Oligonucleotide conjugates

Abstract

Disclosed herein are oligonucleotide conjugates. An example conjugate includes an oligonucleotide; a binding ligand; an endosomal escape portion comprising an endosomal escape moiety; and a linker attached to the endosomal escape portion, wherein: the oligonucleotide is attached to the endosomal escape portion, the linker attaching the endosomal escape portion to the binding ligand; or the binding ligand is attached to the oligonucleotide, the linker attaching the endosomal escape portion to the oligonucleotide. Also disclosed are compositions including the conjugate and methods of using the conjugate.

Description

Attorney Docket No.: 093386-0056-WO01 OLIGONUCLEOTIDE CONJUGATES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 678,492 filed on August 1, 2024, which is incorporated fully herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant no. R01 CA260958 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present disclosure relates to oligonucleotide conjugates and their use in biomedical applications, such as drug delivery. INTRODUCTION

[0004] RNA interference mediated by small interfering RNA (siRNA) holds great promise as molecularly-targeted therapy in cancer, particularly for traditionally “undruggable” oncogenes. Numerous delivery systems have been developed to convey siRNA payloads to tumor cells, though traditional siRNA nanocarriers rely upon cationic lipids or polymers that can lead to systemic toxicity or inflammation, and none have yet been translated into clinical practice for oncology. As an alternative, direct siRNA conjugates are being developed in which the siRNA molecule itself is elaborated with covalent modifications that mediate delivery. Indeed, five of the six FDA- approved siRNA therapeutics are direct RNA conjugates that target hepatocytes. However, unlike traditional cationic delivery vehicles, current siRNA conjugates tend to accumulate in endosomal “depots,” where the dilutional effect of rapid cell division in cancer cells is the primary factor limiting the activity of siRNA therapeutics. That is, current siRNA conjugates exhibit slow endosomal release and thus have delayed activity and / or decreased potency. Accordingly, siRNA conjugates that may be used to more effectively treat diseases characterized by rapidly-dividing cells, e.g., cancers and autoimmune diseases, are needed.Attorney Docket No.: 093386-0056-WO01 SUMMARY

[0005] In one aspect, disclosed are conjugates comprising: an oligonucleotide; a binding ligand; an endosomal escape portion comprising an endosomal escape moiety; and a linker attached to the endosomal escape portion, wherein: the oligonucleotide is attached to the endosomal escape portion, the linker attaching the endosomal escape portion to the binding ligand; or the binding ligand is attached to the oligonucleotide, the linker attaching the endosomal escape portion to the oligonucleotide.

[0006] In another aspect, disclosed are conjugates comprising: an oligonucleotide; an endosomal escape portion attached to the oligonucleotide, the endosomal escape portion comprising an endosomal escape moiety; a lipophilic ligand capable of binding albumin; and a linker attaching the endosomal escape portion to the lipophilic ligand capable of binding albumin, the linker comprising: a branching molecule attached to the endosomal escape portion, and a hydrophilic spacer attaching the branching molecule to the lipophilic ligand, the branching molecule comprising: at least one branch point having at least two independent branches.

[0007] In another aspect, disclosed are conjugates comprising: an oligonucleotide; a saccharide ligand attached to the oligonucleotide, the saccharide ligand comprising N-acetylgalactosamine (GalNAc); and an endosomal escape portion comprising 1 to 10 endosomal escape moieties; and a linker attaching the endosomal escape portion to the oligonucleotide, the linker comprising: a branching molecule attached to the endosomal escape portion, the branching molecule comprising: at least one branch point having at least two independent branches.

[0008] In another aspect, disclosed are compositions including a disclosed conjugate and one or more pharmaceutically acceptable excipients.

[0009] In another aspect, disclosed are methods of gene silencing, the method including administering a disclosed conjugate to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] FIG. 1A is a schematic showing the synthesis of an example chloroquine (CQ) phosphoramidite reagent.Attorney Docket No.: 093386-0056-WO01

[0012] FIG. 1B is a cartoon schematic depicting the general elements that comprise example conjugate siRNA-CQ-L2.

[0013] FIG. 1C is a schematic summarizing the on-column solid-phase synthesis of example conjugate siRNA-CQ-L2.

[0014] FIG.1D shows the chemical structure of example conjugate siRNA-CQ-L2.

[0015] FIG.2A is a series of plots showing the fluorescence of Nile Red incubated with varying concentrations of siRNA-L2or siRNA-CQ-L2. Datapoints represent average of two technical replicates. Lower and upper curves indicate logarithmic regression fits for the 0.01-1.25 M ranges and 5-20 M ranges, respectively. Intersection of these curves was taken to be the inflection point representing the critical micelle concentration (CMC).

[0016] FIG.2B shows biolayer interferometry curves for binding of varying concentrations of siRNA-L2 or siRNA-CQ-L2 to immobilized biotinylated human serum albumin, showing association (left) and dissociation (right) phases, separated by the dotted line.

[0017] FIG. 3A shows microscopy images demonstrating the effect of free CQ on siRNA-L2release into cytoplasm. Scale bar = 20 m.

[0018] FIG.3B shows bar graphs quantifying the area of Gal8 puncta in Gal8-YFP MDA-MB- 231 cells incubated with media only, lipofectamine RNAiMax (positive control), siRNA-L2alone, siRNA-L2 with free 3 M CQ, or siRNA-CQ1-3-L2. Asterisks indicate statistical significance by one-way ANOVA: *: p<0.05, ***: p<0.005; ****: p<0.001.

[0019] FIG. 3C shows representative confocal microscopy images of Gal8-YFP MDA-MB- 231 cells incubated with the specified conditions. Scale bars = 50 m.

[0020] FIGS.4A-D show flow cytometry analysis of uptake of Cy5-labeled siRNA-L2without or with free chloroquine or Cy5-labeled siRNA-CQ-L2in MDA-MB-231 cells.

[0021] FIG. 4A is a bar graph showing geometric means of Cy5 fluorescence (n=4 per condition) following 1 hour of incubation with 100 nM of siRNA conjugates or media only at 37 °C. Asterisks indicate statistical significance by one-way ANOVA with Dunnett’s post-test comparing experimental groups against siRNA-L2. *: p<0.05, **: p<0.01; ****: p<0.001.

[0022] FIG.4B shows representative Cy5 fluorescence histograms corresponding to FIG.4A.

[0023] FIG. 4C is a bar graph showing geometric means of Cy5 fluorescence (n=4 per condition) following 4 hours of incubation with 100 nM of siRNA conjugates or media only at 37Attorney Docket No.: 093386-0056-WO01 °C. Asterisks indicate statistical significance by one-way ANOVA with Dunnett’s post-test comparing experimental groups against siRNA-L2. *: p<0.05, **: p<0.01; ****: p<0.001.

[0024] FIG.4D shows representative Cy5 fluorescence histograms corresponding to FIG.4C.

[0025] FIG. 4E is a bar graph showing firefly luciferase silencing by luciferase mRNA- targeting siRNA-L2 or siRNA-CQ-L2 following 24 hour incubation of Luc-expressing MDA-MB- 231 cells at the specified doses.

[0026] FIG. 4F is a bar graph showing cell viability measured by CellTiter Glo following 24 hour incubation of non-Luc-expressing MDA-MB-231 cells (to avoid conflict with the luminescent viability assay) with siRNA-L2or siRNA-CQ-L2at the specified doses. Asterisks indicate statistical significance by two-way ANOVA with Dunnett’s post-test comparing experimental groups against siRNA-L2. **: p<0.01.

[0027] FIG. 5A is a series of plots showing blood concentrations of siRNA-L2 and siRNA-CQ3-L2 after intravenous injection, assessed by PNA assay.

[0028] FIG. 5B is a bar graph showing organ biodistribution of siRNA-L2vs. siRNA-CQ2-L2or siRNA-CQ3-L2at 48 hours, as quantified by PNA assay.

[0029] FIG. 6A is a liquid chromatography-mass spectrometry (LCMS) chromatogram confirming the purity and molecular weight of example conjugate siRNA-CQ1-L2.

[0030] FIG. 6B is a liquid chromatography-mass spectrometry (LCMS) chromatogram confirming the purity and molecular weight of example conjugate siRNA-CQ2-L2 sense strand targeting luciferase.

[0031] FIG. 6C is a liquid chromatography-mass spectrometry (LCMS) chromatogram confirming the purity and molecular weight of example conjugate siRNA-CQ3-L+.

[0032] FIG. 7 is a series of plots showing kinetics of Gal8 focus formation in response to siRNA-CQ1-L2vs. lipofectamine RNAiMax transfection reagent, assessed as total Gal8 fluorescent intensity normalized to total cytoplasmic area, over the course of 24 hours.

[0033] FIG.8 is a bar graph showing flow cytometry analysis of uptake of Cy5-labeled siRNA- L2 without or with free chloroquine or Cy5-labeled siRNA-CQ-L2 in MDA-MB-231 cells incubated at 4 °C for 2 hours to assess for nonspecific binding. Geometric means of Cy5 fluorescence (n=4 per condition) are shown.Attorney Docket No.: 093386-0056-WO01

[0034] FIG. 9A is a series of plots showing white blood cell counts 48 hours following i.v. injection with siRNA-L2 vs. siRNA-CQ2-L2 or siRNA-CQ3-L2 dosed at 5 mg siRNA / kg body weight.

[0035] FIG. 9B is a series of plots showing hemoglobin 48 hours following i.v. injection with siRNA-L2 vs. siRNA-CQ2-L2 or siRNA-CQ3-L2 dosed at 5 mg siRNA / kg body weight.

[0036] FIG. 9C is a series of plots showing platelet counts 48 hours following i.v. injection with siRNA-L2vs. siRNA-CQ2-L2or siRNA-CQ3-L2dosed at 5 mg siRNA / kg body weight.

[0037] FIG.9D is a series of plots showing creatinine (all values were reported as <0.2 mg / dL) 48 hours following i.v. injection with siRNA-L2vs. siRNA-CQ2-L2or siRNA-CQ3-L2dosed at 5 mg siRNA / kg body weight.

[0038] FIG. 9E is a series of plots showing alanine aminotransferase 48 hours following i.v. injection with siRNA-L2 vs. siRNA-CQ2-L2 or siRNA-CQ3-L2 dosed at 5 mg siRNA / kg body weight.

[0039] FIG. 9F is a series of plots showing aspartate aminotransferase 48 hours following i.v. injection with siRNA-L2vs. siRNA-CQ2-L2or siRNA-CQ3-L2dosed at 5 mg siRNA / kg body weight.

[0040] FIG.9G is a series of plots showing total bilirubin 48 hours following i.v. injection with siRNA-L2 vs. siRNA-CQ2-L2 or siRNA-CQ3-L2 dosed at 5 mg siRNA / kg body weight.

[0041] FIG.10 shows hematoxylin and eosin (H&E) histology 48 hours following i.v. injection with siRNA-L2 vs. siRNA-Q2-L2 or siRNA-CQ3-L2 dosed at 5 mg siRNA / kg body weight. Scale bar = 100 m.

[0042] FIG. 11A is a schematic showing the synthesis of an example imidazole phosphoramidite reagent.

[0043] FIG. 11B is a schematic showing the synthesis of an example benzimidazole phosphoramidite reagent.

[0044] FIG. 12A shows the chemical structure of example imidazole siRNA conjugate (imidazole)3-EG18-L2.

[0045] FIG. 12B shows the chemical structure of example benzimidazole siRNA conjugate (benzimidazole)3-EG18-L2.

[0046] FIG. 13A is a liquid chromatography-mass spectrometry (LCMS) chromatogram confirming the purity and molecular weight of example conjugate siRNA-(imidazole)1-EG18-L2.Attorney Docket No.: 093386-0056-WO01

[0047] FIG. 13B is a liquid chromatography-mass spectrometry (LCMS) chromatogram confirming the purity and molecular weight of example conjugate siRNA-(benzimidazole)3-EG18- L2.

[0048] FIG. 14A shows an agarose gel electrophoresis of siRNA-EG18-L2, siRNA- (chloroquine)3-EG18-L2, siRNA-(imidazole)3-EG18-L2, and siRNA-benzimidazole3-EG18-L2, with or without human serum albumin (HSA), demonstrating binding of all siRNA conjugates to albumin.

[0049] FIG. 14B is a bar graph showing results from a YFP-Galectin 8 (Gal8) assay demonstrating enhanced endosomal escape with chloroquine, imidazole, and benzimidazole- containing siRNA-EG18-L2 conjugates, compared to the Lipofectamine RNAiMax positive control.

[0050] FIG. 14C shows representative microscopy images from a Gal8 assay performed with the same treatment groups.

[0051] FIG.15 is a bar graph showing luciferase mRNA knockdown results at 48 hours with 1 M negative control siRNA-EG18-L2(siNC EG18), luciferase-targeted siRNA-EG18-L2(siLuc EG18), siLuc-imidazole-EG18-L2(Imid-EG18), or siLuc-benzimidazole-EG18-L2(benz-EG18) without any transfection reagents (“carrier free”).

[0052] FIG. 16 shows cartoon schematics depicting example siRNA conjugates described herein (e.g., siRNA-EE3-L2, L-siRNA-EE2, L-siRNA-EE3, and L-siRNA-EE3-L2). The cartoon schematics are intended to show the relative positions of the binding ligand (L) and the endosomal escape moieties (EEs) with respect to the siRNA (i.e., the schematics do not show specific bonds / linkages involved in the connectivity between the conjugate elements). DETAILED DESCRIPTION 1. Definitions

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar orAttorney Docket No.: 093386-0056-WO01 equivalent to those described herein can be used in practice or testing of the disclosed technology. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0054] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0055] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0056] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0057] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about”Attorney Docket No.: 093386-0056-WO01 may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0058] The term “alkenyl” refers to a straight or branched, unsaturated hydrocarbon chain containing at least one carbon-carbon double bond.

[0059] The term “alkyl” refers to a straight or branched, saturated hydrocarbon chain. The term “C1-C3alkyl” means a straight or branched chain hydrocarbon containing from 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl.

[0060] The term “alkynyl” refers to a type of alkyl group in which the first two atoms of the alkyl group form a triple bond. That is, an alkynyl group begins with the atoms -C=C--R, wherein R refers to the remaining portions of the alkynyl group, which may be the same or different. Non- limiting examples of an alkynyl group include -C=CH, -C=CH3 and -C=CCH2CH3. The “R” portion of the alkynyl moiety may be branched, straight chain, or cyclic.

[0061] The term “amino,” as used herein, refers to –NH2.

[0062] The term “attached” refers to two moieties being attached through a bond where there can be intervening moieties or molecules in between. For example, the branching molecule can be attached to the oligonucleotide by having an intervening moiety, such as a second linker, between the oligonucleotide and the linker. Attached can also include “directly attached,” which refers to two moieties being attached through a bond with no other intervening moieties or molecules.

[0063] The term “carboxyl” refers to the group –C(=O)OR, wherein R is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl, any of which may be optionally substituted, e.g., with one or more substituents.

[0064] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0065] The term “hydroxyl” refers to an –OH group.

[0066] The term “oligonucleotide” refers to a polymer of nucleotides. The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide,” may be used interchangeably herein. Typically, a polynucleotide comprises at least three nucleotides. Oligonucleotides can be single stranded or double stranded. The polymer may include natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thithymidine, inosine, pyrrolo-Attorney Docket No.: 093386-0056-WO01 pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyriboses, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5’-N-phosphoramidite linkages). The oligonucleotide can have a modified backbone, such as having the sugar phosphate backbone being replaced with a peptide or peptide-like backbone (e.g., peptide nucleic acids). The oligonucleotide can also includemodified bases that contain a methylene bridge bond between the 2 oxygen and the 4 carbon ofthe pentose ring (e.g., locked nucleic acids). Example oligonucleotides include, but are not limited to, DNA and RNA, such as RNAi, siRNA, or shRNA.

[0067] An oligonucleotide includes a 5’ end and a 3’ end. Oligonucleotides are said to have “5' ends” and “3' ends” because nucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one nucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Thus, an end of an oligonucleotide can be referred to as the “5' end or terminus” if its 5' phosphate is not linked to the 3' oxygen of a nucleotide pentose ring and as the “3' end or terminus” if its 3' oxygen is not linked to a 5' phosphate of a subsequent nucleotide pentose ring.

[0068] The term “linker” refers to a moiety attaching two chemical groups together (e.g., a moiety attaching (1) an endosomal escape portion to (2) a binding ligand or an oligonucleotide). Example linkers may have 30 atoms or less in length. In some embodiments, the linkers are 1-15 carbon atoms in length, such as 1-12 carbon atoms, or 1-10 carbon atoms, or 5-10 carbon atoms in length. Example ligands may also be multivalent, e.g., to accommodate a multivalent binding ligand. Various types of bonds can be present in linkers contemplated herein. Example bonds include, but are not limited to, phosphorothioate bonds, phosphodiester bonds, cleavable linkers (e.g., deoxythymidine (dT)), and / or pH-cleavable bonds (e.g., ketal). Other types of bonds (e.g., amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas) may also be used in embodiments of the present disclosure. In some instances, the linker itself is a bond, including, but not limited to a phosphorothioate bond, a phosphodiester bond, cleavable linkers (e.g., deoxythymidine (dT)), and / or pH-cleavable bonds (e.g., ketal).Attorney Docket No.: 093386-0056-WO01

[0069] The term “specifically binds,” as used herein, is generally meant that a molecule binds to a target molecule when it binds to that target molecule more readily than it would bind to a random, unrelated target.

[0070] The terms “subject” or “subject in need thereof” refer to a target of administration, which optionally displays symptoms related to a particular disease, pathological condition, disorder, or the like. The subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0071] The terms “treatment” or “treating” refer to the medical management of a subject with the intent to heal, cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. 2. Conjugates

[0072] Described herein are conjugates including an oligonucleotide, a binding ligand, and an endosomal escape portion. Compared to existing oligonucleotide conjugates, the conjugates described herein mediate more efficient cytoplasmic delivery, thus providing improved therapeutic efficacy and potency.

[0073] The oligonucleotide, binding ligand, and endosomal escape portion can be attached to one another at various positions. For example, a linker may attach the endosomal escape portionAttorney Docket No.: 093386-0056-WO01 to the binding ligand or to the oligonucleotide. As depicted in FIG.16, in some embodiments, the endosomal escape portion can be attached to the 5’ terminus of the oligonucleotide. As further depicted in FIG. 16, in some embodiments, the binding ligand can be attached to the 3’ terminus of the oligonucleotide.

[0074] The endosomal escape portion, the binding ligand, and the oligonucleotide can also be attached to one another through various types of linkages. For example, the endosomal escape portion, the binding ligand, and / or the oligonucleotide can be attached through phosphorothioate bonds, phosphodiester bonds, a cleavable linker (e.g., deoxythymidine (dT), pH-cleavable bond such as ketal), or a combination thereof. In some embodiments, the endosomal escape portion, the binding ligand, and / or the oligonucleotide are attached through phosphorothioate bonds.

[0075] In some embodiments, the conjugate includes about 20% to about 80% phosphorothioate linkages based on the total amount of phosphate-based linkages in the conjugate, such as about 25% to about 75% phosphorothioate linkages, about 30% to about 70% phosphorothioate linkages, about 35% to about 55% phosphorothioate linkages, or about 40% to about 50% phosphorothioate linkages - based on the total amount of phosphate-based linkages in the conjugate. The combination of phosphodiester linkages and phosphorothioate linkages can be referred to as the total amount of phosphate-based linkages, and is not inclusive of potential phosphorylation of sequences, e.g., to avoid deactivation of phosphatases.

[0076] In some embodiments, the conjugate may include an oligonucleotide; an endosomal escape portion attached to the oligonucleotide, the endosomal escape portion comprising an endosomal escape moiety; a lipophilic ligand capable of binding albumin; and a linker attaching the endosomal escape portion to the lipophilic ligand capable of binding albumin, the linker comprising: a branching molecule attached to the endosomal escape portion, and a hydrophilic spacer attaching the branching molecule to the lipophilic ligand, the branching molecule comprising: at least one branch point having at least two independent branches.

[0077] In other embodiments, the conjugate may include an oligonucleotide; a saccharide ligand attached to the oligonucleotide, the saccharide ligand comprising N-acetylgalactosamine (GalNAc); and an endosomal escape portion comprising 1 to 10 endosomal escape moieties; and a linker attaching the endosomal escape portion to the oligonucleotide, the linker comprising: a branching molecule attached to the endosomal escape portion, the branching molecule comprising: at least one branch point having at least two independent branches.Attorney Docket No.: 093386-0056-WO01

[0078] The conjugate’s arrangement and composition can provide advantageous benefits, such as being able to bind albumin or liver cells while also minimizing its propensity to self-assemble into micelles. For example, when the conjugate includes a lipophilic ligand, the conjugate can have a binding affinity (Kd) to albumin of less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 500 nM, less than 250 nM, or less than 100 nM. In some embodiments, the conjugate has a Kdto albumin of greater than 0.1 nM, greater than 0.2 nM, greater than 0.4 nM, greater than 0.5 nM, greater than 0.6 nM, greater than 0.7 nM, greater than 0.8 nM, greater than 0.9 nM, greater than 1 nM, or greater than 5 nM. In some embodiments, the conjugate has a Kdto albumin of about 1 μM to about 10 μM, such as about 1 μM to about 9 μM, about 2 μM to about 8 μM, about 3 μM to about 7 μM, or about 4 μM to about 6 μM. The conjugate can reversibly bind albumin. In some embodiments, the conjugate does not covalently bind to albumin.

[0079] In some embodiments, when the conjugate includes a saccharide ligand (e.g., comprising N-acetylgalactosamine (GalNAc)), the conjugate can have a binding affinity to a liver cell receptor (e.g., the asialoglycoprotein receptor (ASGPR)) of less than 10 nM, less than 9 nM, less than 8 nM; less than 7 nM; less than 6 nM; less than 5 nM; less than 4 nM; less than 3 nM; less than 2 nM; or less than 1 nM. In some embodiments, the conjugate has a Kdto a liver cell receptor of greater than 0.1 nM, greater than 0.2 nM, greater than 0.4 nM, greater than 0.5 nM, greater than 0.6 nM, greater than 0.7 nM, greater than 0.8 nM, greater than 0.9 nM, greater than 1 nM, or greater than 5 nM. In some embodiments, the conjugate has a Kd to a liver cell receptor of about 1 nM to about 10 nM, such as about 1 nM to about 9 nM, about 2 nM to about 8 nM, about 3 nM to about 7 nM, or about 4 nM to about 6 nM. The conjugate can reversibly bind a liver cell receptor. In some embodiments, the conjugate does not covalently bind to a liver cell receptor. A. Oligonucleotide

[0080] Various oligonucleotides may be used in the conjugates described herein. The oligonucleotide can instill a therapeutic and / or beneficial property to the conjugate. The oligonucleotide can be single stranded or double stranded. An example of a single stranded oligonucleotide includes, but is not limited to, single stranded antisense oligonucleotides (e.g., single stranded antisense DNA and / or RNA). The oligonucleotide can include DNA, RNA, a synthetic mimic of DNA or RNA, or a combination thereof. In some embodiments, theAttorney Docket No.: 093386-0056-WO01 oligonucleotide includes DNA, RNA, or a synthetic mimic of DNA or RNA. In some embodiments, the oligonucleotide includes RNA. In some embodiments, the oligonucleotide is RNA. Example RNAs include, but are not limited to, siRNA, miRNA, and an antisense RNA. In some embodiments, the oligonucleotide includes siRNA, miRNA or single stranded antisense RNA.

[0081] The oligonucleotide can be attached at various positions to other portions of the conjugate. For example, the oligonucleotide can have the binding ligand attached to it 5’ end (e.g., through the linker and the endosomal escape portion) or to its 3’ end (e.g., directly attached). The benefits realized from the end modifications of the disclosed conjugates (e.g., attaching the endosomal escape moiety to the 5’ end of the oligonucleotide and / or attaching the binding ligand to the 3’ end of the oligonucleotide) can be used with any desirable oligonucleotide. That is, the oligonucleotide of the conjugate is sequence agnostic. This modification can be added to either single or double stranded oligonucleotides that contain either natural or modified nucleotide bases. The single or double stranded nucleotides can also be of variable length, such as 10 to 30 bases in length.

[0082] For an example process of selecting an oligonucleotide, such as siRNA, the sequence can be first determined using publicly available prediction algorithms. These algorithms can generate many candidate sequences for targeting any given gene. These potential sequences are first screened for on-target gene silencing potency in vitro (without chemical modifications). After identification of one or more potent sequences, chemical modifications can be added to the sequence, and it can be rescreened for in vitro gene silencing activity prior to screening for albumin binding affinity and pharmacokinetic / pharmacodynamic behaviors in vivo. The disclosed albumin binding end chemistry may be successfully integrated with multiple sequences targeting any single gene and may also be adapted for delivery of sequences against theoretically any gene of interest.

[0083] The oligonucleotide can include a plurality of stabilizing modifications. Examples of stabilizing modifications include, but are not limited to, phosphorothioate linkages, 2’F modification, 2’OMe modification, and combinations of 2’F and 2’OMe modifications (e.g., zipper pattern). In addition, the oligonucleotide can include both phosphodiester linkages and phosphorothioate linkages. In some embodiments, the oligonucleotide includes a plurality of phosphorothioate linkages. In some embodiments, the oligonucleotide includes phosphorothioate linkages at its terminal end(s). In some embodiments, the oligonucleotide includes about 1% toAttorney Docket No.: 093386-0056-WO01 about 30% phosphorothioate linkages based on a total amount of phosphate-based linkages in the oligonucleotide, such as about 10% to about 25% phosphorothioate linkages or about 15% to about 24% phosphorothioate linkages – based on a total amount of phosphate-based linkages in the oligonucleotide. In some embodiments, the oligonucleotide includes about 21% phosphorothioate linkages based on a total amount of phosphate-based linkages in the oligonucleotide.

[0084] The oligonucleotide can have a varying amount of nucleotides. For example, the oligonucleotide can have about 15 to about 40 nucleotides, such as about 16 to about 38 nucleotides, about 15 to about 35 nucleotides, about 18 to about 32 nucleotides, about 18 to about 30 nucleotides, or about 20 to about 35 nucleotides.

[0085] In some embodiments, the oligonucleotide includes a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof. B. Binding Ligand

[0086] The term “binding ligand” refers to a ligand that is capable of binding (e.g., specifically binding) a particular target site (e.g., a specific protein, receptor etc.). Various binding ligands may be used in the conjugates described herein. The particular binding ligand used may vary based on the conjugate and / or the intended target site. Example target sites include, but are not limited to, albumin, liver cells (e.g., hepatocytes) tumor cells, and macrophages.

[0087] The binding ligand may bind to its corresponding target site with high specificity, e.g., a binding affinity (Kd) of less than 100 nM. Example target sites for binding ligands described herein include, but are not limited to, albumin, liver cells (e.g., hepatocytes), tumor cells, and macrophages. Binding ligands can bind to their corresponding target sites through various covalent and / or non-covalent interactions. Example non-covalent interactions include, but are not limited to, hydrogen bonds, ionic interactions, and hydrophobic effects.

[0088] In various embodiments, the binding ligand is multivalent. In some embodiments, the binding ligand is divalent. Example binding ligands include, but are not limited to, lipophilic ligands, saccharide ligands, and folate ligands. In some embodiments, the binding ligand comprises a lipophilic ligand or a saccharide ligand. Various aspects of lipophilic ligands and saccharide ligands are described below.Attorney Docket No.: 093386-0056-WO01 a. Lipophilic Ligand

[0089] When present, the lipophilic ligand may be capable of binding albumin and thus can instill in the conjugate the ability to bind albumin. The lipophilic ligand can include any lipophilic moiety suitable for binding into a fatty acid pocket of albumin. The arrangement and composition of the lipophilic ligand can provide the conjugate with advantageous properties, such as, but not limited to, imparting hydrophobicity to the conjugate, which may synergize with the endosome escaping portion to potentiate membrane penetration.

[0090] In some embodiments, the lipid can include a C4-C22hydrocarbon chain, such as a C6- C22 hydrocarbon chain, a C12-C22 hydrocarbon chain, a C12-C20 hydrocarbon chain, a C14-C22 hydrocarbon chain, a C16-C22 hydrocarbon chain, or a C16-C20 hydrocarbon chain. In some embodiments, the lipid includes a C18 hydrocarbon chain. The lipid can be saturated or unsaturated. In addition, the lipid may have a terminal end. The terminal end may include a functional group that may aid in binding. In some embodiments, the terminal end of the lipid includes an alkyl, carboxyl, hydroxyl, or amino. In some embodiments, the terminal end of the lipid includes an alkyl or carboxyl. In embodiments where there is more than one lipid, each lipid can include a different functional group at its terminal end. For example, one terminal end can include an alkyl and one terminal end can include a carboxyl. In some embodiments, the terminal end includes an alkyl. In some embodiments, the terminal end of the lipid does not include a hydroxyl or a carboxyl.

[0091] The lipophilic ligand can include more than one lipid. Having more than one lipid can allow for multivalency of the lipophilic ligand and the conjugate thereof. The lipophilic ligand can include at least 2 lipids, at least 3 lipids, at least 4 lipids, at least 5 lipids, at least 6 lipids, at least 7 lipids, or at least 8 lipids. In some embodiments, the lipophilic ligand includes less than 10 lipids, less than 9 lipids, less than 8 lipids, less than 7 lipids, less than 6 lipids, or less than 5 lipids. In some embodiments, the lipophilic ligand includes 1 to 10 lipids, such as 1 to 8 lipids, 1 to 6 lipids, 2 to 8 lipids, 2 to 6 lipids, 2 to 4 lipids, or 2 to 3 lipids.

[0092] In some embodiments, the lipophilic ligand incudes two independent lipids, each lipid including a C12-C22 hydrocarbon chain. In some embodiments, the lipophilic ligand includes two independent lipids, each lipid including a C18 hydrocarbon chain.

[0093] The lipophilic ligand and lipid(s) can be attached to the linker. In some embodiments, the lipophilic ligand and lipid(s) are directly attached to the linker, e.g., a linker comprising one orAttorney Docket No.: 093386-0056-WO01 more spacers and a branching molecule. In some embodiments, the lipophilic ligand includes two individual lipids, each lipid bound to a separate, individual spacer which is bound to a separate branch of the branching molecule. In such embodiments, the lipids may be the same or different. For example, the lipids can both include C18 hydrocarbon chains. Alternatively, in other embodiments, the lipids can include hydrocarbon chains of varying length. In some embodiments, the lipophilic ligand includes two distinct types of lipids. b. Saccharide Ligand

[0094] When present, the saccharide ligand may be capable of binding at least one type of receptor on the surface of a liver cell (e.g., an asialoglycoprotein receptor (ASGPR)) and thus can instill in the conjugate the ability to bind a liver cell. In some embodiments, the saccharide ligand may be capable of binding at least one type of receptor on the surface of a macrophage (e.g., a M2 anti-inflammatory macrophage).

[0095] The saccharide ligand may comprise various types of saccharides. For instance, the saccharide ligand may comprise a monosaccharide, a polysaccharide and / or derivatives thereof (e.g., amino and thio derivatives). In some embodiments, the saccharide ligand comprises glucose, mannose, galactose, fucose, and / or a derivative thereof. In some embodiments, the saccharide ligand comprises an amino saccharide such as, but not limited to, glucosamine, sialic acid, -D- galactosamine, N-acetylgalactosamine (GalNAc, i.e., 2-acetamido-2-deoxy-D-galactopyranose),2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy- -D-glucopyranose ( -muramic acid), 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-deoxy-2-sulfoamino-D-glucopyranose, N-sulfo-D-glucosamine, or N-glycoloyl- -neuraminicacid. In some instances, the saccharide comprises mannose. In some instances, the saccharide comprises N-acetylgalactosamine (GalNAc).

[0096] The saccharide ligand can include more than one saccharide. Having more than one saccharide can allow for multivalency of the saccharide ligand and the conjugate thereof. The saccharide ligand can include at least 2 saccharides at least 3 saccharides at least 4 saccharides at least 5 saccharides at least 6 saccharides at least 7 saccharides or at least 8 saccharides In some embodiments, the saccharide ligand includes less than 10 saccharides less than 9 saccharides less than 8 saccharides less than 7 saccharides less than 6 saccharides or less than 5 saccharides InAttorney Docket No.: 093386-0056-WO01 some embodiments, the saccharide ligand includes 1 to 10 saccharides such as 1 to 8 saccharides 1 to 6 saccharides 2 to 8 saccharides 2 to 6 saccharides 2 to 4 saccharides or 2 to 3 saccharides.

[0097] In some embodiments, the saccharide ligand incudes two or three independent saccharides each saccharide including an amino saccharide. In some embodiments, the saccharide ligand includes two or three independent saccharides, wherein each saccharide includes GalNac.

[0098] The saccharide ligand and saccharide(s) can be attached to the linker. In some embodiments, the saccharide ligand and saccharide(s) are directly attached to the linker, e.g., a linker comprising one or more spacers and a branching molecule. In some embodiments, the saccharide ligand includes two or three individual saccharides each saccharide bound to a separate, individual spacer which is bound to a separate branch of the branching molecule. In such embodiments, the saccharides may be the same or different. For example, the saccharides can include the same saccharide (e.g., GalNac). Alternatively, in other embodiments, the saccharides can include two or three distinct types of saccharide. C. Endosomal Escape (EE) Portion

[0099] The “endosomal escape (EE) portion” of the conjugates described herein is a conjugate portion comprising an endosomal escape (EE) moiety. The term “endosomal escape moiety” refers to a moiety which promotes the release of cargo (e.g., a therapeutic oligonucleotide) from a vesicle of the endocytic pathway (e.g., endosome and lysosome) so that the cargo may be delivered to its intended intracellular target. Endosomal escape moieties employ various mechanisms to promote the release of cargo from endosomal vesicles. For example, in various instances, endosomal escape moieties having weakly basic properties (e.g., aminoquinolines, imidazoles, benzimidazoles, etc.) become protonated in the acidic endosomal environment, thereby inducing a “proton sponge effect.” This effect causes an influx of ions and water into the endosome, leading to osmotic swelling and eventual rupture, thereby releasing the cargo. Additionally, endosomal escape moieties may insert themselves within the endosomal / lysosomal membrane, thereby disrupting the membrane

[0100] The number of endosomal escape moieties may vary. In some embodiments, the endosomal escape portion comprises 1 to 10 endosomal escape moieties. In some embodiments, the endosomal escape portion comprises 1 to 8, 2 to 6, or 2 to 4 endosomal escape moieties. In some embodiments, the endosomal escape portion comprises no less than 2 endosomal escapeAttorney Docket No.: 093386-0056-WO01 moieties, no less than 3 endosomal escape moieties, or no less than 4 endosomal escape moieties. In some embodiments, the endosomal escape portion comprises no more than 10 endosomal escape moieties, no more than 8 endosomal escape moieties, or no more than 4 endosomal escape moieties.

[0101] The types of endosomal escape moieties may also vary. Various example endosomal escape moieties suitable for use in the presently disclosed conjugates are described in Ravindar L., et al., Eur. J. Med. Chem. 2024, 264:116043, which is herein incorporated by reference in its entirety. In various embodiments, each endosomal escape moiety independently comprises an aminoquinoline, an imidazole, or a benzimidazole. Example aminoquinolines include, but are not limited to, 4-aminoquinolines such as chloroquine, amodiaquine, hydroxy chloroquine, and amopyroquine. In some embodiments, at least one endosomal escape moiety in the conjugate comprises chloroquine. D. Linker

[0102] The endosomal escape portion can be attached to the binding ligand or to the oligonucleotide through a linker. The arrangement and composition of the linker can provide the conjugate with advantageous properties, such as, but not limited to, improved binding to the target site (e.g., albumin, a liver cell, etc.), decreased propensity to self-assembly into micelles, and improved pharmacokinetics.

[0103] In various embodiments, the linker may comprise or consist of one or more phosphorothioate bonds, phosphodiester bonds, cleavable linkers (e.g., deoxythymidine (dT)), and / or pH-cleavable bonds (e.g., ketal). In some embodiments, the linker comprises one or more phosphorothioate bonds.

[0104] In some embodiments, the linker includes a branching molecule. In some embodiments, the linker includes a branching molecule and spacer. Various aspects of example branching molecules and spacers are described below. a. Branching Molecule

[0105] When present, the branching molecule can be any suitable molecule that allows for branching of the conjugate. The branching molecule can be included in the conjugate as a way to introduce multifunctionality, such as multivalency, to the conjugate. Example branchingAttorney Docket No.: 093386-0056-WO01 molecules include, but are not limited to, a phosphoramidite (e.g., symmetrical branching CED phosphoramidite), a tri-valent splitter, or a tetra-valent splitter.

[0106] In some embodiments, the branching molecule can be positioned between the endosomal escape portion and the binding ligand or the oligonucleotide. In some embodiments, the branching molecule directly attaches the endosomal escape portion to the oligonucleotide or the binding ligand. In other embodiments, the branching molecule attaches the endosomal escape portion to a spacer, wherein the spacer is attached to the binding ligand or the oligonucleotide. The branching molecule can be directly attached, or conjugated, to the oligonucleotide, binding ligand, endosomal escape portion, and / or spacer through a phosphorothioate bond, phosphodiester, or cleavable linker (e.g., deoxythymidine (dT), pH-cleavable bond such as ketal). Although, in some embodiments, there are no intervening moieties or molecules when directly attached, as will be appreciated by those skilled in the art, the placement of the branch point within the branching molecule may be adjusted based upon the structure of the branching molecule itself.

[0107] The branching molecule can have multiple, independent branch points, each branch point having at least two independent branches. For example, the branching molecule can have at least 2 branch points, at least 3 branch points, at least 4 branch points, or at least 5 branch points. In some embodiments, the branching molecule has less than 7 branch points, less than 6 branch points, less than 5 branch points, or less than 4 branch points. In some embodiments, the branching molecule has 1 to 5 branch points, such as 1 to 4 branch points, 1 to 3 branch points, or 1 to 2 branch points.

[0108] Each branch point can have multiple, independent branches. For example, each branch point can have at least 2 branches, at least 3 branches, at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, or at least 10 branches. In some embodiments, each branching point can have less than 12 branches, less than 11 branches, less than 10 branches, less than 9 branches, less than 8 branches, less than 7 branches, less than 6 branches, less than 5 branches, or less than 4 branches. In some embodiments, each branch point has 2 to 12 branches, such as 2 to 10 branches, 2 to 8 branches, 2 to 6 branches, or 2 to 4 branches. In some embodiments, each branch point has 2 branches.

[0109] In some embodiments, the branching molecule can have independent branches that are attached to independent binding ligand components (e.g., lipids of the lipophilic ligand). For example, in some instances, when the binding ligand is the lipophilic ligand, the branchingAttorney Docket No.: 093386-0056-WO01 molecule can have 2 independent branches that are attached to 2 independent lipids or 3 independent branches that are attached to 3 independent lipids. Alternatively, in some embodiments, not every branch is attached to a binding ligand component. For example, in some instances, when the binding ligand is the lipophilic ligand, the branching molecule can have 4 branches, where only 2 of the 4 branches are attached to a lipid.

[0110] In some embodiments, the branching molecule can have independent branches that are attached to independent endosomal escape moieties. For example, the branching molecule can have 2 independent branches that are attached to 2 independent endosomal escape moieties or 3 independent branches that are attached to 3 independent endosomal escape moieties. b. Spacer

[0111] When present, the spacer can include any suitable compound for attaching the binding ligand, oligonucleotide, or endosomal escape portion to the linker. In some embodiments, when the branching molecule and spacer are both present, the spacer may be attached to the branching molecule. As discussed above, the branching molecule can include a branching point having at least two independent branches. Each branch of the branching molecule can be attached to an individual spacer. For example, depending on the conjugate, each spacer can be individually attached to an individual binding ligand component (e.g., a lipid) or endosomal escape moiety. In some embodiments, the spacer attaches the branching molecule to the binding ligand or oligonucleotide through phosphorothioate bonds.

[0112] In various embodiments, the spacer is a hydrophilic spacer comprising one or more hydrophobic compounds. Examples of suitable hydrophilic compounds include, but are not limited to, ethylene glycol, zwitterionic linkers, peptoids (e.g., poly(sarcosine)), amino acids, poly(ethylene glycol) substitutes including, but not limited to, poly(glycerols), poly(oxazoline), poly(acrylamide), poly (N-acryloyl morpholine, poly(N,N-dimethyl acrylamide), poly(2- hydroxypropyl methacrylamide), poly(2-hydroxyethyl methacrylamide), and other similar hydrophilic spacer molecules and / or polymers.

[0113] When present, the hydrophilic spacer can include at least one hydrophilic block. For example, the hydrophilic spacer can include 1 to 100 hydrophilic blocks, such as 1 to 50 hydrophilic blocks, 1 to 20 hydrophilic blocks, as 1 to 18 hydrophilic blocks, 2 to 15 hydrophilic blocks, 3 to 10 hydrophilic blocks, 2 to 10 hydrophilic blocks, 1 to 15 hydrophilic blocks, 1 to 10Attorney Docket No.: 093386-0056-WO01 hydrophilic blocks, 2 to 8 hydrophilic blocks, 2 to 6 hydrophilic blocks, or 1 to 7 hydrophilic blocks. In some embodiments, the hydrophilic spacer includes 5 hydrophilic blocks. In some embodiments, the hydrophilic blocks can be attached to each other through phosphorothioate bonds, phosphodiester, or a cleavable linker (e.g., deoxythymidine (dT), pH-cleavable bond such as ketal). In some embodiments, each of the hydrophilic blocks are attached to each other through phosphorothioate linkages.

[0114] The hydrophilic block can include repeats of the hydrophilic compound. In some embodiments, the hydrophilic spacer includes 1 to 100 hydrophilic blocks (as described above), with each of the repeating blocks including 1 to 150 repeats of the hydrophilic compound, such as 1 to 100 repeats of the hydrophilic compound, 2 to 50 repeats of the hydrophilic compound, 1 to 45 repeats of the hydrophilic compound, 1 to 30 repeats of the hydrophilic compound, 2 to 20 repeats of the hydrophilic compound, or 2 to 10 repeats of the hydrophilic compound. In some embodiments, each hydrophilic block includes less than 150 repeats of the hydrophilic compound, less than 100 repeats of the hydrophilic compound, less than 75 repeats of the hydrophilic compound, less than 50 repeats of the hydrophilic compound, less than 45 repeats of the hydrophilic compound, less than 40 repeats of the hydrophilic compound, or less than 35 repeats of the hydrophilic compound. In some embodiments, each hydrophilic block includes greater than 2 repeats of the hydrophilic compound, greater than 3 repeats of the hydrophilic compound, greater than 4 repeats of the hydrophilic compound, greater than 5 repeats of the hydrophilic compound, greater than 6 repeats of the hydrophilic compound, greater than 7 repeats of the hydrophilic compound, or greater than 8 repeats of the hydrophilic compound.

[0115] In some embodiments, the hydrophilic spacer includes 1 to 10 hydrophilic blocks, with each block including 1 to 15 repeats of the hydrophilic compound. In some embodiments, the hydrophilic spacer includes 1 to 10 hydrophilic blocks, with each block including 1 to 10 repeats of the hydrophilic compound. In some embodiments, the hydrophilic spacer includes 1 to 6 hydrophilic blocks, with each block including 2 to 10 repeats of the hydrophilic compound. In some embodiments, the hydrophilic spacer includes 2 to 6 hydrophilic blocks, with each block including 3 to 8 repeats of the hydrophilic compound.

[0116] The hydrophilic compound can be included in different variations as part of the hydrophilic block. For example, the hydrophilic spacer can include 1 block including 150 repeats of the hydrophilic compound, 2 blocks each including 50 repeats of the hydrophilic compound, 5Attorney Docket No.: 093386-0056-WO01 blocks each including 6 repeats of the hydrophilic compound, 2 blocks – one block including 5 repeats of the hydrophilic compound and the other block including 10 repeats of the hydrophilic compound, or any combination of blocks and repeats as disclosed herein.

[0117] In some embodiments, the hydrophilic spacer includes a plurality of ethylene glycol repeats. For example, the hydrophilic spacer can include 1 to 150 ethylene glycol repeats, 1 to 120 ethylene glycol repeats, 1 to 100 ethylene glycol repeats, 1 to 90 ethylene glycol repeats, 1 to 80 ethylene glycol repeats, 1 to 70 ethylene glycol repeats, 1 to 60 ethylene glycol repeats, 1 to 50 ethylene glycol repeats, 1 to 40 ethylene glycol repeats, 1 to 30 ethylene glycol repeats, 2 to 150 ethylene glycol repeats, 3 to 150 ethylene glycol repeats, 4 to 150 ethylene glycol repeats, 5 to 150 ethylene glycol repeats, 6 to 150 ethylene glycol repeats, 7 to 150 ethylene glycol repeats, 8 to 150 ethylene glycol repeats, 9 to 150 ethylene glycol repeats, 10 to 150 ethylene glycol repeats, 10 to 140 ethylene glycol repeats, 10 to 130 ethylene glycol repeats, 10 to 120 ethylene glycol repeats, 10 to 110 ethylene glycol repeats, 10 to 100 ethylene glycol repeats, 10 to 90 ethylene glycol repeats, 10 to 80 ethylene glycol repeats, 10 to 70 ethylene glycol repeats, 10 to 60 ethylene glycol repeats, 6 to 60 ethylene glycol repeats, 6 to 40 ethylene glycol repeats, 8 to 35 ethylene glycol repeats, or 10 to 32 ethylene glycol repeats. The ethylene glycol repeats can be included as a hydrophilic block in different variations as described above.

[0118] In some embodiments, the hydrophilic spacer includes 1 and 10 hexaethylene glycol blocks (e.g., blocks of six ethylene glycol repeats). In some embodiments, the hydrophilic spacer includes 1 and 5 hexaethylene glycol blocks. In some embodiments, the hexaethylene glycol blocks are attached to each other through phosphorothioate bonds, phosphodiester, or cleavable linker (e.g., deoxythymidine (dT), pH-cleavable bond such as ketal).

[0119] In some embodiments, the length of the hydrophilic spacer may be adjusted to provide desired properties. For example, ethylene glycol spacers with 18 ethylene glycol repeats can provide a higher binding to albumin. Alternatively, shorter spacers can yield conjugates with increased hydrophobicity, which can result in more tendency to bind lipoprotein complexes in the blood. E. Synthesis of Conjugates

[0120] Also provided herein are methods of synthesizing the conjugates. In some embodiments, the method includes solid phase synthesis where the full molecule is made / grown from a solidAttorney Docket No.: 093386-0056-WO01 support, as opposed to solution phase conjugation of the oligonucleotide to the endosomal escape portions / binding ligand moieties post-solid phase synthesis.

[0121] In some embodiments, the endosomal escape moiety can be installed in the conjugates using an endosomal escape (EE)-modified phosphoramidite reagent. In various embodiments, EE- modified phosphoramidite reagents may be synthesized as shown below in Scheme 1. Further description of the synthesis of the conjugates can be found in the Examples disclosed herein. Scheme 1.

[0122] As shown in Scheme 1 above, the reaction of an EE-modified compound of formula i may be reacted with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite under suitable reaction conditions (e.g., in the presence of base and solvent) to provide EE-modified phosphoramidites of formula ii. 3. Uses of the Conjugates A. Compositions

[0123] Also disclosed herein are compositions that include the conjugate and one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include, but are not limited to, buffering agents (e.g., phosphate buffered saline, artificial cerebrospinal fluid (aCSF), etc.), carbohydrates (e.g., glucose, trehalose, starch, etc.) solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, or a combination thereof. In some embodiments, the composition does not include a carrier composition, such as a polymer- or lipid-based formulation. The description of the conjugate, oligonucleotide, binding ligand, endosomal escape portion, and linker above may be applied to the disclosed compositions. B. Administration

[0124] The composition can be administered prophylactically or therapeutically. In prophylactic administration, the composition can be administered in an amount sufficient to induce a response. In therapeutic applications, the composition can be administered to a subject in needAttorney Docket No.: 093386-0056-WO01 thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the conjugate regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0125] The composition may be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0126] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and subjects treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies.

[0127] Dosage amount and interval may be adjusted individually to provide plasma levels of the biologically active agent which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each agent but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, assays well known to those in the art can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions can be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, such as between 30-90% or between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0128] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also varyAttorney Docket No.: 093386-0056-WO01 according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine. C. Methods of Gene Silencing

[0129] Further provided herein are methods of gene silencing using the conjugate or composition thereof to a subject in need thereof. Albumin is the most abundant protein in human serum and is naturally rescued from renal or tissue clearance because it is recycled by the FcRn receptor, leading to recycling back into the blood stream. Due to its extended half-life in the body of approximately 19 days, its natural ability to transport fatty acids and other hydrophobic cargo, and its accumulation at sites of inflammation and vascular leakiness, albumin is an appealing drug carrier. Notably, albumin is also preferentially taken up and used as an amino acid source by cancer cells to meet their high energy needs. Accordingly, in some embodiments, the improved albumin binding of the conjugates disclosed herein provides improved drug delivery and / or uptake by cancer cells.

[0130] While oligonucleotide-based therapeutics can be limited by rapid renal clearance, nuclease degradation, and inability to target / penetrate cells of interest, the conjugates disclosed herein can provide improved circulation half-life, can shield the oligonucleotide from nucleases, and / or can provide extrahepatic delivery of the oligonucleotides. In addition, these advantages can be done without an associated carrier composition, such as a polymer or lipid formulation. Accordingly, in some embodiments the conjugate or composition thereof is administered without an associated carrier composition. In various embodiments, the conjugate or composition thereof can be administered intravenously.

[0131] The method can be used to treat cancer. For example, cells in nutrient starved microenvironments characteristic of tumors can preferentially internalize albumin, thus can provide a mechanism for cell entry and targeted delivery thereto. In some embodiments, the method includes delivering an oligonucleotide to an extrahepatic target by administering one or more of the conjugates disclosed herein to a subject in need thereof. In some embodiments, the method includes treating cancer by administering one or more of the conjugates disclosed herein to a subject in need thereof. Example cancers include, but are not limited to, solid tumor malignancies, such as breast, pancreas, hepatic, bile duct (cholangiocarcinoma), and lung.Attorney Docket No.: 093386-0056-WO01

[0132] The description of the conjugate, oligonucleotide, binding ligand, endosomal escape portion, and linker above may be applied to the disclosed methods. 4. Examples

[0133] Without limiting the scope of the instant disclosure, various experimental examples of embodiments discussed above were prepared and the results are discussed below. Example 1 Materials & Methods

[0134] Desulfation of Hydroxychloroquine. Hydroxychloroquine sulfate (TCI) was desulfated according to previously described methods. Briefly, 6.0 g of hydroxychloroquine sulfate wasdissolved in 40 mL of water. 5.0 mL ofNH4OH was added dropwise while stirring vigorously.After 30 minutes, 40 mL of dichloromethane was added. After 10 more minutes, the solution was transferred to a separatory funnel, and the organic layer was collected and washed with brine, then dried with sodium sulfate. Dichloromethane was evaporated in vacuo, yielding hydroxychloroquine.

[0135] Synthesis of Chloroquine Phosphoramidite. Desulfated hydroxychloroquine (2 mmol) was dissolved in 3 mL of anhydrous dichloromethane. 2.8 mmol of N,N-diisopropylethylamine (DIPEA, Millipore Sigma) was added while stirring at room temperature. 1.8 mmol of 2- cyanoethyl N,N-diisopropylchlorophosphoramidite (Millipore Sigma) was added dropwise and the reaction was stirred at room temperature for 1 hour under nitrogen. The reaction mixture was extracted against brine x2 and then dried with sodium sulfate for 30 minutes followed by evaporation of dichloromethane in vacuo. The product was resuspended in 3:1 anhydrous dichloromethane:acetonitrile and transferred into an oven-dried glass vial for use on a Mermade oligonucleotide synthesizer.

[0136] NMR Confirmation of Chloroquine Phosphoramidite. 1H-NMR (400 MHz, DCM-d2) 1.00 (s, 3H), 1.02 (s, 3H), 1.04 (s, 3H), 1.17 (s, 3H), 1.19 (s, 3H), 1.20 (s, 3H), 1.60–1.63 (m, 2H), 1.65–1.69 (m, 2H), 2.52–2.71 (m, 11H), 3.60-3.64 (m, 2H), 3.74-3.80 (m, 3H), 6.48 (d, J =8.5 Hz, 1H), 7.43 (dd, J = 9.0, 2.2 Hz, 1H), 7.82 (dd, J = 8.9, 2.1Hz, 1H), 7.92 (d, J = 2.5 Hz, 1H),8.51 (d, J = 8.4 Hz, 1H).31P-NMR (400 MHz, DCM-d2) 147.57 (s, 1P).Attorney Docket No.: 093386-0056-WO01

[0137] Synthesis of Chloroquine-modified siRNA-L2. The on-column synthesis of the siRNA-L2backbone on aMermade oligonucleotide synthesizer has been previously described. Briefly, thesense strand of the siRNA sequence is synthesized using Zipper modifications on the RNA bases (alternating 2’ O-methyl and 2’-fluoro modifications), along with phosphorothioate linkages between the three terminal bases on both the 5’ and 3’ ends of both strands. The EG18-L2tail, consisting of a symmetrical brancher (ChemGene) followed by 3 repeats of (ethylene glycol)6 anda terminal stearic acid (C18) on each branch, is appended to the 5’ end of the sense(passenger)siRNA strand, with each group linked by a phosphorothioate bond for stability. In contrast to the previously published structure, the addition of 1-3 instances of an asymmetric brancher(ChemGene) has been interpolated, in which one arm bears a conventional tritylprotecting group while the other is protected with alevulinyl group (FIG. 1B). Initially, the tritylarm is extended with the remainder of the L2tail (symmetric brancher followed by 3 instances ofethylene glycol (EG)6and terminal stearyl groups, all linked byphosphorothioate bonds. Then, thependant levulinyl-protected hydroxyl group was deprotected with hydrazine hydrate (Millipore Sigma) in 1:1 acetic acid:pyridine, washed with 1:1 acetic acid:pyridine followed by anhydrous acetonitrile x4, and finally conjugated the chloroquine phosphoramidite. The siRNA-CQ-L2conjugates were cleaved from the columns using 1:1 ammonium hydroxide:methylamine followed by desalting, HPLC purification (Shimadzu), and removal of organic and aqueous solvents using a Thermo SpeedVac SPD120, as previously described. siRNA-CQ-L2 was resuspended in sterilesaline and molecular weight was confirmed using liquidchromatography-mass spectrometry(LCMS) as previously described.

[0138] Synthesis of Chloroquine-modified GalNAc-siRNA. Oligonucleotide syntheses were performed using standard solid-phase chemistry with a MerMade 12 automated RNA synthesizer (BioAutomation) on controlled pore glass with a universal support (1 or 10 mol scale, 1000 Åpore), using 2 -F and 2 -OMe phosphoramidites with standard protecting groups (Glen Research).GalNac is introduced on the 3’ end using GalNAc Trivalent TEG 1000 Å CPG (Gene Link). A symmetric brancher (ChemGenes) or a trebler (Glen Research) were added on the 5’ end, to yield GalNac siRNA-CQ2 and -CQ3, respectively, after addition of the chloroquine phosphoroamidite. Oligonucleotide conjugates were cleaved from the columns using 1:1 ammonium hydroxide:methylamine (40% in H2O, Millipore Sigma) followed by desalting and IEX purification (Source 15Q anion-exchange column, Cytiva). A final desalting is performed beforeAttorney Docket No.: 093386-0056-WO01 sterile filtration and lyophilization. siRNA conjugates were resuspended in sterile saline and molecular weight was confirmed using liquid chromatography-mass spectrometry (LCMS) as previously described.

[0139] Molecular Weight and AlogP Calculations. Molecular weights were calculated using ChemDraw v22.2 (Revvity) and AlogP was calculated using BIOVIA Draw 2022 (Dassault Systèmes).

[0140] Dynamic Light Scattering. Solutions of the specified siRNA-L2formulations werediluted to 2 μM in DPBS and mixed with a final concentration of 2 μM human serum albumin (Millipore Sigma). Hydrodynamic diameter was measured in a ZEN2112 quartz cuvette using a Malvern ZS90 dynamic light scattering instrument and ZS Xplorer software (Malvern Pananalytical).

[0141] Biolayer Interferometry. Binding kinetics of siRNA-CQ-L2to human serum albuminwas measured by biolayerinterferometry (BLI) at 30 °C, 1000 rpm using an Octet RED 96(ForteBio). Biotinylated human serum albumin was loaded for 660 sec on a Streptavidin Dip and Read Biosensor (ForteBio). Biosensor association to siRNA-L2or siRNA-CQ-L2duplexes dilutedto the specified concentrations in DPBS was measured for 400 sec, followed bymeasurements ofdissociation for 400 sec. The binding values were measured using Octet Data Analysis HT Software. Interstep correction was performed by aligning to the dissociation step, and noise filtering was performed. Global analysis was performed to derive constants simultaneously from all tested analyte concentrations.

[0142] Critical Micelle Concentration. In a black 384-well plate (Greiner), a 1:2 serial dilutionof the specified siRNA-L2 conjugates was made in 50 L of DPBS without calcium or magnesium(Gibco), spanning from 20 to 0.01 M. To each well, 5 L of 0.1 mg / mL Nile Red in acetone (TCI) was added. The plate was covered in foil and agitated at 37 °C for 2 hours to evaporate the acetone. Fluorescence was quantified on a Tecan Infinite M1000 Pro plate reader using excitation 535 ± 10 nm and emission 610 ± 10 nm. Logarithmic regression for the lower and upper data points was performed using Microsoft Excel, and the intersection of these curve fits was taken to be the CMC.

[0143] Gal8 Endosomal Escape Assay. Generation of MDA-MB-231 cells expressing the Gal8-YFP fusion protein was performed according to previously described methods. A black 96- well plate was coated with 50 μg / mL rat tail collagen (Gibco) in 20 mM sterile acetic acid for 30Attorney Docket No.: 093386-0056-WO01 min at 37 °C and subsequently rinsed with sterile DPBS. Then, Gal8 MDA-MB-231 cells were plated at 10,000 cells per well in high-glucose DMEM (Gibco) supplemented with 10% FBS and penicillin / streptomycin (Gibco) and allowed to attach for 24 hours. Wells were rinsed with 100 μL of Opti- MEM (Gibco) and then treated with the media only, lipofectamine 2000 (Invitrogen) with luciferase siRNA (100 nM) according to manufacturer instructions, siRNA-EG18-L2at 1 μM, the same supplemented with 3 μM free chloroquine diphosphate (Millipore Sigma), the same supplemented with 50 μM free chloroquine, siRNA-CQ1-EG18-L2at 1 μM, siRNA-CQ1-EG18-L2at 1 μM, or siRNA-CQ1-EG18-L2at 1 μM, n=4 per condition. Afterincubation for 24 hours,wells were supplemented with NucBlue live cell stain (Invitrogen) in Opti-MEM according to manufacturer instructions and imaged on a Nikon Eclipse Ti confocal microscope using Nikon Elements software v. 4.01. Timelapse imaging was performed using an on-stage plate warmersupplied with humidified 5%CO2in room air to maintain physiologic conditions. In thisexperiment, Gal8 MDA-MB-231 cells were treated with Cy5-labeled siRNA-CQ1-L2(1 μM) orlipofectamine RNAiMax (Invitrogen) with unmodified siRNA (100 nM). In allcases, images wereanalyzed using MATLAB R2023a (Mathworks) using an image analysis algorithm as previously described.

[0144] In vitro Toxicity Assessment. MDA-MB-231 cells were plated at 5,000 cells per well in a black collagen-coated plate (see above) and incubated for 24 hours prior to treatment with theindicated siRNA-L2formulations at 250,500, or 1000 nM for 24 hours in Opti-MEM (n=5 percondition). Cell viability was assessed using the CellTiter Glo assay (Promega) according to manufacturer instructions and luminescence was quantified on a Tecan Infinite M1000 Pro plate reader. Relative luminescence was normalized to media-only controls.

[0145] Flow Cytometry for Uptake. MDA-MB-231 cells expressing GFP were plated at 5,000 cells per well in a black 96-well collagen-coated plate and incubated for 24 hours prior to treatmentwith the indicated Cy5-labeled siRNA-L2formulations at 100 nM for either 1 or 4 hours in Opti-MEM. Cells were then trypsinized and resuspended as a single-cell suspension in 200 μL of PBS + 2% FBS in a 96-well U-bottom plate. Flow cytometric analysis was performed on a Guava easyCyte HT flow cytometer (Cytek). Cells were gated based on FSC / SSC and subsequently for GFP positivity. The geometric mean for Cy5 fluorescence was computed using FlowJo v. 10 software.Attorney Docket No.: 093386-0056-WO01

[0146] Reporter Gene Silencing. MDA-MB-231 cells expressing firefly luciferase were plated at 5,000 cells per well in a black 96-well collagen-coated plate and incubated for 24 hours prior totreatment with the indicated siRNA-L2 formulations at 250, 500, or 1000 nM for 24 hours in Opti-MEM (n=5 per condition).150 μg / mL D-Luciferin (Pierce / Thermo-Fisher) was added to the wells and luminescence was quantified on a Tecan Infinite M1000 Pro plate reader. Relative luminescence was normalized to media-only controls.

[0147] Circulation Kinetics (Peptide Nucleic Acid Hybridization Assay). Female NCr / nu mice(Envigo) were injectedwith siRNA- L2or siRNA-CQ-L2at 1 mg / kg body weight (n=4 each). 10L of blood was collected into heparinized capillaries by tail vein nick using a 30-gauge needle at 1 minute, 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours. The peptide nucleic acid hybridization assay was performed in a method adapted from previously described methods. Cy3-labeled peptide nucleic acid probes complementary to the luciferase antisense siRNA strand were purchased from PNA Bio. 5 L of whole blood from each sample was homogenized in 300 L homogenization buffer (QuantiGene Homogenizing Solution, Thermo Fisher) with 0.5 mg / mL Proteinase K. Standards containing 1:2 serial dilutions from 10,000 to 156 fmol of siRNA were diluted into untreated whole blood processed in the same way. Samples were incubated at 65 °C for 1 hour with periodic vortexing every 15 min. Samples were then centrifuged at 15,000 ×g for 15 min.200 L of each sample was carried forward and SDS was precipitated through addition of 20 L 3M KCl. Samples were centrifuged at 4000 ×g for 15 min, transferred to a new tube, and centrifuged again at 4000 ×g for 5 min. 150 L of supernatant was then mixed with 100 L of 50 mM Tris, 10% acetonitrile, pH 8.8 and 1 L of Cy3-PNA probe at 5 M. Samples were then incubated at 90 °C for 15 min followed by 50 °C for 15 min. Samples were mixed thoroughly and 150 L was transferred into a vial for HPLC analysis. HPLC analysis was performed on a Shimadzu X using a DNAPac PA100 anion exchange column (Thermo Fisher). The mobile phase buffers were bufferA (50% water, 50% acetonitrile, 25 mM Tris-HCl pH 8.5, and 1 mM EDTA) and buffer B (800 mMNaClO4in buffer A). Concentrations were determined through measurement of the area under the curve of the fluorescent peak (excitation 550 nm, emission 570 nm) at ~6 minutes corresponding to hybridized probe and antisense strand and converted to absolute units using the standard curve samples.

[0148] Organ Biodistribution. Wild-type FVB mice were injected with saline control, siRNA- L2, siRNA-CQ2-L2, or siRNA-CQ3-L2at 5 mg / kg body weight (both males and females includedAttorney Docket No.: 093386-0056-WO01in each treatment group). After 48hours, mice were euthanized and blood was collected via cardiacpuncture. Whole blood samples were collected in potassium EDTA-coated vials for complete blood count analysis, while serum samples were prepared by allowing blood to clot in an uncoated tube at room temperature for 1 hour followed by centrifugation at 10,000 ×g for 10 mins x2. Blood counts were performed by the Vanderbilt TPSR Core while serum chemistries were performed by Antech GLP. The specified organs were taken during necropsy and fixed in 10% neutral buffered formalin for 72 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard protocols. Slides were scanned using a Leica SCN400 slide scanner by the Vanderbilt Digital Histology Shared Resource. Tissue accumulation of siRNA treatments was performed using the PNA hybridization assay, with ~5- 10 mg of each organ homogenized into 300 L homogenization buffer using stainless steel beads (Qiagen) in a TissueLyser II tissue homogenizer (Qiagen) for 3 minutes and then proceeding identically to the protocol above.

[0149] Statistical Analysis. Data were analyzed using the specified statistical tests in GraphPad Prism 10 (GraphPad / Dotmatics). All animal experiments were performed in accordance with the Vanderbilt Animal Care and Use Program. Example 2 Synthesis of chloroquine phosphoramidite and chloroquine-modified siRNA-lipid conjugates

[0150] In order to incorporate chloroquine directly into the backbone of the siRNA-L2 conjugate using an oligonucleotide synthesizer, a method for synthesizing a chloroquine phosphoramidite was devised. This two-step procedure consists of first desulfating hydroxychloroquine sulfate followed by reaction of hydroxychloroquine with 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (FIG.1A). Successful synthesis of this novel phosphoramidite was confirmed through ESI mass spectrometry and1H NMR.

[0151] Next, this phosphoramidite was used to incorporate chloroquine into the siRNA-L2 conjugate backbone. Recent investigation of the branching structure and length of the ethylene glycolspacer preceding the L2 lipid tails led to identification of a lead formulation termed siRNA-EG18-L2, and hereafter, siRNA-L2 will be used to refer specifically to the EG18 formulation. ThesiRNA-L2conjugate structure on the 5’ end of the siRNA sense strand features a symmetric brancher followed by three consecutive (ethylene glycol)6(EG6) spacers and twin stearic acid tails (L2), all joined by phosphorothioate linkages. To maintain the ability of this tail structure to engageAttorney Docket No.: 093386-0056-WO01 with albumin, while also avoiding modification of either the 3’ end of the sense strand (which is in steric proximity of the 5’ phosphate on the antisense strand) or the antisense strand itself, the chloroquine molecules were integrated between the RNA bases and the EG18-L2 tails on the 5’ end of the sense strand (FIG. 1B). To create this design, on-column solid-phase synthesis of the complete siRNA- L2 structure was performed with additional asymmetric brancher units (n=1, 2, or 3) between the siRNA bases and the symmetric branching tail structure. Subsequent deprotection of the asymmetric levulinyl-protected hydroxyl groups allowed conjugation of pendant chloroquine groups using our chloroquine phosphoramidite (FIG. 1C), resulting in the final chemical structure depicted in FIG. 1D. The final product, termed siRNA-CQn-L2where n=1, 2, or 3, was purified using HPLC, and the correct molecular weight was confirmed using liquid chromatography-mass spectrometry (LCMS, FIGS.6A-6C). Example 3 Physical Properties of Chloroquine-Modified siRNA-Lipid Conjugates

[0152] Next, the physical properties of the siRNA-CQ-L2conjugates were characterized– these properties are summarized in Table 1 below. Table 1. Summary of physical properties of chloroquine-modified siRNA-lipid conjugates. For properties marked with an asterisk (*), the sense strand for luciferase siRNA is included in the analysis.Attorney Docket No.: 093386-0056-WO01

[0153] Given the hydrophobicity of chloroquine, the AlogP values for the extended tail structure and the overall sense strand were calculated, showing that while the tail is predicted to be more hydrophobic, as anticipated, the overall siRNA sense strand remains hydrophilic based on the overall AlogP values, in agreement with our observation that the siRNA-CQ-L2single strands and annealed duplexes are readily soluble in aqueous solution. Accordingly, the critical micelle concentration (CMC) was essentially unchanged from that of the parent siRNA-L2conjugates regardless of the number of chloroquine molecules added, in the range of 3-5 M (Table 1, FIG. 2A). We did not observe aggregates in the complexes formed between human serum albumin (HSA) and siRNA-CQ-L2 at 2 M, though there was a small increase in the hydrodynamic diameter as measured by dynamic light scattering (DLS) indicative of complex formation. Finally, the binding affinity of siRNA-CQ-L2for HSA was measured using biolayer interferometry (BLI, FIG. 2B). A similar but slightly higher KDwas noted in the siRNA-CQ-L2formulations compared to parent siRNA-L2. The association constant was unchanged among all formulations, and this small difference in KDinstead reflected an increase in the dissociation constant (kd). Example 4 Chloroquine-modified siRNA-lipid conjugates mediate active endosomal escape

[0154] The parent siRNA-L2 molecule, when administered without use of a transfection reagent, tends to accumulate in endolysosomal compartments, as visualized at 24 hours using co- localization of Cy5- labeled siRNA-L2 and Lysotracker dye (FIG.3A, left). Co-administration of free chloroquine at 50 M results in disruption of normal endosomal maturation and earlier cytoplasmic accumulation of siRNA-L2(FIG. 3A, right).

[0155] The level of endosomal disruption mediated by siRNA-CQ-L2versus the parent siRNA- L2 conjugate was directly visualized and quantified using the Gal8-YFP assay developedAttorney Docket No.: 093386-0056-WO01 previously in the Duvall lab, in which endosomal disruption leads to localization of fluorescently-tagged Gal8 that can be quantified objectively through image processing (FIGS. 3B-3C). In Gal8-YFP MDA-MB-231 cells, siRNA-L2 produced low levels of Gal8 foci while addition of free CQ or incubation with siRNA-CQ2-L2 or siRNA-CQ3-L2 led to increased Gal8 signal, quantified as total area of Gal8 puncta normalized to the total cytoplasmic area. In contrast to lipofectamine transfection reagent, which produces robust Gal8 foci within a few hours and toxicity if left on cells for over the recommended 4-6 hours, siRNA-CQ-L2produces foci more gradually (FIG.7). Example 5 Chloroquine-modified siRNA-L2conjugates exhibit improved uptake patterns and gene silencing profiles without increasing toxicity in vitro

[0156] Next, the effects of covalent addition of chloroquine on siRNA-L2 function in vitro were determined. First, the uptake of siRNA-CQ-L2 was quantified as compared to parent siRNA-L2 or siRNA-L2 with co- administered free chloroquine at a fixed 100 nM dose. For these studies, siRNA-L2or siRNA-CQ1-3-L2sense strands were annealed to 5’ Cy5-labeled antisense strand, and equal fluorescence of each resulting double stranded siRNA conjugate was confirmed. We treated adherent MDA-MB-231 triple negative breast cancer cells for 1 hour (FIGS. 4A-4B) or 4 hours (FIGS. 4C-4D) at 37 °C. At both timepoints, significant increases in uptake with the CQ2-L2and CQ3-L2 formulations were observed, while free chloroquine and CQ1-L2 only led to increased uptake at the earlier timepoint. The effect seen with free chloroquine suggests that the increases may be attributed to greater retention rather than simply greater internalization. To confirm this, the same treatments were performed for 2 hours at 4 °C to isolate surface binding from internalization or retention; no increases in fluorescent uptake were seen for any of the siRNA- CQ-L2conditions in this setting (FIG.8).

[0157] It was next determined whether these CQ-driven changes in endosome disruption and intracellular retention impact gene silencing potency and efficacy. We have previously observed that optimal knockdown with siRNA-L2 in the absence of any transfection agents (“carrier-free” delivery) is achieved with 1000 nM doses after 72 hours for adherent cells (“forward” delivery, which is more representative of in vivo conditions compared to “reverse” treatment of cells in suspension). It was hypothesized that the extended time to onset and relatively high doseAttorney Docket No.: 093386-0056-WO01 requirements reflects dose sequestration and slow release form endosomes. Therefore, the silencing of reporter firefly luciferase in adherent MDA-MB-231 cells was compared at varying doses in the range of 250-1000 nM at a much earlier 24 hour timepoint, with luminescence normalized to media-only controls. At every dose, there was a trend towards greater knockdown with increasing chloroquine content, and siRNA-CQ3-L2 showed statistically significant improvements in luciferase silencing compared to siRNA-L2at the highest dose (FIG. 4E). This effect was not due to non-specific cell toxicity, as viability of cells treated with any of these conditions was comparable as measured by CellTiter Glo (FIG. 4F). Example 6 Chloroquine-modified siRNA-L2conjugates exhibit similar in vivo pharmacokinetics and safety profile in vivo compared to parent siRNA-L2

[0158] Finally, the in vivo pharmacokinetics and safety profile of chloroquine-modified siRNA- L2conjugates were characterized. Given the superior properties of siRNA-CQ3-L2, this formulation was compared to parent siRNA-L2. The circulation kinetics of unlabeled siRNA-L2and siRNA-CQ3-L2, injected intravenously at 1 mg / kg body weight (by siRNA weight, exclusive of the tail to ensure equal siRNA dose), were quantified using a peptide-nucleic acid (PNA) hybridization assay on whole blood samples. There were no significant differences in the circulation kinetics (FIG. 5A) or calculated primary half-life (0.38 hours for siRNA-L2 and 0.43 hours for siRNA-CQ3-L2).

[0159] To establish the biodistribution and safety profile of siRNA-CQ-L2, complete blood counts were assessed, serum chemistries, organ pathology (by H&E stain), and siRNA accumulation (by PNA assay) 48 hours after an intravenous dose of 5 mg / kg, previously established to be an effective dose in models of triple negative breast cancer, compared to siRNA-L2 or saline injection control. There were no noted abnormalities in blood counts or serum renalor hepatic markers (FIG. 10). Finally, distribution into healthy organs was essentially unchanged compared to parent siRNA-L2, with the highest accumulation being in the liver and spleen, with no statistically significant differences noted (FIG.5B).Attorney Docket No.: 093386-0056-WO01 Example 7 Chloroquine-modified GalNAc-siRNA

[0160] As described above, GalNac-siRNA-CQ2 and GalNac-siRNA-CQ3 conjugates were prepared from and GalNac-modified siRNA (GalNac-SiRNA) and chloroquine phosphoramidite using solid phase synthesis. The GalNac-siRNA-CQ2 and GalNac-siRNA-CQ3 conjugates will be assessed for their ability to target and bind liver cells. Example 8 Conclusions

[0161] In this work, a chloroquine phosphoramidite was successfully synthesized and incorporated into the albumin-binding siRNA-L2 and the liver cell-binding GalNAc-siRNA constructs to generate a direct siRNA conjugate containing a dedicated endosomolytic domain with tunable valency. Placement of the CQ groups between the siRNA strand and the branching tail structure, intentionally keeping the optimized ethylene glycol spacers intact, preserved the albumin-binding property of the constructs. The KDbinding constants were similar for siRNA- CQ-L2regardless of the number of chloroquine molecules, though there was a slight increase in the dissociation constant that is unlikely to have practical consequence, especially given the near- identical circulation kinetics and organ biodistribution profile compared to siRNA-L2. The small difference in albumin dissociation behavior could possibly be related to the influence of chloroquine’s intrinsic weak interaction with albumin. While addition of the chloroquine makes the siRNA-L2 tail more hydrophobic, the overall siRNA duplex remains hydrophilic, and no substantial change in the critical micelle concentration of the conjugates bearing CQ was observed.

[0162] Addition of at least 2 chloroquine molecules on each strand was sufficient to produce a statistically significant improvement in endosomal disruption compared to the parent siRNA-L2conjugate as measured using the Gal8 assay. While the Gal8 foci produced were generally similar in appearance to those seen with a commercial cationic lipid transfection reagent (lipofectamine), a different time course of appearance of these foci was noted, with lipofectamine causing endosomal disruption within 2 hours while siRNA-CQ-L2 produced signal more gradually over 24 hours. This is consistent with the known function of chloroquine as an autophagy inhibitor, preventing lysosomal maturation and fusion, which is a distinct mechanism of action compared to cationic lipids or polymers. Given concerns about excessive endosomal bursting leading to non-Attorney Docket No.: 093386-0056-WO01 specific toxicity, inflammation, and off-target mRNA modulation, this contrasting profile may be favorable. These same formulations with at least 2 chloroquine molecules also resulted in greater cell uptake in vitro, which was not solely attributable to greater non-specific binding to cells as evidenced by the degree of cell association when treated at 4 °C. Correspondingly, a trend towards greater knockdown of reporter luciferase with increasing CQ content was observed, with siRNA- CQ2-L2and siRNA-CQ3-L2producing statistically significantly greater knockdown. In those conditions, ~60% silencing at the protein level was measured after just 24 hours of treatment at 1000 nM, levels of silencing that siRNA-L2typically achieves at 48-72 hours when delivered “forward” over adherent cells, consistent with an acceleration of cytoplasmic delivery with the addition of sufficient quantities of chloroquine. While relatively high doses were still required to achieve significant silencing, siRNA-CQ-L2 did not cause any change in cell viability at the doses and timepoints tested.

[0163] In mouse models, similar circulation kinetics was observed with the siRNA-CQ-L2 conjugates compared to the parent siRNA-L2, consistent with intact albumin binding. Of note, circulation kinetics were measured using a peptide-nucleic acid hybridization assay, which avoids potential confounding effects of adding fluorescent dyes for tracking the siRNA. In contrast to prior pharmacokinetic measurements for siRNA- L2made using intravital microscopy, absolute concentrations of unlabeled siRNA in the blood were established, with the t0 values of ~1 μM consistent with the 1 mg / kg (~2 nmol for a 25 g mouse) dose in a circulating blood volume of ~8% body weight (2 mL for a 25 g mouse); this accuracy may permit more accurate future modeling of exposure of tumors to the siRNA conjugate therapeutics.

[0164] No significant differences in healthy organ biodistribution of siRNA-CQ2-L2or siRNA- CQ3-L2compared to siRNA-L2were observed. The propensity for accumulation in the liver and spleen has been previously established and is consistent with reticuloendothelial system clearanceof the siRNA-albumin complexes, in contrast with renal clearance of free siRNA. Moreimportantly, a shift in organ distribution as a result of the positive charges added by the chloroquinemolecules as not observed, which has been described in the context of lipid nanoparticles.Chloroquine bears two amines with pKa values (8.4 and 10.2) above the pH of blood and thus will on average carry two positive charges per molecule in physiologic conditions; however, the net charge of the conjugate and siRNA-albumin complex as a whole remains overwhelmingly negative.Attorney Docket No.: 093386-0056-WO01

[0165] The general synthetic approach devised for the synthesis of siRNA-CQ-L2is highly modular. The number of endosomolytic moiety repeats can be precisely defined, as well as the position of these repeats relative to other parts of the conjugate. Further, the specific endosomolytic moiety can be easily exchanged. Aside from the straightforward single-step phosphoramidite synthesis reaction, which is generally applicable to any hydroxyl-containing compound of interest, production of these siRNA conjugates is entirely an on-column solid phase synthesis. Thus, in the future, the approach can be rapidly adapted to explore not only other endosomolytic configurations but also other functionalities such as targeting or stimulus-responsive systems.

[0166] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure.

[0167] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the technology, may be made without departing from the spirit and scope thereof.

[0168] For reasons of completeness, various embodiments are set out in the following numbered clauses:

[0169] Clause 1. A conjugate comprising: an oligonucleotide; a binding ligand; an endosomal escape portion comprising an endosomal escape moiety; and a linker attached to the endosomal escape portion, wherein: the oligonucleotide is attached to the endosomal escape portion, the linker attaching the endosomal escape portion to the binding ligand; or the binding ligand is attached to the oligonucleotide, the linker attaching the endosomal escape portion to the oligonucleotide.

[0170] Clause 2. The conjugate of clause 1, wherein the linker comprises a branching molecule attached to the endosomal escape portion.

[0171] Clause 3. The conjugate of clause 2, wherein the branching molecule includes at least one branch point having at least two independent branches.

[0172] Clause 4. The conjugate of any one of clauses 1-3, wherein the endosomal escape portion comprises 1 to 10 endosomal escape moieties.

[0173] Clause 5. The conjugate of clause 4, wherein each endosomal escape moiety independently comprises an aminoquinoline, an imidazole, or a benzimidazole.Attorney Docket No.: 093386-0056-WO01

[0174] Clause 6. The conjugate of any one of clauses 1-5, wherein the binding ligand is multivalent.

[0175] Clause 7. The conjugate of any one of clauses 1-6, wherein the binding ligand is: a lipophilic ligand capable of binding albumin; or a saccharide ligand capable of binding a liver cell.

[0176] Clause 8. The conjugate of any one of clauses 1-7, wherein the oligonucleotide is attached to the endosomal escape portion, the linker attaching the endosomal escape portion to the binding ligand.

[0177] Clause 9. The conjugate of any one of clauses 1-8, wherein the binding ligand is a lipophilic ligand capable of binding albumin.

[0178] Clause 10. The conjugate of clause 9, wherein the lipophilic ligand comprises a lipid including a C4-C22 hydrocarbon chain.

[0179] Clause 11. The conjugate of clause 9 or 10, wherein the lipophilic ligand comprises two independent lipids, each lipid including a C4-C22 hydrocarbon chain.

[0180] Clause 12. The conjugate of any one of clauses 1-7, wherein the binding ligand is attached to the oligonucleotide, the linker attaching the endosomal escape portion to the oligonucleotide.

[0181] Clause 13. The conjugate of clause 12, wherein the binding ligand is a saccharide ligand capable of binding a liver cell receptor.

[0182] Clause 14. The conjugate of clause 13, wherein the saccharide ligand comprises N- acetylgalactosamine (GalNAc).

[0183] Clause 15. The conjugate of any one of clauses 1-14, wherein the branching molecule is attached to the binding ligand through a phosphorothioate linkage.

[0184] Clause 16. The conjugate of any one of clauses 1-15, wherein a spacer attaches the branching molecule to the binding ligand or the oligonucleotide.

[0185] Clause 17. The conjugate of clause 16, wherein the spacer is a hydrophilic spacer comprising 1 to 100 hydrophilic blocks.

[0186] Clause 18. The conjugate of clause 17, wherein each hydrophilic block comprises 1 to 150 repeats of a hydrophilic compound.

[0187] Clause 19. The conjugate of clause 18, wherein the hydrophilic compound comprises ethylene glycol, a zwitterionic linker, a peptoid, an amino acid, poly(glycerol), poly(oxazoline),Attorney Docket No.: 093386-0056-WO01 poly(acrylamide), poly(N-acryloyl morpholine), poly(N,N-dimethyl acrylamide), poly(2- hydroxypropyl methacrylamide), poly(2-hydroxyethyl methacrylamide), or a combination thereof.

[0188] Clause 20. The conjugate of any one of clauses 16-19, wherein the hydrophilic spacer includes 1 to 6 hydrophilic blocks, each hydrophilic block including 2 to 10 repeats of ethylene glycol.

[0189] Clause 21. The conjugate of any one of clauses 1-20, wherein the oligonucleotide comprises DNA, RNA, synthetic mimics of DNA or RNA, or a combination thereof.

[0190] Clause 22. The conjugate of any one of clauses 1-21, wherein the oligonucleotide comprises siRNA, miRNA or a single stranded antisense oligonucleotide.

[0191] Clause 23. The conjugate of any one of clauses 1-22, wherein the oligonucleotide comprises a plurality of phosphorothioate linkages.

[0192] Clause 24. The conjugate of any one of clauses 1-23, wherein the oligonucleotide has about 15 nucleotides to about 40 nucleotides.

[0193] Clause 25. A conjugate comprising: an oligonucleotide; an endosomal escape portion attached to the oligonucleotide, the endosomal escape portion comprising an endosomal escape moiety; a lipophilic ligand capable of binding albumin; and a linker attaching the endosomal escape portion to the lipophilic ligand capable of binding albumin, the linker comprising: a branching molecule attached to the endosomal escape portion, and a hydrophilic spacer attaching the branching molecule to the lipophilic ligand, the branching molecule comprising: at least one branch point having at least two independent branches.

[0194] Clause 26. The conjugate of clause 25, wherein the endosomal escape portion comprises 2 to 4 endosomal escape moieties.

[0195] Clause 27. The conjugate of clause 26, wherein at least one endosomal escape moiety comprises chloroquine.

[0196] Clause 28. The conjugate of any one of clauses 25-27, wherein the conjugate has a binding affinity (Kd) to albumin of less than 10 μM.

[0197] Clause 29. A conjugate comprising: an oligonucleotide; a saccharide ligand attached to the oligonucleotide, the saccharide ligand comprising N-acetylgalactosamine (GalNAc); and an endosomal escape portion comprising 1 to 10 endosomal escape moieties; and a linker attaching the endosomal escape portion to the oligonucleotide, the linker comprising: a branching moleculeAttorney Docket No.: 093386-0056-WO01 attached to the endosomal escape portion, the branching molecule comprising: at least one branch point having at least two independent branches.

[0198] Clause 30. The conjugate of clause 29, wherein the endosomal escape portion comprises 2 to 4 endosomal escape moieties.

[0199] Clause 31. The conjugate of clause 29 or 30, wherein at least one endosomal escape moiety comprises chloroquine.

[0200] Clause 32. A composition comprising: the conjugate of any one of clauses 1-31; and one or more pharmaceutically acceptable excipients.

[0201] Clause 33. A method of gene silencing, the method comprising administering the conjugate of clause 1 to a subject in need thereof.

[0202] Clause 34. The method of clause 33, wherein the conjugate is administered intravenously.

[0203] Clause 35. The method of clause 33 or 34, wherein the conjugate binds albumin or a liver cell following administration. 5. Sequences

Claims

Attorney Docket No.: 093386-0056-WO01 CLAIMS What is claimed is:

1. A conjugate comprising: an oligonucleotide; a binding ligand; an endosomal escape portion comprising an endosomal escape moiety; and a linker attached to the endosomal escape portion, wherein: the oligonucleotide is attached to the endosomal escape portion, the linker attaching the endosomal escape portion to the binding ligand; or the binding ligand is attached to the oligonucleotide, the linker attaching the endosomal escape portion to the oligonucleotide.

2. The conjugate of claim 1, wherein the linker comprises a branching molecule attached to the endosomal escape portion.

3. The conjugate of claim 2, wherein the branching molecule includes at least one branch point having at least two independent branches.

4. The conjugate of claim 1, wherein the endosomal escape portion comprises 1 to 10 endosomal escape moieties.

5. The conjugate of claim 4, wherein each endosomal escape moiety independently comprises an aminoquinoline, an imidazole, or a benzimidazole.

6. The conjugate of claim 1, wherein the binding ligand is multivalent.

7. The conjugate of claim 1, wherein the binding ligand is: a lipophilic ligand capable of binding albumin; or a saccharide ligand capable of binding a liver cell.Attorney Docket No.: 093386-0056-WO01 8. The conjugate of claim 1, wherein the oligonucleotide is attached to the endosomal escape portion, the linker attaching the endosomal escape portion to the binding ligand.

9. The conjugate of claim 8, wherein the binding ligand is a lipophilic ligand capable of binding albumin.

10. The conjugate of claim 9, wherein the lipophilic ligand comprises a lipid including a C4-C22hydrocarbon chain.

11. The conjugate of claim 9, wherein the lipophilic ligand comprises two independent lipids, each lipid including a C4-C22 hydrocarbon chain.

12. The conjugate of claim 1, wherein the binding ligand is attached to the oligonucleotide, the linker attaching the endosomal escape portion to the oligonucleotide.

13. The conjugate of claim 12, wherein the binding ligand is a saccharide ligand capable of binding a liver cell receptor.

14. The conjugate of claim 13, wherein the saccharide ligand comprises N-acetylgalactosamine (GalNAc).

15. The conjugate of claim 1, wherein the branching molecule is attached to the binding ligand through a phosphorothioate linkage.

16. The conjugate of claim 1, wherein a spacer attaches the branching molecule to the binding ligand or the oligonucleotide.

17. The conjugate of claim 16, wherein the spacer is a hydrophilic spacer comprising 1 to 100 hydrophilic blocks.Attorney Docket No.: 093386-0056-WO01 18. The conjugate of claim 17, wherein each hydrophilic block comprises 1 to 150 repeats of a hydrophilic compound.

19. The conjugate of claim 18, wherein the hydrophilic compound comprises ethylene glycol, a zwitterionic linker, a peptoid, an amino acid, poly(glycerol), poly(oxazoline), poly(acrylamide), poly(N-acryloyl morpholine), poly(N,N-dimethyl acrylamide), poly(2-hydroxypropyl methacrylamide), poly(2-hydroxyethyl methacrylamide), or a combination thereof.

20. The conjugate of claim 19, wherein the hydrophilic spacer includes 1 to 6 hydrophilic blocks, each hydrophilic block including 2 to 10 repeats of ethylene glycol.

21. The conjugate of claim 1, wherein the oligonucleotide comprises DNA, RNA, synthetic mimics of DNA or RNA, or a combination thereof.

22. The conjugate of claim 1, wherein the oligonucleotide comprises siRNA, miRNA or a single stranded antisense oligonucleotide.

23. The conjugate of claim 1, wherein the oligonucleotide comprises a plurality of phosphorothioate linkages.

24. The conjugate of claim 1, wherein the oligonucleotide has about 15 nucleotides to about 40 nucleotides.

25. A conjugate comprising: an oligonucleotide; an endosomal escape portion attached to the oligonucleotide, the endosomal escape portion comprising an endosomal escape moiety; a lipophilic ligand capable of binding albumin; and a linker attaching the endosomal escape portion to the lipophilic ligand capable of binding albumin, the linker comprising: a branching molecule attached to the endosomal escape portion, andAttorney Docket No.: 093386-0056-WO01 a hydrophilic spacer attaching the branching molecule to the lipophilic ligand, the branching molecule comprising: at least one branch point having at least two independent branches.

26. The conjugate of claim 25, wherein the endosomal escape portion comprises 2 to 4 endosomal escape moieties.

27. The conjugate of claim 26, wherein at least one endosomal escape moiety comprises chloroquine.

28. The conjugate of claim 25, wherein the conjugate has a binding affinity (Kd) to albumin of less than 10 μM.

29. A conjugate comprising: an oligonucleotide; a saccharide ligand attached to the oligonucleotide, the saccharide ligand comprising N- acetylgalactosamine (GalNAc); and an endosomal escape portion comprising 1 to 10 endosomal escape moieties; and a linker attaching the endosomal escape portion to the oligonucleotide, the linker comprising: a branching molecule attached to the endosomal escape portion, the branching molecule comprising: at least one branch point having at least two independent branches.

30. The conjugate of claim 29, wherein the endosomal escape portion comprises 2 to 4 endosomal escape moieties.

31. The conjugate of claim 30, wherein at least one endosomal escape moiety comprises chloroquine. A composition comprising:Attorney Docket No.: 093386-0056-WO01 the conjugate of claim 1; and one or more pharmaceutically acceptable excipients.

33. A method of gene silencing, the method comprising administering the conjugate of claim 1 to a subject in need thereof.

34. The method of claim 33, wherein the conjugate is administered intravenously.

35. The method of claim 33, wherein the conjugate binds albumin or a liver cell following administration.

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