Lipid-polynucleic acid conjugate compositions and uses thereof

Abstract

Disclosed herein are polynucleic acid conjugate compositions comprising a polynucleic acid molecule and one or more targeting moiety such as an asialoglycoprotein receptor targeting moiety and a lipophilic moiety, for targeted delivery of the polynucleic acid molecule to a target cell. Disclosed herein are also pharmaceutical compositions comprising polynucleic acid conjugate composition. Further, provided herein are methods and / or uses of polynucleic acid conjugate composition and pharmaceutical composition comprising polynucleic acid conjugate composition described herein.

Description

LIPID-POLYNUCLEIC ACID CONJUGATE COMPOSITIONS AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 661,488, filed on June 18, 2024, and U.S. Provisional Application No. 63 / 767,378 filed on March 5, 2025, each of which is incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0002] Conjugation of a polynucleic acid molecule with targeting moieties is designed and optimized to increase efficiency of targeted delivery. Various conjugations have been designed and tested, yet there is an ongoing effort to develop polynucleic acid conjugate compositions that improve efficiency of targeted delivery and reduce off-target effects without reducing the efficacy of the polynucleic acid molecule, increasing cytotoxicity, or decreasing stability of the polynucleic acid molecule in vivo.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.SUMMARY OF THE DISCLOSURE

[0004] To meet the need for a more effective targeted delivery of polynucleic acid molecules, disclosed herein, in certain aspects, are polynucleic acid conjugate composition, wherein the polynucleic acid conjugate composition comprises a polynucleic acid molecule and one or more targeting moiety. In some instances, the one or more targeting moiety comprises an asialoglycoprotein receptor targeting moiety, a lipophilic moiety, or a combination thereof. In some instances, the one or more targeting moiety consists of an asialoglycoprotein receptor targeting moiety, a lipophilic moiety, and a combination thereof.

[0005] In one aspect, the present disclosure provides a polynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilic moiety.

[0006] In some instances, the lipophilic moiety is a saturated or unsaturated, linear or branched hydrocarbon chain. In some instances, the saturated or unsaturated, linear or branched hydrocarbon chain is a C8-C30 hydrocarbon chains. In some instances, the saturated orunsaturated linear or branched hydrocarbon chain is C8, C12, C14, C16, C18, C20, or C22 hydrocarbon chains. In some instances, the lipophilic moiety comprises a cholesterol or a tocopherol.

[0007] In some instances, the asialoglycoprotein receptor targeting moiety comprises N- Acetylgalactosamine (GalNAc) or galactose. In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the polynucleic acid molecule via a linker. In some instances, the linker comprises formula (IV), formula (XIII’), or formula (XIII”) below,wherein at least one of Y1 or Y2 in formula (IV) is a nucleotide in the polynucleic acid molecule; wherein Z in formula (XIII’) and formula (XIII”) is -H, -OH, -O-Methyl, -F, or -O- methoxyethyl; R in formula (XIII’) and formula (XIII”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; and the in formula (XIII’) and formula (XIII”) is to connect to the next nucleotide of the polynucleic acid molecule.

[0008] In some instances, the linker and the asialoglycoprotein receptor targeting moietywherein Z in formula (V’) or (VI’) is -H, -OH, -O-Methyl, -F, or -O-m ethoxy ethyl; R in formula(V’) or (VI’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; and the is to connect to the next nucleotide of the polynucleic acid molecule.

[0009] In some instances, the polynucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand).

[0010] In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 5’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 5’ end of the passenger strand. In some instances, the 3’ end of the passenger strand comprises an inverted abasic moiety.

[0011] In some instances, the lipophilic moiety is conjugated to the polynucleic acid molecule via a linking moiety. In some instances, the linking moiety is a C6 linker.

[0012] In some instances, the lipophilic moiety is located between the asialoglycoprotein receptor targeting moiety and the polynucleic acid molecule. In some instances, the polynucleic acid conjugate composition comprises a structure of:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and the is to connect to the 5’ end of the polynucleic acid molecule.

[0013] In some instances, the lipophilic moiety is conjugated to the 3’ end of the passenger strand. In some instances, the 3’ end of the passenger strand comprises an inverted abasic moiety. In some instances, the lipophilic moiety is conjugated to the inverted abasic moiety.

[0014] In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 3’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 5’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 3’ end of the passenger strand.

[0015] In some instances, the lipophilic moiety is located between the asialoglycoprotein receptor targeting moiety and the polynucleic acid molecule. In some instances, the polynucleic acid conjugate composition comprises a structure of:

[0016] wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and theis to connect to the 3’ end of the polynucleic acid molecule.

[0017] In some instances, the asialoglycoprotein receptor targeting moiety is located between the polynucleic acid molecule and the lipophilic moiety. In some instances, the polynucleic acid conjugate composition comprises a structure of:

[0018] wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and theis to connect to the 3’ end of the polynucleic acid molecule.

[0019] In another aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleic acid conjugate composition described herein, and a pharmaceutically acceptable excipient.

[0020] In various aspects, the present disclosure provides a method of modulating an expression of an mRNA of a target gene in a cell, comprising: contacting the cell with or administering a subject with the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to the mRNA thereby modulating the expression of the mRNA of the target gene.

[0021] In an aspect, the present disclosure provides a method of treating a disease in a subject, comprising: administering a subject with the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to an mRNA of a gene associated with the onset, development, or a symptom of the disease, thereby treating the disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the disclosure are utilized, and the accompanying drawings below.

[0023] FIG. 1 depicts the % change in serum AGT following a single 2.0 mg / kg subcutaneous dose in cynomolgus monkeys as described in Example 2.DETAILED DESCRIPTION OF THE DISCLOSURE

[0024] Polynucleic acid molecules, such as an inhibitory polynucleic acid molecule, can be utilized to suppress expression of target genes. In some instances, for example, an inhibitory polynucleic acid molecule, e.g., short interfering RNA (siRNA), can act in the RNA interference(RNAi) pathway. Short interfering RNA or siRNA is a double-stranded synthetic RNA that comprises two RNA strands, which are an antisense (or guide) strand and a sense (or passenger) strand. These two RNA strands can form a double-stranded siRNA, and, in some instances, with overhangs at one end of one of the strands or both, or in some instances, with blunt ends at 5’ end, 3’ end, or both. When the siRNA is present in the cell, the siRNA-induced gene silencing is initiated by the assembly of the RNA-induced silencing complex (RISC), which comprises the guide strand and other proteins, such as enzymes. The guide strand directs the RISC to complementary sequence region of the target RNA, which, in some instances, initiates the cleavage of the phosphodiester bond between two nucleotides on the target RNA, thereby generating RNA fragments that are further degraded by cellular enzymes, e.g., exonucleases.

[0025] Polynucleic acid molecule, such as an inhibitory polynucleic acid molecule, can be conjugated, linked, or coupled with targeting moiety for targeted delivery of the polynucleic acid molecule. In some instances, conjugations of the polynucleic acid molecule with targeting moiety can increase uptake efficiency and / or improve delivery to the target cells. In some instances, the conjugations of the polynucleic acid molecule with targeting moiety is designed and optimized to increase stability and efficacy of the polynucleic acid molecule as well as to reduce miRNA-like off target effect. In some instances, the conjugations of the polynucleic acid molecule with targeting moiety can suppress off-target effects caused by a guide strand (or an antisense strand) of the polynucleic acid molecule. In some instances, the conjugations of the polynucleic acid molecule with targeting moiety as described herein can improve stability of the polynucleic acid molecule. In some instances, the conjugations of polynucleic acid molecule can improve uptake efficiency of the polynucleic acid molecule by target cells. In some instances, the conjugations of polynucleic acid molecule can suppress or reduce off-target delivery without compromising the efficacy of the polynucleic acid molecule. In some instances, the conjugations of polynucleic acid molecule can suppress or reduce off-target delivery without increasing cytotoxicity of the polynucleic acid molecule. In some instances, the conjugations of polynucleic acid molecule can suppress or reduce off-target delivery without decreasing stability of the inhibitory polynucleic acid molecule.

[0026] In one aspect, the present disclosure provides a polynucleic acid conjugate composition comprising a polynucleic acid molecule and one or more targeting moiety. In some instances, the one or more targeting moiety targets or delivers the polynucleic acid molecule to a preferred tissue, organ, or cell in the body of the subject. In some instances, the one or more targeting moiety comprises an asialoglycoprotein receptor targeting moiety or a lipophilic moiety. In some instances, the one or more targeting moiety consists of an asialoglycoprotein receptor targeting moiety and a lipophilic moiety. In some aspect, the present disclosure provide apolynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilic moiety.

[0027] In one aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleic acid conjugate composition described herein. In another aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleic acid conjugate composition described herein and a pharmaceutically acceptable excipient.

[0028] In another aspect, the present disclosure provides a method of modulating an expression of an mRNA of a target gene in a cell, comprising: contacting the cell with or administering a subject with the polynucleic acid conjugate composition or the pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to the mRNA thereby modulating the expression of the mRNA of the target gene. Also, provided herein is a method of treating a disease in a subject, comprising: administering a subject with the polynucleic acid conjugate composition or the pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to an mRNA of a gene associated with the onset, development, or a symptom of the disease, thereby treating the disease.Definitions

[0029] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0030] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0031] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0032] “Percent (%) sequence identity” or “Percent (%) identity” with respect to the nucleic acid sequences identified herein is defined as the percentage of nucleic acid in a candidate sequence that are identical with the nucleic acid sequence being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0033] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the polynucleic acid molecules, the polynucleic acid molecule conjugates, the pharmaceutical compositions, the methods and other aspects belong.

[0035] As used herein, the term “complementary” indicates a sufficient degree of complementarity between two nucleic acid molecules that bind stably and specifically to avoid nonspecific binding.

[0036] As used herein, the term “polynucleic acid” and the term “polynucleotide” are interchangeably used to refer a chain of nucleotides. The term “nucleotide” includes a sequence “G” , “C” , “A”, “T”, and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base. In some instances, the “nucleotide” can refer to a modified nucleotide (e.g., with modified sugar moiety, modified base, modified internucleotide linkage, or combination thereof, including, but not limited to 2’ -modified nucleotide, LNA, ENA, BNA, UNA, GNA etc.) In some instances, the “nucleotide” can refer to a modified nucleotide with a non-canonical base (e.g. including, but not limited to, 2- thiouridine, 2-thiothymidine, inosine, 2-aminopurine, 2,6-diaminopurine, dihydrouridine, 4- thiouridine, 4-thiothymidine, 2-thiocytidine).

[0037] As used herein, a “subject” can be any mammal, including a human and a non-human primate.

[0038] The term “condition,” as used herein, includes diseases, disorders, and susceptibilities.

[0039] As used herein, the term “treat,” “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another instance, “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another instance, “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0040] The terms “prevent,” “preventing,” and “prevention,” as used herein, refer to a decrease in the occurrence of pathology of a condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition. The prevention may be complete, e.g., the total absence of pathology of a condition in a subject. The prevention may also be partial, such that the occurrence of pathology of a condition in a subject is less than that which would have occurred without the present disclosure.

[0041] “Administering” and its grammatical equivalents as used herein can refer to providing pharmaceutical compositions described herein to a subject or a patient. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the composition to the subject, depending upon the type of disease to be treated or the site of the disease. For example, the composition can be administered, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or via infusion. One or more such routes can be employed.

[0042] The terms “pharmaceutical composition” and its grammatical equivalents as used herein can refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or a therapeutic agent to be administered to a subject, e.g., a human in need thereof.

[0043] The term “pharmaceutically acceptable” and its grammatical equivalents as used herein can refer to an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. “Pharmaceutically acceptable” can refer a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained.

[0044] A “pharmaceutically acceptable excipient” refers to an excipient that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activitythereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0045] The term “therapeutic agent” can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents can also be referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0046] As used herein, the term “sense strand” can be interchangeably used with the term “passenger strand”, and the tern “antisense strand” can be interchangeably used with the term “guide strand”. In some instances, a nucleic acid sequence described herein for a sense strand and a passenger strand can be interchangeably used. Also, in some instances, a nucleic acid sequence described herein for an antisense strand and a guide strand can be interchangeably used.

[0047] As used herein, the term “consecutive nucleotides” refers to a sequence contains a number of consecutive nucleotides from a reference sequence. For example, if a reference sequence is N1N2N3N4N5N6N7, a consecutive nucleotides can be N1N2N3N4 or N3N4N5N6, but a sequence of N1N3N4N5 or N3N4N7 cannot be a consecutive nucleotides.

[0048] As used herein, the term “negative control” refers to a subject or a cell receiving no treatment or placebo.

[0049] It is appreciated that certain features of the polynucleic acid molecules, and / or polynucleic acid molecule conjugates, pharmaceutical composition comprising the polynucleic acid molecules or the polynucleic acid molecule conjugates, methods and other aspects, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the polynucleic acid molecules, and / or polynucleic acid molecule conjugates, pharmaceutical composition comprising the polynucleic acid molecules or the polynucleic acid molecule conjugates, methods and other aspects, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present polynucleic acid molecules, and / or polynucleic acid molecule conjugates, pharmaceutical composition comprising the polynucleic acid molecules orthe polynucleic acid molecule conjugates, methods and other aspects and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Components of a polynucleic acid conjugate

[0050] In one aspect, provided herein is a polynucleic acid conjugate composition comprising a polynucleic acid molecule and one or more targeting moiety. In some instances, the one or more targeting moiety comprises an asialoglycoprotein receptor targeting moiety and / or a lipophilic moiety. In certain aspect, the present disclosure provide a polynucleic acid conjugate comprising a polynucleic acid molecule conjugated with an asialoglycoprotein receptor targeting moiety and a lipophilic moiety.Polynucleic Acid Molecule

[0051] A polynucleic acid molecule, e.g., an inhibitory polynucleic acid molecule, can be utilized to suppress expression of a target gene. In some instances, the inhibitory polynucleic acid molecule comprises a single-stranded inhibitory polynucleic acid molecule. In some aspects, the polynucleic acid molecule is a single-stranded nucleic acid molecule that hybridizes to certain regions of mRNA of the target gene. In some instances, the single-stranded inhibitory polynucleic acid molecule is an antisense oligonucleotide (ASO).

[0052] In some instances, the inhibitory polynucleic acid molecule comprises a double-stranded inhibitory polynucleic acid molecule. In some instances, the inhibitory polynucleic acid molecule comprises a dsRNA or an siRNA. In some instances, the double-stranded inhibitory polynucleic acid molecule comprises a guide strand (an antisense strand) or a passenger strand (a sense strand).

[0053] In some instances, the polynucleic acid molecule is an siRNA. In some instances, the polynucleic acid molecule, e.g., siRNA, comprises a passenger strand (a sense strand) and a guide strand (an antisense strand), and wherein the guide strand hybridizes to a certain region of mRNA of the target gene.

[0054] In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand, each of which is about 15-30, 16-30, 17-30, 18-30, 18-27, 18-25, 18- 23, 19-23, 20-23, or 21-23 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand, each of which is about 15, 16, 17, 18, 19, 20 nucleotides long. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand, each of which is about 21, 22, 23, 24, 25 nucleotides long. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand, each of which is about 26, 27, 28, 29, 30 nucleotides long. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand of 19 nucleotides long, and a guide strand of about 21 nucleotides long. In some aspects, thepolynucleic acid molecule described herein comprises a passenger strand of 21 nucleotides long, and a guide strand of about 23 nucleotides long.

[0055] In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 3’ overhang on the guide strand. In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 5’ overhang on the guide strand. In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 3’ overhang on the passenger strand. In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 5’ overhang on the passenger strand.Modifications of Polynucleic Acid Molecule

[0056] In some instances, the polynucleic acid molecule is modified in one or more different structures of the polynucleotide acid molecule described herein (e.g., modifications on sugar ring(s), backbone(s) or internucleotide linkage(s), base(s)). In some instances, the polynucleic acid molecule comprises a modified nucleotide, which is a nucleotide with modifications on sugar ring(s), backbone(s) or internucleotide linkage(s), and / or base(s). In some instances, the polynucleic acid molecule is further modified to incorporate a modified nucleotide described herein.Sugar -modified nucleotides

[0057] In some aspects, the polynucleotide acid molecule described herein comprises one or more sugar-modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2’-fluoro modified nucleotide. In some instances, the 2’-fluoro modified nucleotide comprises thiomodified base containing nucleotide, e.g., 2'-fluoro-2-thiouridine-3'-phosphate (U3f). In some instances, the sugar-modified nucleotide includes, but is not limited to, a modification at a 2’ hydroxyl group of the ribose moiety. In some instances, the sugar-modified nucleotide includes modification with an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Examples of the 2’ -fluoro modified nucleotide, include, but are not limited to, 2'- fluoroadenosine-3 '-phosphate (Af); 2’-fluorocytidine-3’-phosphate (Cf); 2'-fluoroguanosine-3'- phosphate (Gf); or 2'-fluorouridine-3 '-phosphate (Uf).

[0058] In some aspects, the sugar-modified nucleotide is a 2’-O-methyl modified nucleotide or 2’-alkoxy modified nucleotide (e.g., 2’-methoxy modified nucleotide). In some instances, 2' hydroxyl group modification includes 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA). In some instances, the alkyl moiety comprises a hetero substitution. In some instances, the carbonof the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some aspects, the sugar-modified nucleotide is a 2’- amino modified nucleotide. In some aspects, the sugar- modified nucleotide is a 2’- azido modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2’- deoxy modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2’-O-methoxythyl (2’ -MOE). In some aspects, the sugar-modified nucleotide is a locked nucleic acid (LNA). In some aspects, the sugar-modified nucleotide is an ethylene-bridged nucleic acid (ENA). In some aspects, the sugar-modified nucleotide is a (S)-constrained ethyl (cEt). In some aspects, the sugar-modified nucleotide is a tricyclo-DNA (tcDNA). In some aspects, the sugar-modified nucleotide is a 2’-NH2 nucleic acid. Examples of the 2’-O-methyl modified nucleotide include, but are not limited to, 2’ -O-methyladenosine-3’ -phosphate (a); 2’- O-methylcytidine-3 ’-phosphate (c); 2 ’-O-methylguanosine-3’ -phosphate (g); or 2’-O- methyluridine-3’ -phosphate (u).

[0059] In some instances, the 2’-O-methyl modified nucleotide comprises 5 ’(E) vinylphosphonate-2-O-methyluridine-3 ’phosphate (vpu). In some instances, the amidite structure of 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) is shown as below:

[0060] In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3 ’phosphate (vpu) is incorporated into the polynucleic acid molecule. In some instances, the 5 ’(E) vinylphosphonate- 2-O-methyluridine-3 ’phosphate (vpu) is incorporated into the siRNA. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3 ’phosphate (vpu) is incorporated into a guide strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3 ’phosphate (vpu) is incorporated into a passenger strand. In some instances, the 5 ’(E) vinylphosphonate-2-O- methyluridine-3 ’phosphate (vpu) is located at the 5’ end of a guide strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3 ’phosphate (vpu) is located at the 5’ end of a passenger strand. In some instances, an incorporated 5 ’(E) vinylphosphonate-2-O- methyluridine-3 ’phosphate (vpu) has a structure shown as below:

[0061] In some instances, the 5 ’(E) vinylphosphonate-2-O-methyluridine-3 ’phosphate (vpu) substitutes one or more nucleotide in siRNA. In some instances, the 5 ’(E) vinylphosphonate-2- O-methyluridine-3 ’phosphate (vpu) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 5 ’(E) vinylphosphonate-2-O-methyluridine- 3 ’phosphate (vpu) substitutes one or more nucleotide in a passenger strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’ phosphate (vpu) substitutes one or more the nucleotide in a guide strand.

[0062] In some aspects, the polynucleotide acid molecule described herein comprises one or more sugarphosphate-modified nucleotide. In some aspects, the modified sugarphosphate is phosphorodiamidate morpholino (PMO). In some aspects, the modified sugarphosphate is phosphoramidate. In some instances, the heterocyclic substitution includes imidazole, and pyrrolidino. In some aspects, the modified sugarphosphate is thiophosphoramidate. In some aspects, the modified sugarphosphate is peptide nucleic acid (PNA).Modi fied backbones or internucleotide linkages

[0063] In some aspects, the polynucleotide acid molecule described herein comprises one or more backbone-modified nucleotide. In some aspects, the modified backbone is a methylphosphonate. In some aspects, the modified backbone is phosphorothioate (s). In some aspects, the modified backbone is a guanidinopropyl phosphoramidate. In some aspects, the modified backbone is a mesyl-phosphoramidate (MsPA) linkages. In some instances, the modified backbone comprises one or more of phosphorodithioates, methylphosphonates, 5’- alkylenephosphonates, 5 ’-methylphosphonate, 3 ’-alkylene phosphonates, borontrifluoridates, borano phosphate esters and selenophosphates of 3’-5’ linkage or 2’-5' linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate linkages, alkyl phosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphoramidates.

[0064] In some aspects, the modified backbone is phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage is a stereochemically enriched phosphorothioateinternucleotide linkage. In certain aspects, the strand contains at least one stereochemically enriched phosphorothioate internucleotide linkage. In some aspects, the strand comprises at least 1, 2, 3 stereochemically enriched phosphorothioate internucleotide linkages. In some aspects, the strand comprises only 1, 2, 3, or 4 stereochemically enriched phosphorothioate internucleotide linkages. In further aspects, at least one (e.g., one or two) stereochemically enriched phosphorothioate internucleotide linkage is disposed between two consecutive nucleosides that are two of six 5’ end nucleosides of the strand. In yet further aspects, at least one (e.g., one or two) stereochemically enriched phosphorothioate internucleotide linkage is disposed between two consecutive nucleosides that are two of six 3’ end nucleosides of the strand. In still further aspects, one stereochemically enriched phosphorothioate is covalently bonded to the first nucleoside and the second nucleoside from the 5’ end within the strand. In some aspects, one stereochemically enriched phosphorothioate internucleotide linkage is covalently bonded to the twenty first nucleoside and the twenty second nucleoside from the 5’ end within the strand. In certain aspects, one stereochemically enriched phosphorothioate internucleotide linkage is covalently bonded to the twenty second nucleoside and the twenty third nucleoside from the 5’ end within the strand. In particular aspects, the stereochemically enriched phosphorothioate internucleotide linkage has Rp stereochemical identity. In certain aspects, the stereochemically enriched phosphorothioate internucleotide linkage has 5p stereochemical identity. In some instances, the modified backbone connects the linker and the targeting moieties to the polynucleic acid molecule.

[0065] In some aspects, the modified backbone comprises 3 ’phosphorothioate, Rp diastereomer ( ?p-enriched phosphorothioate or Rps), 3 'phosphorothioate, Sp diastereomer fS'p-enriched phosphorothioate or Sps), or combinations thereof. In some instances, an incorporated 3 ’phosphorothioate, Rp diastereomer ( ?p-enriched phosphorothioate or Rps) has a structure shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:

[0066] In some instances, an incorporated Sp diastereomer (Sp-enriched phosphorothioate or Sps) has a structure shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:

[0067] In some aspects, the polynucleic acid molecule described herein comprises one or more (e.g., from 1 to 20, from 1 to 10, or from 1 to 5) stereochemically enriched (e.g., internucleoside) phosphorothioates (e.g., having diastereomeric excess of at least 10%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., up to about 99%, for the P-stereogenic center). The polynucleic acid molecule described herein comprises one or more (e.g., from 1 to 20, from 1 to 10, or from 1 to 5; e.g., intemucleoside) phosphorodithioates. The phosphorodithioates may be non-P-stereogenic in the polynucleic acid molecule described herein. Phosphorothioates and phosphorodithioates may enhance the stability of the polynucleic acid molecule described herein to exonuclease activity of serum. Non-P-stereogenic phosphorodithioates may simplify the synthesis of the polynucleic acid molecule described herein by reducing the number of possible diastereomers. Typically, the phosphorothioate or phosphorodithioate may connect two contiguous nucleosides within the six 3’ end nucleosides and the six 5’ end nucleosides of the polynucleic acid molecule described herein.

[0068] In some aspects, the stereochemically enriched phosphorothioate internucleotide linkages (e.g., 7?p-enriched phosphorothioate or 5p-enriched phosphorothioate) connect the three nucleosides at the 5’ end of the nucleic acid (positions 1, 2, 3 from the 5’ end) and three nucleosides at the 3’ end (positions 1, 2, 3 from the 3’ end) of the nucleic acid. In some aspects, the stereochemically enriched phosphorothioate internucleotide linkages (e.g., 7?p-enriched phosphorothioate or 5p-enriched phosphorothioate) connect the three nucleosides at the 5’ end of the guide strand and three nucleosides at the 3’ end of the guide strand. In certain aspect, the stereochemically enriched phosphorothioate internucleotide linkage (e.g., k-enriched phosphorothioate or 5p-enriched phosphorothioate) may be covalently bonded to the first nucleoside (e.g., the 3 ’-carbon atom of the first nucleoside) and the second nucleoside (e.g., the5 ’-carbon atom of the second nucleoside) from the 5’ end of the guide strand. In some instances, the stereochemically enriched phosphorothioate (e.g., Ap-enriched phosphorothioate or 5p-enriched phosphorothioate) may be covalently bonded to the second nucleoside (e.g., the 3 ’-carbon atom of the second nucleoside) and the third nucleoside (e.g., the 5 ’-carbon atom of the third nucleoside) from the 5’ end of the guide strand. Additionally or alternatively, the stereochemically enriched phosphorothioate (e.g., Ap-enriched phosphorothioate or 5p-enriched phosphorothioate) may be covalently bonded to the 21stnucleoside from the 5’ end or the 3rdnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 21stnucleoside from the 5’ end, or the 3 ’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 22ndnucleoside from the 5’ end, or the 5 ’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand. Further, additionally or alternatively, the stereochemically enriched phosphorothioate (e.g., 5p- enriched phosphorothioate or Ap-enriched phosphorothioate) may be covalently bonded to the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 22ndnucleoside from the 5’ end, or the 3 ’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 23rdnucleoside from the 5’ end, or the 5 ’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand.

[0069] Combinations of a specific 5’ stereochemically enriched phosphorothioate internucleotide linkage and a specific 3’ stereochemically enriched phosphorothioate internucleotide linkage in specific locations within the double stranded siRNA can provide beneficial effect to the function of the siRNA. For example, specific 5’ stereochemically enriched phosphorothioate internucleotide linkages and specific 3’ stereochemically enriched phosphorothioate intemucleotide linkages in specific locations within the double stranded siRNA can produce superior efficacy and / or duration of action, e.g., as measured by the reduction in the activity of the target relative to a reference guide strand that lacks the combination of a 5’ stereochemically enriched phosphorothioate and a 3’ stereochemically enriched phosphorothioate internucleotide linkages. In some instances, the double stranded siRNA comprises: (i) Ap-enriched phosphorothioate or A'p-enriched phosphorothioate covalently bonded to the first nucleoside from the 5’ end (e.g., the 3 ’-carbon atom of the first nucleoside from the 5’ end) and the second nucleoside from the 5’ end (e.g., the 5 ’-carbon atom of the second nucleoside from the 5’ end); (ii) Ap-enriched phosphorothioate or 5p-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3’ carbon atom of the second nucleoside from the 5’ end) and the third nucleoside from the 5’ end (e.g., the 5 ’-carbon atom of the 3rdnucleoside from the 5 ’-end); (iii) Ap-enriched phosphorothioate or5p-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rdnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 21stnucleoside from the 5’ end, or the 3 ’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 22ndnucleoside from the 5’ end or the 5 ’-carbon atom of the 2ndnucleoside from the 3’ end); and (iv) 7?p-enriched phosphorothioate or 5p-enriched phosphorothioate covalently bonded to the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3 ’-carbon atom of the 22ndnucleoside from the 5’ end, or the 3 ’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’-end or the 1stnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 23rd nucleoside from the 5’ end, or the 5’-carbon atom of the 1stnucleoside from the 3’ end) in the guide strand

[0070] In some instances, the modified backbone is phosphorodithioate, e.g., 3’phosphorodithioate (ss). In some instances, an incorporated 3 ’phosphorodithioate (ss) has a structure shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:5’"T" Base

[0071] In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkage and one or more phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss).Base-modified nucleotides

[0072] In some aspects, the polynucleic acid molecule described herein comprises one or more purine modification. In some aspects, the purine modification described herein is 2,6- diaminopurine. In some aspects, the purine modification described herein is 3 -deaza-adenine. In some aspects, the purine modification described herein is 7-deaza-guanine. In some aspects, the purine modification described herein is 8-azido-adenine.

[0073] In some aspects, the purine modification comprises a modified guanine (e.g., inosine) or one or more of any types of unnatural nucleic acids.

[0074] In some aspects, the polynucleic acid molecule described herein comprises one or more pyrimidine modification. In some aspects, the pyrimidine modification described herein is 2-thio-thymidine. In some aspects, the pyrimidine modification described herein is 5- carboxamide-uracil. In some aspects, the pyrimidine modification described herein is 5-methyl- cytosine. In some aspects, the pyrimidine modification described herein is 5-ethynyl uracil.

[0075] In some embodiment, the polynucleic acid molecule described herein comprises an abasic substitution. In those cases where a hybridized polynucleotide construct is contemplated for use as siRNA, a reduction of miRNA-like off-target effects is desirable. The inclusion of one or more (e.g., one or two) abasic substitutions in the hybridized polynucleotide constructs may reduce or even eliminate miRNA-like off-target effects, as the abasic substitutions lack nucleobases that are capable of engaging in base-pairing interactions and alleviate steric hindrance. Thus, the polynucleic acid molecule disclosed herein comprises one or more (e.g., one or two) abasic substitutions. In some aspects, abasic substitution is at the 5thnucleotide from the 5’ end of the guide strand described herein. In some aspects, abasic substitution is at the 7thnucleotide from the 5’ end of the guide strand described herein.

[0076] When the polynucleic acid molecule disclosed herein comprises two or more of the abasic substitutions, their structures may be same or different. In certain aspects, a passenger strand contains one abasic substitution (e.g., a guide strand may be free of abasic substitutions). In other aspects, a guide strand contains one abasic substitution (e.g., a passenger strand may be free of abasic substitutions). In yet other aspects, a guide strand contains one abasic substitution, and a passenger strand contains one abasic substitution. In further aspects, a passenger strand includes an abasic substitution between a nucleoside number (x) and a nucleoside number (x+1), where x is an integer from 2 to 7. In yet further aspects, a guide strand includes an abasic substitution between a nucleoside number (x) and a nucleoside number (x+1), where x is an integer from 2 to 7.

[0077] The abasic substitution may be of formula (III):whereL is a sugar analogue, or is substituted with a heteroacyl from A, U ,C, G, or is any other substituted nucleic acid (e.g., locked or unlocked nucleic acid, glycol nucleic acid, etc.; each X4is independently O or S; each X5is independently O, S, NH, or a bond; each R9is independently H, optionally substituted Ci-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted (C1-9 heterocyclyl)-Ci-6-alkyl,optionally substituted (Ce-io aryl)-Ci-6-alkyl, optionally substituted (C3-8 cycloalkyl)-Ci-6-alkyl, - LinkA(-T)P, or a conjugation moiety; each LinkA is independently a multivalent linker (e.g., including -C(O)-N(H)-); each T is independently an auxiliary moiety;R10is a bond to a 3 ’-carbon atom of a nucleoside (x) in the strand;R11is a bond to a 5’-oxygen atom of a nucleoside (x+1) in the strand; p is an integer from 1 to 6; and t is an integer from 1 to 6.

[0078] In some aspects, the abasic substitution described herein is attached to the guide strand of the polynucleic acid molecule described herein. In particular aspects, an abasic substitution (e.g., an internucleotide, abasic spacer of formula (III) in which t is 1) may be included in the guide strand described herein (e.g., within the seed region of the guide strand). In some aspects, an abasic substitution (e.g., an intemucleotide, abasic spacer of formula (III) in which t is 1) may be bonded to the 3’ carbon atom of the second, third, fourth, or fifth nucleoside from the 5’ end of the guide strand described herein. In certain aspects, an abasic substitution (e.g., an internucleotide, abasic spacer of formula (III) in which t is 1) may be bonded to the 3’ carbon atom of the thirteenth, fourteenth, fifteenth, or sixteenth nucleoside from the 5’ end of the guide strand described herein. In some aspects, an abasic substitution fourth, fifth, sixth, seventh, eighth, and / or ninth nucleoside from the 5’ end of the guide strand described herein.Targeting Moiety

[0079] In certain aspects, the polynucleic acid molecule described herein is conjugated with one or more targeting moiety to form a polynucleic acid conjugate composition. In some instances, the one or more targeting moiety is selected based on its ability to target the conjugate composition described herein to a desired cell population, tissue, or an organ selectively or preferably. In some instances, the one or more targeting moiety targets the cell, tissue, or an organ that expresses the corresponding binding partner (e.g., either the corresponding receptor or ligand) of the targeting moiety. For example, the polynucleic acid molecule conjugated with N- acetyl galactosamine (GalNAc) can target hepatocytes expressing asialoglycoprotein receptor (ASGPR).

[0080] In some instances, the one or more targeting moiety is designed to improve targeted delivery to the target cells or tissues. In some instances, the one or more targeting moiety comprises an asialoglycoprotein receptor targeting moiety or a lipophilic moiety. In some instances, the one or more targeting moiety consists of an asialoglycoprotein receptor targeting moiety and a lipophilic moiety.Asialoglycoprotein Receptor Targeting Moiety

[0081] In some instances, the one or more targeting moiety comprises an asialoglycoprotein receptor targeting moiety. In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the polynucleic acid molecule to improve targeted delivery to hepatocytes. Asialoglycoprotein receptors (ASGPR), which is primarily expressed on hepatocytes and minimally expressed on non-hepatic cells, facilitates internalization by clatherin-mediated endocytosis. In some instances, the asialoglycoprotein receptor exhibits high affinity for the asialoglycoprotein receptor targeting moiety. In some instances, the asialoglycoprotein receptor exhibits high affinity for carbohydrates, e.g., galactose, N-Acetylgalactosamine (GalNAc), or glucose, glycoproteins, glycopeptides, or derivatives thereof.

[0082] In some instances, the asialoglycoprotein receptor targeting moiety comprises carbohydrate. In some instances, the carbohydrate comprises a monosaccharide or a polysaccharide. In some instances, the monosaccharide comprises galactose, glucose, or derivatives thereof. In some instances, the galactose derivative comprises N- Acetylgalactosamine (GalNAc). In some instances, asialoglycoprotein receptor targeting moiety comprises galactose modified polymers, galactose-containing glycoprotein, or galactose- containing glycopeptide. In some instances, the asialoglycoprotein receptor targeting moiety comprises N-Acetylgalactosamine (GalNAc) or galactose.

[0083] In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the polynucleic acid molecule via a linker. In some instances, the linker comprises formula (I), (II), (IV), (IV’), (IV”), (IV’”), or (IV””) as described herein.

[0084] In some aspects, the polynucleic acid molecules described herein comprises a passenger strand or a guide strand bonded to at least one group of formula (I):or a salt thereof, or a stereoisomer thereof, where each X1is independently O or S; each X2is independently O, S, NH, or a bond;MOIETY is optionally substituted C2-10 alkane-tetrayl or a group -M1-M2-M3-, wherein each M1and each M3is independently absent or optionally substituted C1-6 alkylene, and M2isoptionally substituted C3-9 heterocycle-tetrayl, optionally substituted Ce-io arene-tetrayl, or optionally substituted C3-8 cycloalkane-tetrayl; each R1and each R2is independently H, optionally substituted Ci-16 alkyl, optionally substituted C2-16 heteroalkyl, a conjugation moiety, or -LinkA(-T)P, provided that at least one R1or at least one R2is a conjugation moiety or -LinkA(-T)P; each R3is independently H, optionally substituted Ci-16 alkyl, optionally substituted C2-16 heteroalkyl, optionally substituted C2-16 alkenyl, optionally substituted C2-16 alkynyl, optionally substituted (C1-9 heterocyclyl)-Ci-6-alkyl, optionally substituted (Ce-io aryl)-Ci-6-alkyl, optionally substituted (C3-8 cycloalkyl)-Ci-6-alkyl, a conjugation moiety, or -LinkA(-T)P;R4is H, optionally substituted C1-6 alkyl, -LinkA(-T)P, or -Sol; each LinkA is independently a multivalent linker (e.g., including -C(O)-N(H)- (e.g., at least one multivalent linker including -C(O)-N(H)- bonded to T)); each T is independently an auxiliary moiety;Sol is solid support; m is an integer from 1 to 6; each n is independently 0 or 1; each p is independently an integer from 1 to 6; and q is an integer from 0 to 3.The at least one group of formula (I) may be bonded to a 5’ end, 3’ end, internucleoside phosphate, intemucleoside phosphorothioate, or internucleoside phosphorodithioate of the polynucleotide. When the at least one group of formula (I) is bonded to the intemucleoside phosphate, intemucleoside phosphorothioate, or intemucleoside phosphorodithioate, q is 0. The polynucleotide construct contains no more than one Sol.

[0085] Group -LinkA- can include, but not limit to, from 0 to 3 multivalent monomers (e.g., optionally substituted Cl -6 alkane-triyl, optionally substituted Cl -6 alkane-tetrayl, or trivalent nitrogen atom) and one or more divalent monomers (e.g., from 1 to 40), where each divalent monomer is independently optionally substituted Cl -6 alkylene; optionally substituted C2-6 alkenylene; optionally substituted C2-6 alkynylene; optionally substituted C3-8 cycloalkylene; optionally substituted C3-8 cycloalkenylene; optionally substituted C6-14 arylene; optionally substituted Cl -9 heteroarylene having 1 to 4 heteroatoms selected from N, O, and S; optionally substituted Cl -9 heterocyclylene having 1 to 4 heteroatoms selected from N, O, and S; imino; optionally substituted N; O; or S(0)m, wherein m is 0, 1, or 2. In some aspects, each monomer is independently optionally substituted Cl-6 alkylene; optionally substituted C3-8 cycloalkylene; optionally substituted C3-8 cycloalkenylene; optionally substituted C6-14 arylene; optionally substituted Cl -9 heteroarylene having 1 to 4 heteroatoms selected from N, O,and S; optionally substituted Cl -9 heterocyclylene having 1 to 4 heteroatoms selected from N, O, and S; imino; optionally substituted N; O; or S(0)m, where m is 0, 1, or 2 (e.g., m is 2). In certain aspects, each monomer is independently optionally substituted Cl -6 alkylene; optionally substituted C3-8 cycloalkylene; optionally substituted C3-8 cycloalkenylene; optionally substituted C6-14 arylene; optionally substituted Cl-9 heteroarylene having 1 to 4 heteroatoms selected from N, O, and S; optionally substituted Cl-9 heterocyclylene having 1 to 4 heteroatoms selected from N, O, and S; optionally substituted N; O; or S(0)m, where m is 0, 1, or 2 (e.g., m is 2). The non-bioreversible linker connecting the auxiliary moiety to the conjugating moiety or to the reaction product thereof can include from 2 to 500 (e.g., from 2 to 300 or from 2 to 200) of such monomers. Group -LinkA- may include a poly(alkylene oxide) (e.g., polyethylene oxide, polypropylene oxide, poly(trimethylene oxide), polybutylene oxide, poly(tetramethylene oxide), and diblock or triblock co-polymers thereof). In some aspects, the non-bioreversible linker includes polyethylene oxide (e.g., poly(ethylene oxide) having a molecular weight of less than 1 kDa).

[0086] Group -LinkA(-T)p in formula (I) may be prepared by a process described in the sections below. In some instances, -LinkA(-T)p is of formula (II):_Q1_Q2([_Q3-Q4_Q5]s_Q6_T)p,(II) where each s is independently an integer from 0 to 20 (e.g., from 0 to 10), where the repeating units are the same or different;Q1is a conjugation linker (e.g., [-Q3-Q4-Q5]s-Qc-, where Qcis optionally substituted C2-12 heteroalkylene (e.g., a heteroalkylene containing -C(O)-N(H)-, -N(H)-C(O)-, -S(O)2- N(H)-, or -N(H)-S(O)2-), optionally substituted C1-12 thioheterocyclylene (e.g.,substituted C1-12 heterocyclylene (e.g., l,2,3-triazole-l,4-diylcyclobut-3-ene-l,2-dione-3,4-diyl, or pyrid-2-yl hydrazone);Q2is a linear group (e.g., [-Q3-Q4-Q5]s-), if p is 1, or a branched group (e.g., [-Q3-Q4- Q5]S-Q7([-Q3-Q4-Q5]S-(Q7)PI)P2, where pl is 0 or 1, p2 is 0, 1, 2, or 3), if p is an integer from 2 to 6; each Q3and each Q6is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; each Q4is independently absent, optionally substituted C1-12 alkylene, optionally substituted C2-12 alkenylene, optionally substituted C2-12 alkynylene, optionally substituted C2-12 heteroalkylene, optionally substituted Ce-io arylene, optionally substituted C1-9 heteroarylene, or optionally substituted C1-9 heterocyclylene; each Q5is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -CH2-, -C(O)O-, - OC(O)--C(O)NH-, -NH-C(O)-, -NH-CH(Ra)-C(O)-, or -C(O)-CH(Ra)-NH-; each Q7is independently optionally substituted C1-6 alkane-triyl, optionally substituted C1-6 alkane-tetrayl, optionally substituted C2-6 heteroalkane-triyl, or optionally substituted C2-6 heteroalkane-tetrayl; and each Rais independently H or an amino acid side chain; provided that at least one of Q3, Q4, and Q5is present.

[0087] In some aspects, each Q4is independently absent, optionally substituted C1-12 alkylene, optionally substituted C2-12 alkenylene, optionally substituted C2-12 alkynylene, optionally substituted C2-12 heteroalkylene, or optionally substituted C1-9 heterocyclylene. In certain aspects, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0088] Thus, in formula (II), LinkA may include a single branching point, if each pl is 0, or multiple branching points, if at least one pl is 1.

[0089] In formula (II), Q1may be -O-QL-QC-, where QLis optionally substituted C2-12 heteroalkylene, optionally substituted C1-12 alkylene, or -(optionally substituted C1-6 alkylene)- (optionally substituted Ce-io arylene)-. In some aspects, QLis optionally substituted C2-12 heteroalkylene or optionally substituted C1-12 alkylene. In formula (II), Qcmay be:

[0090] In formula (II), Q2may be a linear group of formula [-Q3-Q4-Q5]s-, where Q3, Q4, and Q5are as defined for formula (II). Alternatively, Q2may be a branched group [-Q3-Q4-Q5]s- Q7([-Q3-Q4-Q5]s-(Q7)pi)P2, where each Q7is independently optionally substituted C1-6 alkane-triyl, optionally substituted Ci-6 alkane-tetrayl, optionally substituted C2-6 heteroalkane-triyl, or optionally substituted C2-6 heteroalkane-tetrayl; where pl is 0 or 1; p2 is 0, 1, 2, or 3; where, when pl is 0, LinkA is a trivalent or tetraval ent linker, and, when pl is 1, LinkA is a tetraval ent, pentavalent, or hexavalent linker.In certain aspects, pl is 0.In some aspects, Q7is:

[0091] Compounds that may be used in the preparation of group -LinkA(-T)p in formula (I) are described herein as well as in WO 2015 / 188197. Non-limiting examples of -LinkA include:whereR18is a bond to MOIETY, each R19is independently a bond to auxiliary moiety, each m5 is independently an integer from 1 to 20, each m6 is independently an integer from 1 to 10, m7 is an integer from 1 to 6, and each X6is independently O or S.In formula (II), when the conjugation linker is of formula [-Q3-Q4-Q5]s-Qc-, -Q2([-Q3- Q4-Q5]S-Q6-T)Pmay be:(xxv) whereR20is a bond to Qcin Q1, each R19is independently a bond to an auxiliary moiety, each m5 is independently an integer from 1 to 20, each m6 is independently an integer from 1 to 10, m7 is an integer from 1 to 6, and each X6is independently O or S.

[0092] In some aspects, the linker described herein is cleavable. In some aspects, the linker described herein is non-cleavable.

[0093] In some aspects, the polynucleic acid molecule described herein comprises a guide strand or a passenger strand bonded to a linker of formula (IV),(IV), wherein at least one of Y1 or Y2 is a nucleotide in the polynucleic acid molecule, and wherein the is connected to an asialoglycoprotein receptor targeting moiety, e.g., GalNAc.In some instances, the linker comprises formula (IV). In some instances, the linker comprises formula (IV), wherein the at least one of Y1 or Y2 is a nucleotide in the polynucleic acid molecule.

[0094] In some instances, the Y1 is the last nucleotide on the 3’ end or the first nucleotide on the 5’ end of one of the strands of the polynucleic acid molecule. In some instances, the Y1 is the last nucleotide on the 3’ end or the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule . In some instances, the Y1 is the last nucleotide on the 3’ end or the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule, and the Y2 is a 3 -hydroxy -propoxy group. In some instances, the Y2 is the first nucleotide on the 5’ end or the last nucleotide on the 3’ end of one of the strands of the polynucleic acid molecule. In some instances, the Y2 is the first nucleotide on the 5’ end or the last nucleotide on the 3’ end of the passenger strand of the polynucleic acid molecule. In some instances, the Y2 is the first nucleotide on the 5’ end or the last nucleotide on the 3’ end of the passenger strand of thepolynucleic acid molecule, and the Y1 is a 3 -hydroxy -propoxy group. In other instances, the Y1 and Y2 are two consecutive nucleotides in one of the strands of the polynucleic acid molecule.

[0095] In some instances, the polynucleic acid molecule described herein comprises a guide strand or a passenger strand coupled or bonded to a linker of formula (XIII’) or formula (XIII”),wherein Z in formula (XIII’) and formula (XIII”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), R in formula (XIII’) and formula (XIII”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and the is to connect to the next nucleotide of the polynucleic acid molecule.

[0096] In some instances, the linker (e.g., formula (XIII”)) is conjugated to the 5’ end of the passenger strand via a phosphodiester internucleotide linkage. In some instances, the linker (e.g., formula (XIII’)) is conjugated to the 5’ end of the passenger strand via a phosphorothioate internucleotide linkage. In some instances, the linker (e.g., formula (XIII’) or formula (XIII”)) is conjugated to the 5’ end of the passenger strand via a phosphorodithioate internucleotide linkage.

[0097] In some aspects, the polynucleic acid molecule described herein comprises a guide strand or a passenger strand bonded to a linker of formula (IV’),wherein Z in formula (IV’) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl); R in formula (IV’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; wherein theis connected to an asialoglycoprotein receptor targeting moiety, e.g., GalNAc; and wherein Y is to connect to the next nucleotide of the polynucleic acid molecule.

[0098] In some aspects, the polynucleic acid molecule described herein comprises a guide strand or a passenger strand bonded to a linker of formula (IV”),Wherein Z in formula (IV”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl); R in formula (IV”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; wherein theconnected to an asialoglycoprotein receptor targeting moiety, e.g., GalNAc; and wherein Y is to connect to the next nucleotide of the polynucleic acid molecule.

[0099] In some aspects, the polynucleic acid molecule described herein comprises a guide strand or a passenger strand bonded to a linker of formula (IV’”),wherein Z in formula (IV’”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl); R in formula (IV’”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; wherein theis connected to an asialoglycoprotein receptor targeting moiety, e.g., GalNAc; and wherein Y is to connect to the next nucleotide of the polynucleic acid molecule.

[0100] In some aspects, the targeting moiety described herein is conjugated to 3’ end of the passenger strand (e.g., formula (IV’)). In some aspects, the targeting moiety described herein is conjugated to 5’ end of the passenger strand (e.g., formula (IV”) or (IV’”)). In some aspects, the targeting moiety described herein is conjugated to 3’ end of the guide strand (e.g., formula (IV’)). In some aspects, the targeting moiety described herein is conjugated to 5’ end of the guide strand (e.g., formula (IV”) or (IV’”)).

[0101] In some aspects, the linker conjugated with one or more targeting moieties as shown in Formula (IV”) or (IV’”) is added to the first nucleotide on the 5’ end of the passenger strand.

[0102] In some instances, the targeting moiety comprises one or more (e.g., from 1 to 6) asialoglycoprotein receptor targeting moieties (e.g., GalNAc). In some instances, the one or more (e.g., from 1 to 6) asialoglycoprotein receptor targeting moieties (e.g., GalNAc) are conjugated with linker comprising formula (I), (II), (IV), (IV’), (IV”), or (IV’”).

[0103] In some instances, one or more (e.g., from 1 to 6) targeting moieties can be linked to MOIETY or to X2 in Formula (V’, V”, or V”’) through -LinkA-

[0104] In some aspects, the targeting moiety includes, but is not limited to, one or more (e.g., from 1 to 6 or from 1 to 3) asialoglycoprotein receptor ligands (e.g., GalNAc). In some aspects, an asialoglycoprotein receptor ligand (e.g., GalNAc) is attached to -LinkA- through an anomeric carbon (e.g., where the anomeric carbon is the carbon atom in an acetal or a hemiaminal). In some aspects, an asialoglycoprotein receptor ligand (e.g., GalNAc) comprises an anomeric carbon bonded to trivalent, tetravalent linker, pentavalent, or hexavalent linker, wherein the anomeric carbon is part of a hemiaminal group. An asialoglycoprotein receptorligand (e.g., GalNAc) attached to a linker through a hemiaminal may produce a hybridized polynucleotide construct having superior efficacy in gene silencing as compared to hybridized polynucleotide constructs having the asialoglycoprotein receptor ligand (e.g., GalNAc) attached to a linker through an acetal.

[0105] In some instances, the linker and asialoglycoprotein receptor targeting moiety comprises Formula (V), (V’), (V”), (V’”), (V””), (V’””), (V”””), or (VI’). In some instances, the linker and asialoglycoprotein receptor targeting moiety comprise Formula (V’), (V””), (V’””), (V”””), or (VI’).

[0106] In some aspects, the linker and three asialoglycoprotein receptor targeting moieties, each of which comprises GalNAc, are as shown in Formula (V):wherein one of Y1 or Y2 is nucleotide, or wherein both Y1 and Y2 are nucleotides and Y1 and Y2 are consecutive or neighboring nucleotides from the polynucleic acid molecule described herein.

[0107] In some aspects, the linker and three asialoglycoprotein receptor targeting moieties, each of which comprises GalNAc, are as shown in Formula (V’):wherein Z in formula (V’) corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), R in formula (V’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and wherein the is to connect to the next nucleotide of the polynucleic acid molecule.

[0108] In some aspects, the linker and three asialoglycoprotein receptor targeting moieties, each of which comprises GalNAc, are as shown in Formula (V”):Wherein Z in formula (V”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), R in formula (V”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and wherein theis to connect to the next nucleotide of the polynucleic acid molecule.

[0109] In some aspects, the linker and three asialoglycoprotein receptor targeting moieties, each of which comprises GalNAc, are as shown in Formula (V’”):wherein Z in formula (V’”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), and R in formula (V’”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and wherein the is to connect to the next nucleotide of the polynucleic acid molecule.

[0110] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (pl)(X3)(X3)-GalNAc structure comprising triantennary GalNAc moieties via one phosphotriester group (pl) and two(X3) linkers (see Formula (VI’)) as shown below,wherein Z in formula (VI’) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), R in formula (VI’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and wherein theis to connect to the next nucleotide of the polynucleic acid molecule.[OHl] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 3 is conjugated with (pl)(X3)(X3)-GalNAc structure comprising triantennary GalNAc moiety via one phosphotriester group (pl) and two (X3) linkers (see Formula (VI’)). Examples of formula (VI’) include, but are not limited to, a(pl)(X3)(X3)- GalNAc (see Formula (VI’ -a)) as shown below,wherein the is to connect to the next nucleotide of the polynucleic acid molecule.

[0112] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (pl)(X3)-GalNAc structure comprising diantennary GalNAc moiety via one phosphotriester group (pl) and one (X3) linker (see Formula (VI”)) as shown below,wherein Z in formula (VI”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), R in formula (VI”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and wherein theis to connect to the next nucleotide of the polynucleic acid molecule.

[0113] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 6 is conjugated with (pl)(X3)-GalNAc structure comprising diantennary GalNAc moiety via one phosphotriester group (pl) and one (X3) linker (see Formula (VI”)).

[0114] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (pl)(X3)(X3)(X3)- GalNAc structure comprising tetraantennary GalNAc moiety via one phosphotriester group (pl) and three (X3) linkers (see Formula (VI’”)) as shown below,wherein Z in formula (VI’”) is a moiety that corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-Methyl, -F, or -O-methoxyethyl), R in formula (VI’”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and wherein theis to connect to the next nucleotide of the polynucleic acid molecule.

[0115] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 6 is conjugated with (pl)(X3)(X3)(X3)-GalNAc structure comprising diantennary GalNAc moiety via one phosphotriester group (pl) and three (X3) linkers (see Formula (VI’”)).

[0116] In some instances, the conjugate comprising one or more asialoglycoprotein receptor targeting moieties (e.g., GalNAc) and a linker as described in Formula (V), (V’), (V”), (V’”), (V””), (V’””), (V”””), or (VI’) comprises one asialoglycoprotein receptor targeting moiety, so the conjugate comprises a structure of Formula (V), (V’), (V”), (V’”), (V””), (V’””),(V”””), or (VI’) with any two of the targeting moieties removed. In some instances, the conjugate described herein comprises two asialoglycoprotein receptor targeting moieties, so the conjugate described herein comprises a structure of Formula (V), (V’), (V”), (V’”), (V””), (V’””), (V”””), or (VI’) with any one of the targeting moieties removed.

[0117] In some aspects, the linker and the targeting moieties described herein are conjugated to 3’ end of the passenger strand (e.g., as shown in Formula (V), (V’), (V””), (V’””), (V”””)). In some aspects, the linker and the targeting moieties described herein are conjugated to 5’ end of the passenger strand (e.g., as shown in Formula (V’), (V”), or (V’”)). In some aspects, the linker and the targeting moieties described herein are conjugated to 3’ end of the guide strand (e.g., as shown in Formula (V), (V’), (V””), (V’””), (V”””)). In some aspects, the linker and the targeting moieties described herein are conjugated to 5’ end of the guide strand (e.g., as shown in Formula (V’), (V”), or (V’”)).

[0118] In some instances, the 3’ end of passenger strand (or sense strand) from Table 1, Table 3, Table 5, or Table 6 is conjugated with X2-GalNAc (see Formula (V), (V’), (V””), (V’””), (V”””)). In some instances, the 5’ end of passenger strand (or sense strand) from Table 1, Table 3, Table 5, or Table 6 is conjugated with X2-GalNAc (see Formula (V), (V”), (V’”)). In some instances, the 3’ end of guide strand from Table 1, Table 3, Table 5, or Table 6 is conjugated with X2-GalNAc (see Formula (V), (V’), (V’ ” ’), (V’ ” ”), (V’ ’””)). In some instances, the 5’ end of guide strand from Table 1, Table 3, Table 5, or Table 6 is conjugated with X2-GalNAc (see Formula (V), (V”), or (V’”)).

[0119] In some instances, the linker conjugated with one or more GalNAc as shown in Formula (V), (V’), (V””), (V’””), (V”””) is added to the first nucleotide on the 3’ end of the passenger strand. In some aspects, the linker conjugated with one or more GalNAc as shown in Formula (V), (V”) or (V’”) is added to the first nucleotide on the 5’ end of the passenger strand. In some instances, the linker conjugated with one or more GalNAc as shown in Formula (V), (V’), (V””), (V’””), (V”””) is added to the first nucleotide on the 3’ end of the guide strand. In some aspects, the linker conjugated with one or more GalNAc as shown in Formula (V), (V”) or (V’”) is added to the first nucleotide on the 5’ end of the guide strand.Lipophilic Moiety

[0120] In some aspects, the one or more targeting moiety comprises a lipophilic moiety. In some instances, the lipophilic moiety comprises lipid or derivative thereof. In some instances, the lipid or derivative thereof comprises saturated hydrocarbon chain or unsaturated hydrocarbon chain. In some instances, the lipid or derivative thereof comprises linear hydrocarbon chain or branched hydrocarbon chain. In some instances, the lipophilic moiety is a saturated or unsaturated, linear or branched hydrocarbon chain.

[0121] In some instances, the lipid or derivative thereof comprises short-chain hydrocarbon chain (e.g., five or less carbons), medium-chain hydrocarbon chain (e.g., 6 to 12 carbons), long- chain hydrocarbon chain (e.g., 13 to 21 carbons), or very long chain hydrocarbon chain (e.g., 22 or more carbons).

[0122] In some instances, the saturated or unsaturated, linear or branched hydrocarbon chain is a C8-C30 hydrocarbon chains. In some instances, the saturated or unsaturated, linear or branched hydrocarbon chain is C8, C12, C14, C16, C18, C20, or C22 hydrocarbon chains.

[0123] In some instances, the lipophilic moiety comprises a cholesterol or a tocopherol.

[0124] In some aspects, the lipophilic moiety is conjugated to the polynucleic acid molecule via a linking moiety. In some instances, the linking moiety is a spacer or a linker. In some instances, the linking moiety comprises a linear carbon chain linker. In some instances, the linear carbon chain linker comprises a short chain linker (e.g., three or less carbons), a medium chain linker (e.g., four to nine carbons), or a long chain linker (e.g., 10 or more carbons). In some instances, the linear carbon chain linker comprises C6 linker.

[0125] In some aspects, the lipophilic moiety is conjugated to the polynucleic acid molecule. In some instances, the lipophilic moiety is conjugated to the 5’ end or at the 3’ end of the passenger strand.

[0126] In some instances, the lipophilic moiety is conjugated to the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule. In some instances, the lipophilic moiety is conjugated to the first nucleotide on the 3’ end of the passenger strand of the polynucleic acid molecule.

[0127] In some instances, the lipophilic moiety and the linking moiety are conjugated to the first nucleotide from the 5’ end of the passenger strand of the polynucleic acid molecule. In some instances, the lipophilic moiety and the linking moiety are conjugated to the first nucleotide from the 3’ end of the passenger strand of the polynucleic acid molecule.

[0128] In some instances, the lipophilic moiety comprises Formula (VII), (VII’), (VII”), (VII’”), (VII””), or (VII’””) as described herein. In some instances, the polynucleic acid molecule described herein comprises a guide strand or a passenger strand bonded to a linker of Formula (VII):wherein nl is a 1-15; n2 is 1-30; R is CH3, COOH, or OH; and thecis to connect to the 5’ end and / or the 3’ end of the guide strand or the passenger strand.

[0129] In some instances, nl is 3-15, 3-10, 3-8, 4-7, or about 6. In some instances, n2 is 3-30, 3- 25, 5-25, 8-25, 10-22, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some instances, the passenger strand is connected to a linker of Formula (VII). In some instances, the 5’ end of the passenger strand is connected to a linker of Formula (VII). In some instances, the 3’ end of the passenger strand is connected to a linker of Formula (VII). In some instances, the linker is a linear carbon chain linker. In some instances, the linear carbon chain linker comprises a short chain linker (e.g., three or less carbons), a medium chain linker (e.g., four to nine carbons), or a long chain linker (e.g., 10 or more carbons). In some instances, the linear carbon chain linker comprises C6 linker. In some instances, the hydrocarbon chain comprises shortchain hydrocarbon chain (e.g., five or less carbons), medium-chain hydrocarbon chain (e.g., 6 to 12 carbons), long-chain hydrocarbon chain (e.g., 13 to 21 carbons), or very long chain hydrocarbon chain (e.g., 22 or more carbons). In some instances, the hydrocarbon chain is a saturated or unsaturated, linear or branched hydrocarbon chain. In some instances, the saturated or unsaturated, linear or branched hydrocarbon chain is a C8-C30 hydrocarbon chains. In some instances, the saturated or unsaturated, linear or branched hydrocarbon chain is C8, C12, C14, Cl 6, Cl 8, C20, or C22 hydrocarbon chains.

[0130] In some instances, the 5’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (C16a) (see Formula (VII ’)) as shown below:

[0131] In some instances, the 5’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (Cl 8a) (see Formula (VII”)) as shown below:

[0132] In some instances, the 5’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (C22a) (see Formula (VII’”)) as shown below:

[0133] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (C22a) (see Formula (VII””)) as shown below:

[0134] In some instances, the 5’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (TPc)(C6) (see

[0135] In some instances, the 3’ end of passenger strand (sense strand) of a polynucleic acid molecule from Table 1, Table 3, Table 5, or Table 6 is conjugated with (C18b)(X2) (see Formula (VII”””) as shown below:Polynucleic acid conjugate composition

[0136] In some aspects, provided herein is a polynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilicmoiety. In some aspects, the polynucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand). In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 5’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 5’ end of the passenger strand. In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 5’ end of the passenger strand, and the lipophilic moiety is conjugated to the 5’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the polynucleic acid molecule, e.g., passenger strand, and / or the asialoglycoprotein receptor targeting moiety via a linking moiety. In some instances, the lipophilic moiety is conjugated to the polynucleic acid molecule via a linking moiety. In some instances, the linking moiety comprises a linear carbon chain linker. In some instances, the linear carbon chain linker comprises a short chain linker (e.g., three or less carbons), a medium chain linker (e.g., four to nine carbons), or a long chain linker (e.g., 10 or more carbons). In some instances, the linear carbon chain linker comprises C6 linker. In some instances, the linking moiety is a C6 linker.

[0137] In some instances, both the asialoglycoprotein receptor targeting moiety and the lipophilic moiety are conjugated to the 5’ end of the polynucleic acid molecule, e.g., passenger strand, and the lipophilic moiety is located between the asialoglycoprotein receptor targeting moiety and the polynucleic acid molecule. In some instances, the 5’ end of the polynucleic acid conjugate composition comprises a structure of Formula (VIII), as shown below:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and the is to connect to the 5’ end of the polynucleic acid molecule, e.g., guide strand or passenger strand. In some instances, the one or more linker with asialoglycoprotein receptor targeting moieties comprises Formula (V’ ”)), Formula (V’), Formula (VI’), Formula (VI”), or Formula (VI’”).

[0138] For example, in some instances, the 5’ end of the polynucleic acid conjugate composition comprises a structure of 5’(Xl)(C22b) (see Formula (VIII’)), as shown below:lipophilic moiety are conjugated to the 5’ end of the polynucleic acid molecule, e.g., passenger strand, the asialoglycoprotein receptor targeting moiety is located between the polynucleic acid molecule and the lipophilic moiety. In some instances, the 5’ end of the polynucleic acid conjugate composition comprises a structure of Formula (IX), as shown below:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and the is to connect to the 5’ end of the polynucleic acid molecule, e.g., guide strand or passenger strand. In some instances, the one or more linker with asialoglycoprotein receptor targeting moieties comprises Formula (V’”)), Formula (V’), Formula (VI’), Formula (VI”), or Formula (VI’”).

[0140] In an aspect, provided herein is a polynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilic moiety. In some aspects, the polynucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand). In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 5’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 3’ end of thepassenger strand. In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 5’ end of the passenger strand, and the lipophilic moiety is conjugated to the 3’ end of the passenger strand.

[0141] In some aspects, provided herein is a polynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilic moiety. In some aspects, the polynucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand). In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 3’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 5’ end of the passenger strand. In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 3’ end of the passenger strand, and the lipophilic moiety is conjugated to the 5’ end of the passenger strand.

[0142] In some aspects, provided herein is a polynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilic moiety. In some aspects, the polynucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand). In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 3’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the 3’ end of the passenger strand. In some instances, the asialoglycoprotein receptor targeting moiety is conjugated to the 3’ end of the passenger strand, and the lipophilic moiety is conjugated to the 3’ end of the passenger strand. In some instances, the lipophilic moiety is conjugated to the polynucleic acid molecule, e.g., passenger strand, and / or the asialoglycoprotein receptor targeting moiety via a linking moiety. In some instances, the lipophilic moiety is conjugated to the polynucleic acid molecule via a linking moiety. In some instances, the linking moiety comprises a linear carbon chain linker. In some instances, the linear carbon chain linker comprises a short chain linker (e.g., three or less carbons), a medium chain linker (e.g., four to nine carbons), or a long chain linker (e.g., 10 or more carbons). In some instances, the linear carbon chain linker comprises C6 linker. In some instances, the linking moiety is a C6 linker.

[0143] In some instances, if both the asialoglycoprotein receptor targeting moiety and the lipophilic moiety are conjugated to the 3’ end of the polynucleic acid molecule, e.g., passenger strand, the lipophilic moiety is located between the asialoglycoprotein receptor targeting moiety and the polynucleic acid molecule. In some instances, the 3’ end of the polynucleic acid conjugate composition comprises a structure of Formula (X), as shown below:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and the is to connect to the 3’ end of the guide or passenger strand. In some instances, the one or more linker with asialoglycoprotein receptor targeting moieties comprises Formula (V’”)), Formula (V’), Formula (VI’), Formula (VI”), or Formula (VI’”).

[0144] For example, in some instances, the 3’ end of the polynucleic acid conjugate composition comprises a structure of 3’(C22b)(X2) (see Formula (X’)), as shown below:

[0145] In some instances, if both the asialoglycoprotein receptor targeting moiety and the lipophilic moiety are conjugated to the 3’ end of the polynucleic acid molecule, e.g., passenger strand, the asialoglycoprotein receptor targeting moiety is located between the polynucleic acid molecule and the lipophilic moiety. In some instances, the 3’ end of the polynucleic acid conjugate composition comprises a structure of Formula (XI), as shown below:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and the is to connect to the 3’ end of the polynucleic acid molecule, e.g., guide strand or passenger strand. In some instances, the one or more linker with asialoglycoprotein receptor targeting moieties comprises Formula (V’”)), Formula (V’), Formula (VI’), Formula (VI”), or Formula (VI’”). .

[0146] For example, in some instances, the 3’ end of the polynucleic acid conjugate composition comprises a structure of 3’(X2)(C22b) see Formula (XI’)), as shown below:

[0147] In some instances, the polynucleic acid molecule further comprises an inverted abasic moiety. In some instances, the inverted abasic moiety comprises an inverted abasic deoxyribonucleotide (invAb). In some instances, the inverted abasic moiety comprises a structure of Formula (XII), as shown below:wherein R is a moiety that corresponds to one of the sugar modifications described herein (e.g., - H, -OH, -O-Methyl, -F, or -O-methoxyethyl); wherein R’ is abasic or others; and wherein A is - O or -S; and wherein A’ is -O or -S.

[0148] In some instances, the polynucleic acid molecule, e.g., the passenger strand, comprises an inverted abasic moiety at the 5’ end. In some instances, the polynucleic acid molecule, e.g., the passenger strand, comprises an inverted abasic moiety at the 3’ end. In some instances, the 5’ end of the passenger strand comprises an inverted abasic moiety. In some instances, the 3’ end of the passenger strand comprises an inverted abasic moiety.

[0149] In some instances, wherein the asialoglycoprotein receptor targeting moiety and the lipophilic moiety are conjugated to the 5’ end of the passenger strand, the 3’ end of the passenger strand comprises an inverted abasic moiety. In some instances, wherein the asialoglycoprotein receptor targeting moiety and the lipophilic moiety are conjugated to the 3’ end of the passenger strand, the 5’ end of the passenger strand comprises an inverted abasic moiety

[0150] In some instances, wherein the lipophilic moiety is conjugated to the 5’ end of the passenger strand, the 5’ end of the passenger strand further comprises an inverted abasic moiety. In some instances, wherein the lipophilic moiety and the inverted abasic moiety are located at the 5’ end of the passenger strand, the lipophilic moiety is conjugated to the inverted abasic moiety.

[0151] In some instances, wherein the lipophilic moiety is conjugated to the 3’ end of the passenger strand, the 3’ end of the passenger strand comprises an inverted abasic moiety. In some instances, wherein the lipophilic moiety and the inverted abasic moiety are located at the 3’ end of the passenger strand, the lipophilic moiety is conjugated to the inverted abasic moiety.Pharmaceutical compositions

[0152] Delivery of the polynucleic acid conjugate composition described herein can be achieved by contacting a cell with the construct using a variety of methods. In particular aspects, the polynucleic acid conjugate composition described herein is formulated with various excipients, vehicles, and carriers, as described more fully elsewhere herein.

[0153] In some aspects, provided herein is a pharmaceutical composition comprising a polynucleic acid conjugate composition described herein and a pharmaceutically acceptable excipient. A pharmaceutical composition described herein can be prepared to include a hybridized polynucleotide construct disclosed herein, into a form suitable for administration to a subject using carriers, excipients, and vehicles. Frequently used excipients include, but are not limited to, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol, and polyhydric alcohols. Intravenous vehicles include, but are not limited to, fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents, and inert gases. Other pharmaceutically acceptable vehicles include, but are not limited to, aqueous solutions, nontoxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics.

[0154] The pharmaceutical compositions described herein may be administered locally or systemically. The therapeutically effective amounts may vary according to factors, such as the degree of infection in a subject, the age, sex, and weight of the individual. Dosage regimes can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0155] The pharmaceutical composition can be administered in a convenient manner, such as by injection (e.g., subcutaneous, intravenous, intraorbital, and the like), oral administration, ophthalmic application, inhalation, topical application, or rectal administration. Depending on the route of administration, the pharmaceutical composition can be coated with a material to protect the pharmaceutical composition from the action of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition. The pharmaceutical composition can also be administered parenterally or intraperitoneally. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0156] Pharmaceutical compositions suitable for injectable use include, but are not limited to, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The composition typically is sterile and fluid to the extent that easy syringability exists. In some instances, the composition is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size, in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride are used in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0157] Sterile injectable solutions can be prepared by incorporating the pharmaceutical composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the pharmaceutical composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0158] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of pharmaceutical composition is calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification for the dosage unit forms are related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieve. The principal pharmaceutical composition is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable vehicle in an acceptable dosage unit. In the case ofcompositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the ingredients.

[0159] The pharmaceutical composition can be orally administered, for example, in a carrier, e.g., in an enteric-coated unit dosage form. The pharmaceutical composition and other ingredients can also be enclosed in a hard or soft-shell gelatin capsule or compressed into tablets. For oral therapeutic administration, the pharmaceutical composition can be incorporated with excipients and used in the form of ingestible tablets, troches, capsules, pills, wafers, and the like. Such compositions and preparations may contain at least 1% by weight of active compound. The percentage of the compositions and preparations can, of course, be varied and can conveniently be between about 5% to about 80% of the weight of the unit. The tablets, troches, pills, capsules, and the like can also contain the following: a binder, such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid, and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar, or both. A syrup or elixir can contain the agent, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring, such as cherry or orange flavor. Any material used in preparing any dosage unit form may be of pharmaceutically acceptable purity and substantially non-toxic in the amounts employed. In addition, the pharmaceutical composition can be incorporated into sustained-release preparations and formulations.

[0160] The pharmaceutical composition described herein may comprise one or more permeation enhancer that facilitates bioavailability of the polynucleic acid molecule described herein. WO 2000 / 67798, Muranishi, 1990, Crit. Rev. Ther. Drug Carrier Systems, 7, 1, Lee et al., 1991, Crit. Rev. Ther. Drug Carrier Systems, 8, 91 are herein incorporated by reference in its entirety. In some aspects, the permeation enhancer is intestinal. In some aspects, the permeation enhancer is transdermal. In some aspects, the permeation enhancer is to facilitate crossing the brain-blood barrier. In some aspects, the permeation enhancer improves the permeability in the oral, nasal, buccal, pulmonary, vaginal, or corneal delivery model. In some aspects, the permeation enhancer is a fatty acid or a derivative thereof. In some aspects, the permeation enhancer is a surfactant or a derivative thereof. In some aspects, the permeation enhancer is a bile salt or a derivative thereof. In some aspects, the permeation enhancer is a chelating agent or a derivative thereof. In some aspects, the permeation enhancer is a non-chelating non-surfactant or aderivative thereof. In some aspects, the permeation enhancer is an ester or a derivative thereof. In some aspects, the permeation enhancer is an ether or a derivative thereof. In some aspects, the permeation enhancer is arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, 1- dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof. In one specific aspect, the permeation enhancer is sodium caprate (CIO). In some aspects, the permeation enhancer is chenodeoxycholic acid (CDCA), ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate or sodium glycodihydrofusidate. In some aspects, the permeation enhancer is polyoxyethylene-9-lauryl ether, or polyoxyethylene-20-cetyl ether.

[0161] For the pharmaceutical composition comprising a polynucleic acid conjugate composition described herein, suitable pharmaceutically acceptable salts include, but are not limited to, (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (ii) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; and (iii) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like.

[0162] While the hybridized polynucleotide constructs described herein may not require the use of excipients for delivery to the target cell, the use of excipients may be advantageous in some aspects. Thus, for delivery to the target cell, the hybridized polynucleic acid molecule described herein can non-covalently bind an excipient to form a complex. The excipient can be used to alter biodistribution after delivery, to enhance uptake, to increase half-life or stability of the strands in the hybridized polynucleotide constructs (e.g., improve nuclease resistance), and / or to increase targeting to a particular cell or tissue type.

[0163] Exemplary excipients include, but are not limited to, a condensing agent (e.g., an agent capable of attracting or binding a nucleic acid through ionic or electrostatic interactions); a fusogenic agent (e.g., an agent capable of fusing and / or being transported through a cell membrane); a protein to target a particular cell or tissue type (e.g., thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, or any other protein); a lipid; a lipopolysaccharide; a lipid micelle or a liposome (e.g., formed from phospholipids, such as phosphotidylcholine, fattyacids, glycolipids, ceramides, glycerides, cholesterols, or any combination thereof); a nanoparticle (e.g., silica, lipid, carbohydrate, or other pharmaceutically-acceptable polymer nanoparticle); a polyplex formed from cationic polymers and an anionic agent (e.g., a CRO), where exemplary cationic polymers include polyamines (e.g., polylysine, polyarginine, polyamidoamine, and polyethylene imine); cholesterol; a dendrimer (e.g., a polyamidoamine (PAMAM) dendrimer); a serum protein (e.g., human serum albumin (HSA) or low-density lipoprotein (LDL)); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); a lipid; a synthetic polymer, (e.g., polylysine (PLL), polyethylenimine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolic) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropyl acrylamide polymer, pseudopeptide-polyamine, peptidomimetic polyamine, or polyamine); a cationic moiety (e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or alpha helical peptide); a multivalent sugar (e.g., multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N- acetyl -glucosamine, multivalent mannose, or multivalent fucose); a vitamin (e.g., vitamin A, vitamin E, vitamin K, vitamin B, folic acid, vitamin B 12, riboflavin, biotin, or pyridoxal); a cofactor; or a drug to disrupt cellular cytoskeleton to increase uptake (e.g., taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin).

[0164] Other therapeutic agents as described herein may be added in a pharmaceutical composition described herein in combination with a polynucleic acid conjugate composition described herein.Methods

[0165] In an aspect, provided herein is a method of utilizing a polynucleic acid conjugate composition or a pharmaceutical composition described herein. In some aspects, described herein is a method of modulating an expression of an mRNA of a target gene in a cell, comprising: contacting the cell with or administering a subject with the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein. In some instances, provided herein is a method of modulating an expression of an mRNA of a target gene in a cell, comprising: contacting the cell with or administering a subject with the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to the mRNA thereby modulating the expression of the mRNA of the target gene.

[0166] In some aspects, described herein is a method of modulating expression of a target gene in a subject, comprising: administering to the subject a polynucleic acid conjugate composition described herein or a pharmaceutical composition described herein, thereby modulating the expression of the target gene in the subject. In some instances, the polynucleic acid molecule hybridizes to an mRNA of the target gene, thereby modulating the expression of the target gene.

[0167] In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 10% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 20% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 30% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 40% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 50% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 60% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 70% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 80% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about or at least 90% compared to a negative control. In some aspects, the method described herein reduces expression of a target gene in a subject by about 100% compared to a negative control.

[0168] In some aspects, the method described herein achieves an IC50 value of about 5nM. In some aspects, the method described herein achieves an IC50 value of about lOnM. In some aspects, the method described herein achieves an IC50 value of about 15nM. In some aspects, the method described herein achieves an IC50 value of about 20nM. In some aspects, the method described herein achieves an IC50 value of about 25nM. In some aspects, the method described herein achieves an IC50 value of about 30nM. In some aspects, the method described herein achieves an IC50 value of about 35nM. In some aspects, the method described herein achieves an IC50 value of about 40nM. In some aspects, the method described herein achieves an IC50 value of about 45nM. In some aspects, the method described herein achieves an IC50 value of about 50nM. In some aspects, the method described herein achieves an IC50 value of about 55nM. In some aspects, the method described herein achieves an IC50 value of about 60nM. In some aspects, the method described herein achieves an IC50 value of about 65nM. In some aspects, the method described herein achieves an IC50 value of about 70nM. In someaspects, the method described herein achieves an IC50 value of about 75nM. In some aspects, the method described herein achieves an IC50 value of about 80nM. In some aspects, the method described herein achieves an IC50 value of about 85nM. In some aspects, the method described herein achieves an IC50 value of about 90nM. In some aspects, the method described herein achieves an IC50 value of about 95nM. In some aspects, the method described herein achieves an IC50 value of about lOOnM.

[0169] In some aspects, described herein is a method of preventing, alleviating, or treating a disease in a subject in need thereof, comprising: administering to the subject a polynucleic acid conjugate composition or a pharmaceutical composition described herein, wherein the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein reduces an expression of a target gene in the subject. In some aspects, described herein is a method of preventing, alleviating, or treating a disease in a subject in need thereof, comprising: administering to the subject a polynucleic acid conjugate composition or a pharmaceutical composition described herein, wherein the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein reduces an expression of an mRNA of a target gene in the subject.

[0170] In some aspects, described herein is a method of treating a disease in a subject, comprising: administering a subject with the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein. In some instances, described herein is a method of treating a disease in a subject, comprising: administering a subject with the polynucleic acid conjugate composition described herein or the pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to an mRNA of a gene associated with the onset, development, or a symptom of the disease, thereby treating the disease.EXAMPLES

[0171] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. For all of the sequences presented herein, oligonucleotide structure representation reads from left to right (5' to 3'). Monomer codes present in the oligonucleotide code are linked by 5'-3 ' phosphodiester bonds unless specified (succeeded by 3' intemucleotide linkage reading left to right). Abbreviations of nucleotide monomers used in oligonucleotide structure representation are as follows. “A” stands for Adenosine-3 '-phosphate; “a” stands for 2'- O-methyladenosine-3 '-phosphate; “Af” stands for 2'-fluoroadenosine-3 '-phosphate; "A6" stands for 2'-methoxyethyladenosine-3'phosphate; “dA” stands for 2'-deoxyadenosine-3 '-phosphate;"al" refers to 2-Amino-2'-O-methyladenosine-3'-phosphate; “C” stands for Cytidine-3'-phosphate; “c” stands for 2'-O-methylcytidine-3'-phosphate; “Cf” stands for 2'-fluorocytidine-3'- phosphate; “dC” stands for 2'-deoxycytidine-3 '-phosphate; “G” stands for Guanosine-3 '- phosphate; “g” stands for 2'-O-methylguanosine-3 '-phosphate; “Gf ’ stands for 2'- fluoroguanosine-3 '-phosphate; “dG” stands for 2'-deoxy guanosine-3 '-phosphate; "i" stands for 2'-O-methylinosine-3'-phosphate; “U” stands for Uridine-3 '-phosphate; “u” stands for 2'-O- methyluridine-3 '-phosphate; "U6" stands for 2'-m ethoxy ethyluridine-3 '-phosphate ; “Uf” stands for 2'-fluorouridine-3 '-phosphate; "u3" refers to 2'-O-methyl-2-thiouridine-3 '-phosphate; "U3f" refers to 2'-fluoro-2-thiouridine-3 '-phosphate; “dU” stands for 2'-deoxyuridine-3 '-phosphate; “T” stands for 5 -m ethyluridine-3 '-phosphate; “t” stands for 2'-O-methyl-5-methyluridine-3'- phosphate; “Tf” stands for 2'-fluoro-5-methyluridine-3'-phosphate; “dT” stands for 2’- deoxythymidine-3 ’-phosphate; “s” stands for 3’-phosphorothioate; "ss" stands for 3'- phosphorodithioate; "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer; "(Sps)" stands for 3' phosphorothioate, Sp diastereomer; "(invAb)" stands for inverted abasic deoxyribonucleotide, "vp" stands for 5'vinylphosphonate modified nucleotide; "vpt4" stands for 5'vinylphosphonate- 2'O-methoxyethyl-5-methyluridine-3'phosphate; “vpu” stands for 5'(E) vinylphosphonate-2-O- methyluridine-3 'phosphate.Example 1 - Testing AGT siRNAs in Transgenic Mice

[0172] Sequences of siRNAs evaluated in this example are specified in Table 1 with the 3’ end and / or 5’ end of each passenger / sense strand conjugated with a GalNAc linker and / or a lipophilic moiety. Each modified passenger strand of SEQ ID NOs: 10, 12, and 19-21 is conjugated to a targeting moiety (e.g., GalNAc or galactose) at its 3’ end via a linker (e.g., Formula (V’)). Each modified passenger strand of SEQ ID NOs: 11 and 17 is conjugated to a targeting moiety (e.g., GalNAc or galactose) at its 5’ end via a linker (e.g., Formula (V’”)) and phosphorothioate intemucleotide linkage. Each modified passenger strand of SEQ ID NOs: 12- 13 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 5’ end via a linker (e.g., Formula (VII”’). Modified passenger strand of SEQ ID NO: 19 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 5’ end via a linker (e.g., Formula (VII”). Modified passenger strand of SEQ ID NO: 20 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 5’ end via a linker (e.g., Formula (VII’)). Modified passenger strand of SEQ ID NO: 21 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 5’ end via a linker (e.g., Formula (VII’””)). Each modified passenger strand of SEQ ID NOs: 14 and 17 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 3’ end via a linker (e.g., Formula (VII””)). Modified passenger strand of SEQ ID NO: 15 is conjugated to one or more targeting moiety (e.g., GalNAc or galactose and lipophilic moiety) at its 3’ end via a structure, e.g., Formula (X’).Modified passenger strand of SEQ ID NO: 16 is conjugated to one or more targeting moiety (e.g., GalNAc or galactose and lipophilic moiety) at its 3’ end via a structure, e.g., Formula (XI’). Modified passenger strand of SEQ ID NO: 18 is conjugated to one or more targeting moiety (e.g., GalNAc or galactose and lipophilic moiety) at its 5’ end via a structure, e.g., Formula (VIIF).

[0173] Transgenic AGT mice (HuAoGen Mice, B6.Cg-Tg(hAGT)2041Sig / J, Jackson Laboratory) were assigned to treatment groups (n=5, 2 males, 3 females). Each treatment group was administered a single 0.5 mg / kg subcutaneous injection of AGT siRNA on day 0. Blood samples were collected pre-dose (day 0), and on days 7, 14, 21, 28, 35, and 42. Blood was processed to plasma, and circulating AGT protein expression levels in all plasma samples were analyzed using an AGT ELISA assay (IBL America, #27412). Results for each individual were calculated as a percent change of circulating AGT protein relative to the pre-dose value (Day 0), and group mean values are listed in Table 2.Example 2 - Testing AGT siRNAs in Non-human Primates

[0174] Nucleic acid sequences of siRNAs used for the non-human primate study are provided in Table 3 with the 3’ end and / or 5’ end of each passenger / sense strand conjugated with a GalNAc linker and / or a lipophilic moiety. Each modified passenger strand of SEQ ID NOs: 22-24 is conjugated to a targeting moiety (e.g., GalNAc or galactose) at its 3’ end via a linker (e.g., Formula (V’)). Modified passenger strand of SEQ ID NO: 25 is conjugated to a targeting moiety (e.g., GalNAc or galactose) at its 5’ end via a linker (e.g., Formula (V’”)) and phosphorothioate internucleotide linkage. Modified passenger strand of SEQ ID NO: 23 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 5’ end via a linker (e.g., Formula (VII”)).Modified passenger strand of SEQ ID NO: 24 is conjugated to a targeting moiety (e.g., lipophilic moiety) at its 5’ end via a linker (e.g., Formula (VIF)). Modified passenger strand of SEQ ID NO: 26 is conjugated to a targeting moiety (e.g., GalNAc or galactose) at its 3’ end via a linker (e.g., Formula (VI’)).

[0175] Three male cynomolgus monkeys were assigned to each treatment group. Each treatment group was administered a single 2.0 mg / kg subcutaneous injection of AGT siRNA (as shown in Table 3) on day 1. Blood samples were collected pre-dose (days -14, -7, and day 0), and on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 98, and 112. Blood was processed to serum, and circulating AGT protein expression levels in all serum samples were analyzed using an AGT ELISA assay (IBL America, #27412). Results for each individual were calculated as the % change in circulating AGT protein relative to the day 0 pre-dose timepoint, and group values are presented as mean ± standard deviation in FIG. 1 and listed in Table 4.Example 3 - Testing AGT siRNAs in Non-human Primates

[0176] Exemplary siRNAs with various modifications were designed and synthesized, and these sequences are shown in Table 5. Modified passenger strand of SEQ ID NO: 25 is conjugated to a targeting moiety (e.g., GalNAc or galactose) at its 5’ end via a linker (e.g., Formula (V’”)) and phosphorothioate intemucleotide linkage. Modified passenger strand of SEQ ID NO: 26 is conjugated with a targeting moiety (e.g., GalNAc or galactose) at its 3’ end via a linker (e.g., Formula (VI’)). Modified passenger strand of SEQ ID NO: 27 is conjugated with a targeting moiety at 5’ end via a linker (e.g., Formula (VII’)) and another targeting moiety (e.g., GalNAc or galactose) at the 3’ end via a linker (e.g., Formula (VI’)). Modified passenger strand of SEQ ID NO: 42 is conjugated to a linker (e.g., Formula (XIII’)) and phosphorothioate internucleotide linkage at its 5’ end and is also conjugated with a targeting moiety (e.g., GalNAc or galactose) at its 3’ end via a linker (e.g., Formula (VI’)). Modified passenger strand of SEQ ID NO: 43 is conjugated with a targeting moiety (e.g., GalNAc or galactose) at its 3’ end via a linker (e.g., Formula (VI’)).

[0177] SRS-003815, SRS-003816, SRS-004240 to SRS-004242, SRS-004348 to SRS-004352, SRS-004371, and SRS-004374 were selected for evaluation in cynomolgus monkeys. Three male cynomolgus monkeys were assigned to each treatment group. Each treatment group was administered a single 1.0 mg / kg subcutaneous injection of AGT siRNA (as shown in Table 5) on day 1. Blood samples were collected pre-dose (days -14, -7, and day 0), and on days 7, 14, 21, 28, and 35. Blood samples are also collected weekly to day 84. Blood was processed to serum, and AGT circulating protein levels in all serum samples were analyzed using an AGT ELISA assay (IBL America, #27412). Results for each individual were calculated as the % change in circulating AGT protein relative to the day 0 pre-dose timepoint, and group values are presented as mean ± standard deviation and listed in Table 8.Example 4 - Testing Sodl siRNAs in mice

[0178] Sequences of siRNAs used for the non-human primate study are provided in Table 6 with the 3’ end of each passenger / sense strand conjugated with a GalNAc. Five female C57BL / 6 mice were assigned to each treatment group. Each treatment group was administered a single 3.0 mg / kg subcutaneous injection of mSodl siRNA (as shown in Table 6) on day 1. On Day 21 post-injection, animals were euthanized and liver, quadriceps, gastrocnemius, diaphragm, pgWAT, iWAT, injection site (skin), and heart samples were collected, pre-treated with RNAlater or Trizol, then snap frozen in liquid nitrogen. Expression of mouse Sodl mRNA was assessed in these samples by RT-qPCR, normalizing to mouse Gapdh, and group valueswere normalized to the IxPBS treated control group. The relative expression for each treatment group and for each tissue is listed in Table 7. Results are reported as group mean with standard deviation.

[0179] The results show that liver tissues from mice treated with a polynucleic acid molecule conjugated with an asialoglycoprotein receptor targeting moiety, e.g., GalNAc, and a lipophilic moiety had a decreased expression of the Sodl target gene and the knockdown activity of the polynucleic acid molecule on Sodl expression was maintained, similar to the expression level of Sodl in mice treated with a polynucleic acid molecule conjugated with only the asialoglycoprotein receptor targeting moiety (SRS-003817). Further, the results show that liver tissues from mice treated with a polynucleic acid molecule conjugated with an asialoglycoprotein receptor targeting moiety, e.g., GalNAc, and a lipophilic moiety had a lower expression of Sodl compared to the expression of Sodl in liver tissues obtained from mice treated with a polynucleic acid molecule conjugated with only the lipophilic moiety (SRS- 003824).

[0180] In addition, the results show that certain configurations of conjugations of the polynucleic acid molecule with an asialoglycoprotein receptor targeting moiety, e.g., GalNAc, and a lipophilic moiety can maintain knockdown level of Sodl not only in the liver tissue but also in non-hepatic tissues. For example, the passenger strand of SRS-003820 is conjugated with a lipophilic moiety via Formula (VII”) and the asialoglycoprotein receptor targeting moiety via Formula (VI”). Based on results listed in Table 7, Sodl expression level from liver tissue of mice treated with SRS-003820 was similar to the Sodl expression level from liver tissue of mice treated with a polynucleic acid molecule conjugated with only the asialoglycoprotein receptor targeting moiety (SRS-003817). However, Sodl expression level from non-hepatic tissue of mice treated with SRS-003820 was lower than Sodl expression level from non-hepatic tissues of mice treated with SRS-003817.

[0181] While preferred aspects of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the aspects of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.Table 1. Sequences for Transgenic Mice StudyTable 2. Percent Change in Serum AGT Relative to Day 0 Pre-Dose AverageTable 3. Sequences for Non-Human Primate StudyTable 4. Percent Change in Serum AGT Relative to Day 0 Pre-DoseTable 5. Sequences for Non-Human Primate StudyTable 6. Sequences for Mice StudyTable 7. Relative mSodl Expression per Tissue on Day 21Table 8. Percent Change in Serum AGT Relative to Day 0 Pre-Dose

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A polynucleic acid conjugate composition comprising a polynucleic acid molecule, an asialoglycoprotein receptor targeting moiety, and a lipophilic moiety.

2. The polynucleic acid conjugate composition of claim 1, wherein the lipophilic moiety is a saturated or unsaturated, linear or branched hydrocarbon chain.

3. The polynucleic acid conjugate composition of claim 2, wherein the saturated or unsaturated, linear or branched hydrocarbon chain is a C8-C30 hydrocarbon chains.

4. The polynucleic acid conjugate composition of claim 2 or claim 3, wherein the saturated or unsaturated linear or branched hydrocarbon chain is C8, C12, C14, C16, C18, C20, or C22 hydrocarbon chains.

5. The polynucleic acid conjugate composition of claim 1, wherein the lipophilic moiety comprises a cholesterol or a tocopherol.

6. The polynucleic acid conjugate composition of any one of claims 1-5, wherein the asialoglycoprotein receptor targeting moiety comprises N-Acetylgalactosamine (GalNAc) or galactose.

7. The polynucleic acid conjugate composition of claim 6, wherein the asialoglycoprotein receptor targeting moiety is conjugated to the polynucleic acid molecule via a linker.

8. The polynucleic acid conjugate composition of claim 7, wherein the linker comprises formula (IV), formula (XIII’), or formula (XIII”) below,wherein at least one of Y1 or Y2 in formula (IV) is a nucleotide in the polynucleic acid molecule; wherein Z in formula (XIII’) and formula (XIII”) is -H, -OH, -O-Methyl, -F, or - O-methoxy ethyl; R in formula (XIII’) and formula (XIII”) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; and thein formula (XIII’) and formula (XIII”) is to connect to the next nucleotide of the polynucleic acid molecule.

9. The polynucleic acid conjugate composition of claim 8, wherein the linker and the asialoglycoprotein receptor targeting moiety comprise:orwherein Z in formula (V’) or (VT) is -H, -OH, -O-Methyl, -F, or -O-methoxyethyl; R in formula (V’) or (VI’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others; and the is to connect to the next nucleotide of the polynucleic acid molecule.

10. The polynucleic acid conjugate composition of any one of claims 1-9, wherein the polynucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand).

11. The polynucleic acid conjugate composition of claim 10, wherein the asialoglycoprotein receptor targeting moiety is conjugated to the 5’ end of the passenger strand.

12. The polynucleic acid conjugate composition of any one of claims 10-11, wherein the lipophilic moiety is conjugated to the 5’ end of the passenger strand.

13. The polynucleic acid conjugate composition of any one of claims 10-12, wherein the 3’ end of the passenger strand comprises an inverted abasic moiety.

14. The polynucleic acid conjugate composition of any one of claims 1-13, wherein the lipophilic moiety is conjugated to the polynucleic acid molecule via a linking moiety.

15. The polynucleic acid conjugate composition of claim 14, wherein the linking moiety is a C6 linker.

16. The polynucleic acid conjugate composition of any one of claims 10-15, wherein the lipophilic moiety is located between the asialoglycoprotein receptor targeting moiety and the polynucleic acid molecule.

17. The polynucleic acid conjugate composition of claim 16, wherein the polynucleic acid conjugate composition comprises a structure of:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and theis to connect to the 5’ end of the polynucleic acid molecule.

18. The polynucleic acid conjugate composition of any one of claims 10-11, wherein the lipophilic moiety is conjugated to the 3’ end of the passenger strand.

19. The polynucleic acid conjugate composition of claim 18, wherein the 3’ end of the passenger strand comprises an inverted abasic moiety.

20. The polynucleic acid conjugate composition of claim 19, wherein the lipophilic moiety is conjugated to the inverted abasic moiety.

21. The polynucleic acid conjugate composition of claim 10, wherein the asialoglycoprotein receptor targeting moiety is conjugated to the 3’ end of the passenger strand.

22. The polynucleic acid conjugate composition of claim 21, wherein the lipophilic moiety is conjugated to the 5’ end of the passenger strand.

23. The polynucleic acid conjugate composition of claim 21, wherein the lipophilic moiety is conjugated to the 3’ end of the passenger strand.

24. The polynucleic acid conjugate composition of claim 23, wherein the lipophilic moiety is located between the asialoglycoprotein receptor targeting moiety and the polynucleic acid molecule.

25. The polynucleic acid conjugate composition of claim 24, wherein the polynucleic acid conjugate composition comprises a structure of:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and theis to connect to the 3’ end of the polynucleic acid molecule.

26. The polynucleic acid conjugate composition of claim 23, the asialoglycoprotein receptor targeting moiety is located between the polynucleic acid molecule and the lipophilic moiety.

27. The polynucleic acid conjugate composition of claim 26, wherein the polynucleic acid conjugate composition comprises a structure of:wherein nl is 1-30; R is CH3, COOH, or OH; X is one or more linker with asialoglycoprotein receptor targeting moi eties; and theis to connect to the 3’ end of the polynucleic acid molecule.

28. A pharmaceutical composition comprising a polynucleic acid conjugate composition of any one of claims 1-27, and a pharmaceutically acceptable excipient.

29. A method of modulating an expression of an mRNA of a target gene in a cell, comprising: contacting the cell with or administering a subject with the polynucleic acid conjugate composition of any one of claims 1-27 or the pharmaceutical composition of claim 28, wherein the polynucleic acid molecule hybridizes to the mRNA thereby modulating the expression of the mRNA of the target gene.

0. A method of treating a disease in a subject, comprising: administering a subject with the polynucleic acid conjugate composition of any one of claims 1-27 or the pharmaceutical composition of claim 28, wherein the polynucleic acid molecule hybridizes to an mRNA of a gene associated with the onset, development, or a symptom of the disease, thereby treating the disease.

Images