface cell factors (e.g., megalin and / or cubilin) ​​and / or target RNA are present.

[0485] Indications In some embodiments, the modulated nucleic acid agents of this disclosure (e.g., a conjugate comprising a targeting moiety and one or more modulated nucleic acid compounds disclosed herein) are used to treat and / or prevent symptoms of a disease or disorder disclosed herein.

[0486] In some embodiments, the diseases or disorders for which the regulatory nucleic acid compounds disclosed herein are provided have increased or abnormal expression of cell surface factors such as megalin and / or cubilin.

[0487] In some embodiments, megalin expression has been reported to be abundant, particularly in the following tissues and / or cells: renal tissue, thyroid tissue, parathyroid tissue, inner ear cells, and nervous system tissues. In some embodiments, megalin is expressed (e.g., at relatively high levels) on the surface of renal cells, such as proximal tubular epithelial cells and podocytes.

[0488] In some embodiments, the disease or disorder is selected from glomerular disorders, renal tubular disorders, other renal disorders, congenital metabolic disorders, systemic metabolic disorders, thyroid disorders, parathyroid disorders, inner ear disorders, neurological disorders, viral infections, or a combination thereof.

[0489] In some embodiments, the disease or disorder is or includes glomerular disorders. In some embodiments, the glomerular disorder is selected from lupus nephritis, Goodpasture syndrome, IgA nephropathy, Alport syndrome, glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, minimal change nephrotic syndrome, ApoL1 nephropathy, post-infectious glomerulonephritis, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis syndrome, anti-LRP2 nephropathy, C3 glomerulopathy, or a combination thereof.

[0490] In some embodiments, the disease or disorder is or includes renal tubular disorder. In some embodiments, renal tubular disorder is selected from Fanconi syndrome, cystinuria, Lowe syndrome, Dent syndrome, light chain proximal tubulopathy, Guitelman syndrome, renal tubular acidosis, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, hereditary aminoaciduria, hereditary salt loss disorder, hereditary phosphorus loss disorder, porphyria-associated kidney disease, nephropathy-type cystinosis, autosomal dominant tubulointerstitial kidney disease, or a combination thereof.

[0491] In some embodiments, the disease or disorder is or includes other kidney disorders. In some embodiments, the other kidney disorders are selected from ADPKD, ARPKD, nephronovitis, chronic kidney disease, nephrolithiasis, acute kidney injury, Alagille syndrome, cardiorenal syndrome, renal cell carcinoma, renal osteodystrophy, or a combination thereof.

[0492] In some embodiments, the disease or disorder is or includes a congenital metabolic disorder. In some embodiments, the congenital metabolic disorder is selected from phenylketonuria, urea cycle disorders, maple syrup urine disease, galactosemia, hereditary tyrosinemia, glutaricemia, 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, non-ketotic hyperglycinemia, or a combination thereof.

[0493] In some embodiments, the disease or disorder is or includes a systemic metabolic disorder. In some embodiments, the systemic metabolic disorder is selected from diabetes mellitus, obesity, hypertension, gout, polyneuropathy, hypoglycemia, vitamin B deficiency, cirrhosis, coronary heart disease, stroke, lipodystrophy, or a combination thereof.

[0494] In some embodiments, the disease or disorder is or includes a disorder of the thyroid gland. In some embodiments, the thyroid disorder is selected from Hashimoto's disease, Graves' disease, hypothyroidism, hyperthyroidism, goiter, thyroid nodule, thyroiditis, thyroid cancer, TSH-producing pituitary adenoma, thyroid hormone resistance, MCT8 deficiency, Riedel's thyroiditis, Pendred syndrome, sarcoidosis, McCune-Albright syndrome, familial dysalbuminemia hyperthyroxinemia, thyroxine-binding globulin (TBG) deficiency, or a combination thereof.

[0495] In some embodiments, the disease or disorder is or includes a disorder of the parathyroid gland. In some embodiments, the parathyroid disorder is selected from hyperparathyroidism / hypercalcemia, hypoparathyroidism / hypocalcemia, nephrolithiasis (kidney stones), pancreatitis, granulomatosis, Addison's disease, and pernicious anemia (many of which belong to hyperparathyroidism and hypoparathyroidism).

[0496] In some embodiments, the disease or disorder is or includes a disorder of the inner ear. In some embodiments, the inner ear disorder 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), supraventricular dysplasia syndrome (SCD), drug-induced ototoxicity, otogenic herpes zoster, suppurative labyrinthitis, and vestibular schwannoma.

[0497] In some embodiments, the disease or disorder is or includes a neurological disorder, such as a neurodegenerative disease. In some embodiments, the neurological disorder is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, neuro-AIDS, brain cancer, stroke, brain injury, spinal cord injury, autism, lysosomal storage disorders, fragile X syndrome, hereditary intellectual disability, hereditary ataxia, blindness, paralysis, stroke, traumatic brain injury and spinal cord injury, as well as lysosomal storage disorders, such as MPS I, MPS II, MPS IIIA, MPS IIIB, metachromatic leukodystrophy, Gaucher, Krabbe, Pompe CLN2, Niemann-Pick and Tay-Sachs disease, or a combination thereof.

[0498] In some embodiments, the disease or disorder is or includes a viral infection. In some embodiments, the viral infection includes polyomavirus (e.g., BK virus)-mediated nephropathy.

[0499] Delivery of regulatory nucleic acid agents The delivery of the conjugate modulating nucleic acid agents of this disclosure to cells, for example, to cells within a subject such as a human subject (e.g., a subject susceptible to or diagnosed with metabolic disorders including target RNA-related disorders, e.g., kidney disease, nephropathy, and various other kidney diseases or conditions) can be achieved in a number of different ways. For example, delivery may be carried out by contacting the conjugate modulating nucleic acid agent of this disclosure with cells, either in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition containing the conjugate modulating nucleic acid agent (e.g., polymyxin-conjugated dsRNA) to the subject. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors encoding and directing the expression of the conjugate modulating nucleic acid agent.

[0500] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the conjugate modulated 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 in their entirety by reference). In the case of in vivo delivery, factors to be considered for delivering the conjugate modulated nucleic acid agent include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also demonstrated successful 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; Thakker, 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 enable targeting of conjugate-modulated nucleic acid agents to target tissues and avoid undesirable off-target effects. Conjugate-modulated nucleic acid molecule can be further modified by chemical conjugation to lipophilic groups such as cholesterol, which can promote cellular uptake and prevent degradation. For example, systemic injection of an iRNA agent against ApoB conjugated to a lipophilic cholesterol portion into mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).

[0501] In alternative embodiments, the conjugate-modulating nucleic acid agent may be delivered using a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of the conjugate-modulating nucleic acid agent (the modulating nucleic acid is negatively charged) and also enhance interactions with the negatively charged cell membrane, enabling efficient uptake of the conjugate-modulating nucleic acid agent by cells. Cationic lipids, dendrimers, or polymers may bind to the conjugate-modulating nucleic acid agent or be induced to form vesicles or micelles containing the conjugate-modulating nucleic acid agent (see, for example, Kim SH, et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of the conjugate-modulating nucleic acid agent when administered systemically. Methods for preparing and administering cationic conjugate-modulated nucleic acid agent conjugates are 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 useful for systemic delivery of ASO or iRNA drugs include DOTAP (Sorensen, DR., et al (2003), cited above; Verma, UN, et al., (2003), cited above), "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), and polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Examples include Biotechnol.71659), Arg-Gly-Asp(RGD) peptide (Liu, S.(2006)Mol.Pharm.3:472-487), and polyamidoamine (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 conjugate modulated nucleic acid agent forms a complex with cyclodextrin for systemic administration. Methods for administering iRNA and cyclodextrin and pharmaceutical compositions can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole.

[0502] In some embodiments, the conjugated agent, when delivered to cells, tissues, or organisms, is characterized in that the payload portion is delivered to and / or expressed in 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% more target cells compared to similar cells, tissues, or organisms to which only the unconjugated payload portion was delivered.

[0503] In some embodiments, the conjugate is characterized in that, when delivered to a tissue or organism, the payload portion is delivered to and / or expressed in 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% more target cells compared to non-target cells.

[0504] In some embodiments, the conjugated agent, when delivered to cells, tissues, or organisms, is characterized in that the expression and / or activity of the target of the payload portion is regulated, 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% compared to similar cells, tissues, or organisms other than those to which the unconjugated payload portion was delivered.

[0505] In some embodiments, the disclosure provides a conjugate agent comprising (i) a targeting moiety specific to an internalized cell surface factor, and (ii) a payload moiety containing a nucleic acid agent, wherein the binding moiety and the nucleic acid agent are conjugated to each other by a cleavable linker, so that the conjugate agent is in a first associated state when it is extracellular to a kidney cell and in a second dissociated state when it is intracellular to a cell containing the cell surface factor.

[0506] Medication plan Those skilled in the art will be able to determine the appropriate amount, dose, or dosage of a conjugate to administer to a patient by taking into account factors such as age, weight, general health status, route of administration, nature of symptoms, disease or disorder requiring treatment, and the presence of other drugs, according to known methods. For example, various antibody dosing regimens are described in Hendrikx J et al. (2017) Oncologist 22(10):1212-1221, PMID:28754722, the full contents of which are incorporated herein by reference.

[0507] In some embodiments, the conjugate modulating nucleic acid agent of the present disclosure (e.g., a conjugate agent comprising a targeting moiety and one or more modulating nucleic acid compounds disclosed herein) is administered in a fixed dose, i.e., independently of body weight. In some embodiments, the fixed dose reduces inter-patient variability, e.g., efficacy and / or PK / PD parameters.

[0508] In some embodiments, the modulated nucleic acid agent of this disclosure (e.g., a conjugate agent comprising a targeting moiety and one or more modulated nucleic acid compounds disclosed herein) is administered based on body weight, for example, in a mg / kg dose.

[0509] In some embodiments, the conjugate modulating nucleic acid agent of the Disclosure (e.g., a conjugate agent comprising a targeting moiety and one or more modulating nucleic acid compounds as disclosed herein) is administered in 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, monthly, or at other intervals in between. In some embodiments, the dosing schedule disclosed herein may be repeated one or more times.

[0510] Methods for suppressing target gene expression This disclosure also provides a method for inhibiting the expression of a target gene in cells. The method comprises contacting cells with a conjugate-modulating nucleic acid agent (e.g., a target site-conjugated double-stranded RNA agent) in an amount effective to inhibit the expression of the target gene in the cells, thereby inhibiting the expression of the target gene in the cells.

[0511] Contacting cells with a conjugate-modulating nucleic acid agent (e.g., a targeting partial conjugate double-stranded RNA agent) can be performed in vitro or in vivo. In vivo contact of a conjugate-modulating nucleic acid agent with cells involves contacting the conjugate-modula...

Claims

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

2. The linker is C ２～２２ The nucleic acid conjugate agent according to claim 1, comprising an alkylene or a branched alkylene chain, wherein the carbon atoms of the alkylene chain may be interposed by one or more -O- groups.

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 according to any one of the claims, wherein the nucleic acid conjugate can mediate the enhancement of delivery of the nucleic acid to a target tissue.

6. The nucleic acid conjugate according to any one of claims 1 to 5, characterized in that when the nucleic acid conjugate is delivered to cells, tissues, or targets, an enhancement of the delivery of the nucleic acid to the cells, tissues, or targets is observed compared to a comparator.

7. The nucleic acid conjugate agent according to claim 6, wherein the comparator is similar to the cells, tissues, or subjects described above, except that an unconjugated nucleic acid is delivered.

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

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

10. The nucleic acid conjugate agent according to claim 9, wherein the comparator is similar to the cells, tissues, or subjects described above, except that an unconjugated nucleic acid is delivered.

11. Formula I: It includes a payload portion containing nucleic acids conjugated to the structure, In the formula, R １ is a C3-C7 alkyl or C3-C7 aryl that may be substituted, L is or branch linker, n is an integer from 1 to 16, and AA １ AA ２ AA ３ These are, independently, chemically bonded or as follows: It is an amino acid selected from, Here, X is And, Here, L is an optional linker, and M is, And, Here, Y = O, S, or NSO ２ Me and Z = nucleic acid. The nucleic acid conjugate agent according to any one of the above claims.

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

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

14. The aforementioned R １ but, The conjugate agent according to any one of the above claims.

15. The structure of the above formula I is A conjugate agent according to any one of the above claims, selected from the group consisting of the above.

16. The conjugate agent according to any one of the claims, wherein the conjugate comprises a plurality of structures of formula I, and optionally the nucleic acid conjugate agent comprises at least two, at least three, at least four, or at least five structures of formula I.

17. The multiple structures of formula I described above are conjugated to the payload portion via a branch linker (L), and optionally, the branch linker L is The structure is selected from the group consisting of the following, where compound 1, compound 2, and / or compound 3 independently represent formula I': The conjugate agent according to claim 16, comprising the compound.

18. The conjugating 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 nucleic acid is The conjugate agent according to any one of the claims, which is conjugated to a ligand selected from the group consisting of the following.

21. The conjugate agent according to any one of the claims, wherein the nucleic acid is an antisense sequence element, or comprises an antisense sequence element, and optionally the antisense sequence element is complementary to at least a portion of one or more of the exons, introns, untranslated regions, splice junctions, promoter regions, enhancer regions, or non-coding regions in the target sequence.

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

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

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

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

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

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

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

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

32. The conjugate agent according to claim 31, wherein the DNA is a DNA analog or comprises a DNA analog, and optionally comprises one or more morpholino subunits linked to each other by phosphorus-containing bonds.

33. The conjugate agent according to claim 32, wherein the DNA analog is a phosphorodiamidate morpholino nucleic acid (PMO) or comprises a PMO.

34. The conjugate agent according to claim 33, wherein the PMO comprises about 12 to 40 nucleotides.

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

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

37. The nucleic acids mentioned above are as follows: Modified skeletons, modified nucleic acid bases, modified ribose, modified deoxyribose, or combinations thereof. A conjugate agent according to any one of the claims, comprising a modification including the above.

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

39. The conjugate agent according to any one of the claims, wherein the payload portion is conjugated at the 5' end or 3' end of the payload portion with the remaining portion of the compound of formula I.

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

41. The conjugate agent according to any one of the claims, wherein the nucleic acid comprises one or more phosphorylguanidine-containing skeletons ("PN skeletons") and / or mesylphosphoamidate modifications.

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

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

44. The cell according to claim 43, wherein the cell is selected from kidney cells including the target kidney cell, mammalian cells, human cells expressing megalin, and human cells expressing cubilin.

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

46. A method for regulating the expression of a target gene in a cell, 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 disorder in a subject that is suffering from or is susceptible to such a disease or disorder, 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 cells according to claim 43 or 44.

48. The method according to claim 47, wherein the disease is a disease associated with the expression of a cell surface receptor, and optionally, the disease comprises cells in which both the cell surface receptor and the target recognized by the payload portion are present.

49. A method for improving drug delivery to cells, comprising contacting a system or subject comprising at least one 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.

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 a combination thereof.

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

52. Administering / providing the aforementioned conjugate agent to the aforementioned cells, tissues, or subject is (a) Similar cells, tissues, or subjects, except that the unconjugated payload portion is delivered; (b) non-target cells; or (c) Both (a) and (b) The method according to any one of claims 45 to 51, which delivers the payload portion to at least 5% more target cells compared to the method described above.

53. The method according to claim 52, wherein the target cell is a kidney cell or includes a kidney cell.

54. The method according to claim 52 or 53, wherein the target cells are cells expressing a renal cell surface factor, or include cells expressing a renal cell surface factor, and optionally the renal cell surface factor is megalin or cubilin.

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