Chemically modified oligonucleotides and conjugates and methods thereof

Abstract

Described herein are oligonucleotides that are chemically stabilized and possess desirable properties for therapeutic use, and oligonucleotide polypeptide conjugates comprising the stabilized oligonucleotides for delivery and therapeutic uses.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 618,781, filed January 8, 2024, which is incorporated herein by reference. SEQUENCE LISTING

[0002] The Sequence Listing written in file DNL-022-03-WO SeqListing.xml is 19 kilobytes in size, was created January 6, 2025, and is hereby incorporated by reference. FIELD

[0003] Described herein are oligonucleotides that are chemically stabilized and possess desirable properties for therapeutic use, and oligonucleotide polypeptide conjugates comprising the stabilized oligonucleotides for delivery' and therapeutic uses. BACKGROUND

[0004] To be useful as a therapeutic, antisense oligonucleotides (ASOs) have to satisfy a large number of different requirements. In antisense therapeutics, an ASO should be able to penetrate the cell membrane, and exhibit adequate resistance to extra- and intracellular nucleases. A fundamental property of ASOs is their ability' to recognize and hybridize complementary' sequences. ASOs therefore need to have both high affinity for target RNA to efficiently degrade or splice the bound RNA, and high specificity to avoid the unintentional blocking of the expression of other proteins. These properties of ASOs create a number of problems for their practical use.

[0005] Affinity' and specificity are commonly used to characterize the hybridization properties of an ASO. Affinity is a measure of the binding strength of the ASO to its complementary target sequence (expressed as the thermostability (Tm) of the duplex). Each nucleobase pair in the duplex adds to the thermostability. As such, affinity generally increases with increasing size of the ASO. Specificity is a measure of the ability of the ASO to discriminate between a fully complementary and a mismatched target sequence.

[0006] Given the shortcomings of natural oligonucleotides, there are existing approaches for enhancing stability (e.g., nuclease resistance) and affinity. While there are several types of chemical modifications that are known for modifying antisense oligonucleotides (ASOs), such as ribose 2'-modifications, locked nucleic acids (or locked nucleosides, LNAs), peptide nucleic acids (PNAs), and hexitol nucleic acids (HNAs). often the PK (e.g., cellular uptake and nuclease stability) and PD (e.g., target affinity and selectivity) properties of modified ASOs nevertheless remains inadequate for their therapeutic use. What is therefore needed is a chemical modification patterning process that can be applied to improve the properties of an ASO, either alone or as a part of an oligonucleotide polypeptide conjugate. The present disclosure addresses these and other shortcomings. BRIEF SUMMARY

[0007] Accordingly, in certain embodiments, the subject matter described herein is directed to stabilized oligonucleotide polypeptide conjugates of Formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LN A, MOE and cEt; t is an integer from 2 to 5; each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside; u is an integer from 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X. Y, and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage; wherein each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; and wherein the sum of t, u, and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage. In some embodiments, the oligonucleotide contains a 5' terminal group, wherein the 5' terminal group can be, but is not limited to, a phosphodiester (PO) group, a phosphorothioate (PS) group, a phosphorodithioate (PS2) group, a mesyl-phosphoramidate (MsPA) group, a cyclic phosphoryl guanidine (PN) group, or an O-isopropyl phosphorothioate (OiPS) group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of: PO, PS. and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PN 5' terminal group.

[0008] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0009] In certain embodiments, the subject matter described herein is directed to oligonucleotide polypeptide conjugates that possess desirable properties, such as plasma stability, slow plasma clearance, target knockdown, and target sequence specificity, and are of Formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LN A, MOE and cEt; t is an integer from 2 to 5; each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X. Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; wherein each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; wherein the sum of t, u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage. In some embodiments, the oligonucleotide contains a 5' terminal group, wherein the 5' terminal group can be, but is not limited to, a PO group, a PS group, a PS2 group, a MsPA group, a PN group, or an OiPS group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of: PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains aPN 5' terminal group.

[0010] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0011] In certain embodiments, the subject matter described herein is directed to oligonucleotide polypeptide conjugates that possess desirable properties, such as plasma stability, plasma clearance, target knockdown, and target sequence specificity', and are of Formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LN A, MOE and cEt; t is an integer from 2 to 5; each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X to the polypeptide by a linking group; and, each X, Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; wherein each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate or a phosphodiester; and wherein the sum of t. u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of: PS2, MsPA, PN, and OiPS. In some embodiments, the oligonucleotide contains a 5' terminal group, wherein the 5' terminal group can be, but is not limited to, a PO group, a PS group, a PS2 group, a MsPA group, a PN group, or an OiPS group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of: PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains aPN 5' terminal group.

[0012] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0013] In certain embodiments, the subject matter described herein is directed to stabilized oligonucleotides of formula (IA): (X)t-(Y)u-(Z)v            (IA) wherein: each X is a nucleoside independently selected from the group consisting of LNA. MOE and cEt; t is an integer from 2 to 5; each Y nucleoside is independently a ribonucleoside or a deoxy ribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA. MOE and cEt; v is an integer from 2 to 5; and, each X. Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; wherein each intemucleoside linkage that is not a PS2, MsPA. PN, or OiPS is a phosphorothioate or a natural phosphodiester; and wherein the sum of t, u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2. MsPA, PN, and OiPS; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of: PS2, MsPA, PN, and OiPS. In some embodiments, the oligonucleotide contains a 5' terminal group, wherein the 5' terminal group can be, but is not limited to, a PO group, a PS group, a PS2 group, a MsPA group, a PN group, or an OiPS group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of: PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains aPN 5' terminal group.

[0014] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0015] In certain embodiments, the subject matter described herein is directed to methods of preparing a stabilized oligonucleotide polypeptide conjugate, the methods comprise: (i) determining the in vivo plasma clearance of a first oligonucleotide in a first oligonucleotide polypeptide conjugate; (ii) determining a soft spot in the first oligonucleotide of the first oligonucleotide polypeptide conjugate in an oligonucleotide catabolism assay: (iii) providing a second oligonucleotide polypeptide conjugate comprising a stabilized second oligonucleotide and determining the in vivo plasma clearance of the second oligonucleotide in the second oligonucleotide polypeptide conjugate, wherein the stabilized second oligonucleotide comprises: (a) the same nucleotide sequence as the first oligonucleotide; (b) 1 to 10 stabilizing intemucleoside linkages that are not present in the first oligonucleotide, wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the second oligonucleotide relative to the first oligonucleotide as determined by an oligonucleotide catabolism assay; and (c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; wherein at least one of the 1 to 10 stabilizing intemucleoside linkages not present in the first oligonucleotide is located at or adjacent to the soft spot identified in step (ii); wherein, the in vivo plasma clearance of the second oligonucleotide polypeptide conjugate is determined to be slower than that of the first oligonucleotide polypeptide conj ugate. In vivo plasma clearance can be determined for the oligonucleotide or the oligonucleotide polypeptide conjugate.

[0016] In certain embodiments, the method further comprises: (iv) providing a third oligonucleotide polypeptide conjugate comprising a further stabilized oligonucleotide having (a) one or more additional stabilizing intemucleoside linkages not present in the first or second oligonucleotides, and (b) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodi ester linkages.

[0017] In certain embodiments, the subject matter described herein is directed to methods of preparing a stabilized oligonucleotide polypeptide conjugate, the methods comprise: (i) determining the in vivo plasma clearance of a first oligonucleotide in a first oligonucleotide polypeptide conjugate; (ii) determining a soft spot in the first oligonucleotide of the first oligonucleotide polypeptide conjugate in an oligonucleotide catabolism assay; (iii) providing a one or more stabilized second oligonucleotides comprising: (a) the same nucleotide sequence as the first oligonucleotide; (b) 1 to 10 stabilizing intemucleoside linkages that are not present in the first oligonucleotide, wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the oligonucleotide as determined by an oligonucleotide catabolism assay; and (c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; wherein at least one of the 1 to 10 stabilizing intemucleoside linkages not present in the first oligonucleotide is located at or adjacent to the soft spot identified in step (ii); (iv) providing one or more second oligonucleotide polypeptide conjugates comprising the one or more stabilized second oligonucleotides; and (v) determining the in vivo plasma clearances of the one or more second oligonucleotides in the one or more second oligonucleotide polypeptide conjugates; and (vi) selecting a second oligonucleotide polypeptide conjugate having slower clearance relative to the first oligonucleotide polypeptide conjugate.

[0018] In certain embodiments, the method further comprises: (vi) providing a one or more stabilized third oligonucleotides comprising: (a) the same nucleotide sequence as the first oligonucleotide; (b) 1 to 10 stabilizing intemucleoside linkages that are not present in the stabilized second oligonucleotide of the second oligonucleotide polypeptide conjugate selected in step (v), wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the oligonucleotide as determined by an oligonucleotide catabolism assay; and (c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; wherein at least one of the 1 to 10 stabilizing intemucleoside linkages is not present in the selected stabilized second oligonucleotide; (vii) providing one or more third oligonucleotide polypeptide conjugates comprising the one or more stabilized third oligonucleotides; and (viii) determining the in vivo plasma clearances of the one or more third oligonucleotides in the one or more third oligonucleotide polypeptide conjugates; and (ix) selecting a third oligonucleotide polypeptide conjugate having slower clearance relative to the second oligonucleotide polypeptide conjugate select in step (v).

[0019] In certain embodiments, one or more additional rounds of stabilization are performed by repeating steps (vi)-(ix).

[0020] In certain embodiments, the 1 to 10 stabilizing intemucleoside linkages of step (iii) or step (vi) that are not present in the first or second oligonucleotide comprise a substitution of a phosphorothioate or phosphodi ester for a stabilizing intemucleoside (e.g., an PS2, PN. MsPA. or OiPS). In some embodiments, the 1 to 10 stabilizing intemucleoside linkages of step (iii) or step (vi) that are not present in the first or second oligonucleotide comprises a substitution of one stabilizing intemucleoside linkage for different stabilizing intemucleoside linkage.

[0021] The selected second or third (or subsequent) oligonucleotide polypeptide conjugate can have enhanced pharmacokinetic and pharmacodynamic properties relative to the prior (first or second, respectively) oligonucleotide polypeptide conjugate. Thus, the stabilized oligonucleotides are particularly useful for conjugation to a polypeptide to form an oligonucleotide polypeptide conjugate having enhanced therapeutic potential.

[0022] In certain embodiments, each oligonucleotide is a gapmer. In certain embodiments, the gapmer comprises 16 to 22 nucleotides.

[0023] These and other embodiments are fully described below. BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. 1 illustrates plasma pharmacokinetic profile of modified MAPT ASO OTVs.

[0025] FIG. 2 illustrates single dose brain ASO concentration of modified MAPT ASO OTVs.

[0026] FIG. 3. illustrates multidose brain ASO concentration of modified MAPT ASO OTVs.

[0027] FIG. 4 illustrates target MAPT knockdown after 4 doses modified MAPT ASO OTVs.

[0028] FIG. 5 illustrates target MAPT knockdown after 8 doses of modified MAPT ASO OTVs.

[0029] FIG. 6 illustrates brain ASO concentration of unmodified MAPT ASO OTV.

[0030] FIG. 7 illustrates target MAPT knockdown of unmodified MAPT ASO OTV.

[0031] FIG. 8 illustrates MAPT ASO potency.

[0032] FIG. 9 illustrates ASO stability in brain tissue.

[0033] FIG. 10 depicts certain pharmacokinetic properties of oligonucleotide polypeptide conjugates, identified as 0TVM1, 0TVM2, 0TVM3, 0TVM4, 0TVM5, and OTVM unmod (unmodified) as measured by huIgG assay. Each of the above oligonucleotide polypeptide conjugates were administered to Sprague Dawley rats at a concentration of lOmg / kg by intravenous injection, and a dose volume of 2mL / kg. Plasma was collected at 0.25, 4, 24, 48, and 72 hr after injection. huIgG concentrations were measured in plasma using the methods described herein.

[0034] FIG. 11 depicts certain pharmacokinetic properties of oligonucleotide polypeptide conjugates, identified as 0TVM1, 0TVM2, 0TVM3, 0TVM4, 0TVM5, and OTVM unmod (unmodified) as measured by Total ASO assay. Each of the above oligonucleotide polypeptide conjugates were administered to Sprague Dawley rats at a concentration of lOmg / kg by intravenous injection, and a dose volume of 2mL / kg. Plasma was collected at 0.25, 4, 24, 48, and 72 hr after injection. Total ASO concentrations were measured in plasma using the methods described herein.

[0035] FIG. 12 illustrates in vivo ICV knockdown data for naked ASOs in mice. DETAILED DESCRIPTION 1. Overview

[0036] The subject matter described herein is directed to methods of improving the properties of an oligonucleotide by identifying catabolic sites on the oligonucleotide, and providing a stabilized oligonucleotide that has the same nucleotide sequence but is chemically modified at site-specific intemucleoside linkages to alter the catabolism of the oligonucleotide. An assay described herein allows for the identification of specific loci in an oligonucleotide that are susceptible to catabolic degradation. Once identified, one or more of the loci, or one or more sites adj acent to the loci, can be chemically modified as described herein to result in a stabilized oligonucleotide. While backbone modifications are known in the art and include, but are not limited to. e.g.. phosphorothioate linkages, phosphoramidate linkages, and phosphorodiamidate linkages, as described herein, it is now possible to specifically pattern backbone modifications at targeted loci specifically to enhance properties of the oligonucleotide. It has also been found that the stabilized oligonucleotides can confer enhanced pharmacokinetic and pharmacodynamic properties of oligonucleotide polypeptide conjugates containing the stabilized oligonucleotides as compared to oligonucleotide polypeptide conjugates comprising oligonucleotides having the same sequence but not stabilized as described herein. Thus, the results are stabilized oligonucleotides that are particularly useful for conjugation to a polypeptide to form an oligonucleotide polypeptide conjugate for enhanced therapeutic potentials.

[0037] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. II. Definitions

[0038] As used in the present specification, the following words, phrases, and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0039] As used herein, the singular forms "a." “an,” and “the” include plural referents unless the content clearly dictates otherwise.

[0040] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, ± 5%, ± 1% or ± 0.1%, are within the intended meaning of the recited value.

[0041] As used herein, the term “intemucleoside linkage” refers to the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified intemucleoside linkage” means any intemucleoside linkage other than a natural phosphodiester intemucleoside linkage. As used herein, “oligonucleotide” means a strand of linked nucleosides connected via intemucleoside linkages, wherein each nucleoside and intemucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 16-22 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or intemucleoside linkage is modified, i.e., is not naturally occurring. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or intemucleoside modifications.

[0042] As used herein, “stabilized oligonucleotide’' and “stabilized oligonucleotide polypeptide conjugate” refer to a modified oligonucleotide that has incorporated into its backbone a one or more stabilizing intemucleoside linkages.

[0043] As used herein, a “stabilizing intemucleoside linkage” refers to a modified intemucleoside linkage that is specifically introduced into the backbone of an oligonucleotide of an oligonucleotide polypeptide conjugate to remove a soft spot (e.g., nuclease sensitive site), increase resistance of the oligonucleotide to cleavage, or otherwise modulate one or more pharmacokinetic or pharmacodynamic properties of the oligonucleotide and / or oligonucleotide polypeptide conjugate into which it is introduced, but excludes phosphorothioates and phosphodiesters. A result is a stabilized oligonucleotide and / or oligonucleotide polypeptide conjugate that has improved properties compared to an oligonucleotide or oligonucleotide polypeptide conjugate that has the same oligonucleotide sequence but has not been stabilized as described herein. The methods of stabilization and their resulting stabilized oligonucleotides and oligonucleotide polypeptide conjugates provided herein are improved over those having only phosphorothioate intemucleoside linkages, or a combination of natural phosphodiester and phosphorothioate intemucleoside linkages.

[0044] To be clear, a stabilized oligonucleotide may contain phosphorothioate intemucleoside linkage(s) and natural phosphodiester intemucleoside linkage(s) after stabilization but will have no additional phosphorothioate or natural phosphodiester intemucleoside linkage(s) as a result of stabilization. In certain embodiments provided herein, the oligonucleotides and oligonucleotide polypeptide conjugates that are modified to introduce one or more stabilizing intemucleoside linkages initially do not comprise any stabilizing intemucleoside linkages. In certain embodiments provided herein, the oligonucleotides and oligonucleotide polypeptide conjugates that are modified to introduce one or more stabilizing intemucleoside linkages may have undergone a prior stabilization as described herein, and as such may contain stabilizing intemucleoside linkage(s) before undergoing iterative stabilization(s) as described herein. In certain embodiments provided herein, the oligonucleotides and oligonucleotide polypeptide conjugates that are modified to introduce one or more stabilizing intemucleoside linkages initially comprise phosphorothioate intemucleoside linkages and no natural phosphodiester linkages. In certain embodiments provided herein, the oligonucleotides and oligonucleotide polypeptide conjugates that are modified to introduce one or more stabilizing intemucleoside linkages initially comprise phosphorothioate intemucleoside linkages and natural phosphodiester linkages, with greater than 50% being phosphorothioate intemucleoside linkages. In certain embodiments provided herein, the oligonucleotides and oligonucleotide polypeptide conjugates that are modified to introduce one or more stabilizing intemucleoside linkages initially comprise one, two. three or four natural phosphodiester linkages.

[0045] Examples of modified intemucleoside linkages that can be used as a stabilizing intemucleoside linkage include, but are not limited to, phosphorodithioate, O-isopropyl phosphorothioate, phosphoramidates, phosphonates, phosphotriesters, and phosphoryl guanidines. See Nucleic Acids Res., 47, 5465-5479 (2019); Proc. Natl. Acad. Sci. U.S.A., 116, 1229-1234 (2019); Nucleic Acids Research, 50(10), 5401-5423 (2022); Vasquez G, nucleic acid therapeutics, 32(1), 40-50 (2022); and Molecular Therapy: Nucleic Acids; 29: 176-188 (2022). Additional examples of modified intemucleoside linkages that can be used as a stabilizing intemucleoside linkage include, but are not limited to, modified intemucleoside linkage si, s2, s3, s4. s5, s6, s7, s8. s8, slO. si 1, sl2. s 13, sl4, s 15. s 16, s 17, or s 18 as described in WO 2017210647, the disclosure of which is incorporated by reference in its entirety. Further examples of modified intemucleoside linkages that can be used as a stabilizing intemucleoside linkage include, but are not limited to, modified intemucleoside linkages nOOl, n002, n003, n004, n005, n006, n007, n008, n009, nOlO, n020, n025 or n026 as described in WO 2022099159, the disclosure of which is incorporated by reference in its entirety'.

[0046] A “phosphodiester intemucleoside linkage” is an unmodified intemucleoside linkage. These ty pes of linkages are also referred to a “PO.”

[0047] A “phosphorothioate intemucleoside linkage” is a modified intemucleoside linkage in which one of the two non-bridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom. These types of linkages are also referred to a “PS.”

[0048] A “phosphorodithioate intemucleoside linkage” is a modified intemucleoside linkage in which each of the two non-bridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom. As used herein, “PS2” refers to a phosphorodithioate of the formula: I JVW I O S^P--0-^- SH          .

[0049] A “phosphoramidate intemucleoside linkage” is a modified intemucleoside linkage in which a non-bridging oxygen atom of a phosphodiester intemucleoside linkage is replaced with a -NR2 group. As used herein, “MsPA” refers to a mesyl-phosphoramidate of the formula: I i / WV* I O HN--P--O- O 0

[0050] A “phosphonate intemucleoside linkage'’ is a modified intemucleoside linkage in which a non-bridging oxygen atom of a phosphodiester intemucleoside linkage is replaced with an -R group. See, JACS, 134, 11618-11631 (2012). Other exemplary phosphonates (p-methyl) may be purchased as nucleotide monomers from ChemGenes (Wilmington, MA).

[0051] A “phosphotriester intemucleoside linkage'’ is a modified intemucleoside linkage in which a non-bridging oxygen atom of a phosphodiester intemucleoside linkage is replaced with an -OR group. Exemplary phosphotriesters (O-isopropyl) may be purchased as nucleotide monomers from ChemGenes (Wilmington, MA).

[0052] A “phosphoryl guanidine intemucleoside linkage” is a modified intemucleoside linkage in which a non-bridging oxygen atom of a phosphodiester intemucleoside linkage is replaced with a derivative of guanidine. As used herein. “PN” refers to a cyclic phosphoryl guanidine of the formula:

[0053] An “O-isopropyl phosphorothioate intemucleoside linkage” is a modified intemucleoside linkage in which an isopropyl group is attached to anon-bridging oxygen atom of a phosphorothioate intemucleoside linkage. As used herein. “OiPS” refers to an O-isopropyl phosphorothioate intemucleoside linkage of the formula: / VW" \ O S .

[0054] As used herein, “gapmer” means a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "‘gap” and the external regions may be referred to as the “wings.’’ Unless otherwise indicated, “gapmer” refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gap of a gapmer are unmodified 2'-deoxyribosyl. The wings of a gapmer typically contain sugar substitutions that increase stability or are less susceptible to nuclease degradation relative to the gap section. The term “LNA gapmer” indicates a gapmer having a sugar motif of LNA nucleosides in both wings and a gap of 2'-deoxynucleosides. The term “MOE gapmer” indicates a gapmer having a sugar motif of 2'-M0E nucleosides in both wings and a gap of 2'-deoxynucleosides. Unless otherwise indicated, a gapmer provided herein may comprise one or more modified intemucleoside linkages and / or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications. For example, other 2'-modifications may be used (e.g., a group other than hydrogen or hydroxyl) at the 2'-OH group of a ribosyl sugar moiety. In certain embodiments, the 2'-modification is 2'-fluoro (2'-F) or 2'-O-methoxy (2'-0Me). As used herein, a gapmer has a 5'-Xt-Yu-Zv-3' configuration, where the X region represents the 5' wing, the Y region represents the gap, the Z region represents the 3' wing and t, u, and v represent the number of X, Y, and Z nucleosides, respectively.

[0055] As used herein, “LNA” refers to a bicyclic nucleoside analogue which comprises a bridge between the 2' and 4' position in the ribose ring (2' to 4' bicyclic nucleotide analogue), and is known as “Locked Nucleic Acid” or “Locked Nucleoside.” As used herein. “LNA oligonucleotide,” refers to an oligonucleotide containing one or more such bicyclic nucleoside analogues. Biochemistry, 43(42):13233-13240 (2004). In certain embodiments, a LNA provided herein has the following structure, wherein “Base” is a nucleobase: %

[0056] As used herein. “MOE” means methoxyethyl. “2'-M0E” or “2'-M0E modified sugar” means a 2'-OCH2CH2OCH3 group in place of the 2'-OH group of a ribosyl sugar moiety. As used herein, “MOE nucleoside” refers to a nucleoside comprising a 2'-M0E modified sugar and “MOE oligonucleotide'’ refers to an oligonucleotide comprising one or more 2'-MOE modified sugars.

[0057] As used herein, “constrained ethyl” or “cEt” means a P-D ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4'-carbon and the 2'-carbon of the P-D ribosyl sugar moiety , wherein the bridge has the formula 4'-CH(CH3)—0-2', and wherein the methyl group of the bridge is in the S configuration. As used herein. “cEt nucleoside” refers to a nucleoside comprising a cEt modified sugar and “cEt oligonucleotide” refers to an oligonucleotide comprising one or more cEt modified sugars.

[0058] As used herein, “nucleobase” means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U). or guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. A “5-methylcytosine” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or intemucleoside linkage modification.

[0059] As used herein, “nucleoside” means a compound comprising a nucleobase and a sugar moiety7. The nucleobase and sugar moiety7 are each, independently, unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase. “Linked nucleosides” are nucleosides that are connected in a contiguous sequence (i.e., no additional nucleosides are presented between those that are linked).

[0060] As used herein, “motif means the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or intemucleoside linkages, in an oligonucleotide.

[0061] As used herein, “polypeptide” means a compound or a fragment of a compound consisting of 3 or more amino acids linked together via amide bonds. Specific polypeptides are known in the art and are known to be useful for targeted delivery to cells or tissues.

[0062] As used herein, the term “pharmacokinetic properties,” “PK” and the like refer to known pharmacokinetic parameters, such as drug absorption, bioavailability, distribution, metabolism, serum stability, half-life, in vivo plasma clearance and elimination, and the relation of these to plasma drug concentrations. The pharmacokinetic properties of the oligonucleotides and the oligonucleotide polypeptide conjugates can be readily determined by those of skill in this field using known techniques and / or technique described herein.

[0063] As used herein, the term “pharmacodynamic properties” and the like refer to the physiological action of the drug on the body, i.e., the pharmacological effect(s), e.g., target knockdown, target sequence specificity, and the relationship between drug concentration and the pharmacologic effect(s).

[0064] As used herein, “binding affinity” refers to the strength of the non-covalent interaction between two molecules, e.g., a single binding site on a polypeptide / protein and a target, e.g., transferrin receptor, to which it binds. Thus, for example, the term may refer to 1:1 interactions between a polypeptide / protein and its target, unless otherwise indicated or clear from context. Binding affinity may be quantified by measuring an equilibrium dissociation constant (Kd), which refers to the dissociation rate constant (kd, time'1) divided by the association rate constant (ka, time1 M1). Kd can be determined by measurement of the kinetics of complex formation and dissociation, e.g, using Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, “binding affinity” includes not only formal binding affinities, such as those reflecting 1:1 interactions between a polypeptide / protein and its target, but also apparent affinities for which Kd’s are calculated that may reflect avid binding.

[0065] The phrase “specifically binds” or “selectively binds” to a target, e.g., transferrin receptor, when referring to a polypeptide or protein (including a modified constant domain as described herein), refers to a binding reaction whereby the protein binds to the target with greater affinity, greater avidity, and / or greater duration than it binds to a structurally different target, e.g., a target not in the transferrin receptor family. In typical embodiments, the protein has at least 5-fold. 10-fold, 100-fold, 1000-fold, 10,000-fold or greater affinity for a target molecule or protein (e.g, a transferrin receptor) compared to an unrelated target when assayed under the same affinity assay conditions. In some embodiments, a modified CH3 domain specifically binds to an epitope on a transferrin receptor that is conserved among species, e.g., conserved between non-human primate and human species. In some embodiments, a protein may bind exclusively to a human transferrin receptor.

[0066] Additional definitions may also be provided below as appropriate. III. Stabilized Oligonucleotides and Conjugates Thereof

[0067] In certain embodiments, the subject matter described herein is directed to stabilized oligonucleotides and conjugates comprising a stabilized oligonucleotide bound to polypeptide through a linking group. A. Oligonucleotide Polypeptide Conjugates

[0068] In certain embodiments, the subject matter described herein is directed to stabilized oligonucleotide polypeptide conjugates of Formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LNA. MOE and cEt; t is an integer from 2 to 5; each Y nucleoside is independently a ribonucleoside or a deoxy ribonucleoside; u is an integer from 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X, Y, and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage; wherein each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; and wherein the sum of t, u and v is an integer from 16 to 22.

[0069] In certain embodiments, the subject matter described herein is directed to an oligonucleotide conjugated to a polypeptide, wherein the oligonucleotide is of Formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; t is an integer from 2 to 5; each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside; u is an integer from 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X, Y, and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; and wherein the sum oft, u and v is an integer from 16 to 22.

[0070] In certain embodiments, each stabilizing intemucleoside linkage is independently selected from the group consisting of phosphorodithioate, phosphoramidates, phosphonates, phosphotriesters, and phosphoryl guanidines. In certain embodiments, each the stabilizing intemucleoside linkage is independently selected from the group consisting of PS2, MsPA, PN, and OiPS.

[0071] In certain embodiments, each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0072] In certain embodiments, the sum of t and v is an integer from 4 to 8.

[0073] In certain embodiments, t is 2, 3 or 4. In certain embodiments, t is 3.

[0074] In certain embodiments, v is 2, 3 or 4. In certain embodiments, v is 3.

[0075] In certain embodiments, t is 2, 3. or 4 and v is 2. 3, or 4. In certain embodiments, t is 3 and v is 3.

[0076] In certain embodiments, at least two intemucleoside linkages adjacent to any one or two Zs are each independently a stabilizing intemucleoside linkage. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Zs are each independently a stabilizing intemucleoside linkage. In certain embodiments, at least two intemucleoside linkages adjacent to any one or two Zs are each independently PS2, MsPA, PN, or OiPS. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Zs are each independently PS2. MsPA, PN. or OiPS. In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Zs are each PN. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Zs are each PN. In certain embodiments, each intemucleoside linkage adjacent to a Z nucleoside is a stabilizing intemucleoside linkage. In certain embodiments, each intemucleoside linkage adjacent to a Z nucleoside is a PN.

[0077] In certain embodiments, the two 3' terminal intemucleoside linkages (intemucleoside linkages between nucleosides n and n-1 and between nucleosides n-1 and n-2, wherein n is the number of nucleosides in the oligonucleotide) are stabilizing intemucleoside linkages. In certain embodiments, the three 3' terminal intemucleoside linkages (intemucleoside linkages between nucleosides n and n-1, n-1 and n-2, and n-2 and n-3) are stabilizing intemucleoside linkages. In some embodiments, the two or three 3' terminal stabilizing intemucleoside linkages are each PN.

[0078] In certain embodiments, a least one intemucleoside linkage adjacent a Z nucleoside is a PO. In certain embodiments, one intemucleoside linkage adjacent a Z nucleoside is a PO. In certain embodiments, the PO is between nucleosides n and n-1, between nucleosides n-1 and n-2, or between nucleosides n-2 and n-3, wherein n is the number of nucleosides in the oligonucleotide. In certain embodiments, one intemucleoside linkage adjacent a Z nucleoside is a PO and at least two of the other intemucleoside linkages adjacent to Z nucleosides are stabilizing intemucleoside linkages. In certain embodiments, one intemucleoside linkage adjacent a Z nucleoside is a PO and all of the other intemucleoside linkages adjacent to a Z nucleoside are stabilizing intemucleoside linkages. In certain embodiments, one intemucleoside linkage adjacent a Z nucleoside is a PO and all of the other intemucleoside linkages adjacent to Z nucleosides are PN.

[0079] In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Xs are each independently a stabilizing intemucleoside linkage. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Xs are each independently a stabilizing intemucleoside linkage. In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Xs are each independently PS2, MsPA, PN, or OiPS. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Xs are each independently PS2, MsPA, PN, or OiPS. In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Xs are each PN. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Xs are each PN. In certain embodiments, each intemucleoside linkage adjacent to an X nucleoside is a stabilizing intemucleoside linkage. In certain embodiments, each intemucleoside linkage adjacent to an X nucleoside is a PN.

[0080] In certain embodiments, the 5' terminal intemucleoside linkage (intemucleoside linkages between nucleosides 1 and 2, counting from the 5' terminal nucleoside) is a stabilizing intemucleoside linkage. In certain embodiments, the 5’ terminal intemucleoside linkage is a PN.

[0081] In certain embodiments, the two 5' terminal intemucleoside linkages (intemucleoside linkages betw een nucleosides 1 and 2 and between nucleosides 2 and 3, counting from the 5' terminal nucleoside) are stabilizing intemucleoside linkages. In certain embodiments, the two 5' terminal intemucleoside linkages are PN.

[0082] In certain embodiments, the intemucleoside linkages between nucleosides 2 and 3 and between nucleosides 3 and 4 (counting from the 5' terminal nucleoside) are stabilizing intemucleoside linkages. In certain embodiments, the intemucleoside linkages between nucleosides 2 and 3 and between nucleosides 3 and 4 are PN.

[0083] In certain embodiments, the three 5' terminal intemucleoside linkages (intemucleoside linkages betw een nucleosides 1 and 2, 2 and 3, and 3 and 4, counting from the 5' terminal nucleoside) are stabilizing intemucleoside linkages. In some embodiments, the three 5' terminal stabilizing intemucleoside linkages are each PN.

[0084] In certain embodiments, a least one intemucleoside linkage adjacent an X nucleoside is a PO. In certain embodiments, one intemucleoside linkage adjacent an X nucleoside is a PO. In certain embodiments, the PO is between nucleosides 1 and 2, between nucleosides 2 and 3, or between nucleosides 3 and 4 (counting from the 5' terminal nucleoside). In certain embodiments, one intemucleoside linkage adjacent an X nucleoside is a PO and at least two of the other intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages. In certain embodiments, one intemucleoside linkage adjacent an X nucleoside is a PO intemucleoside linkage and all of the other intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages. In certain embodiments, one intemucleoside linkage adjacent an X nucleoside is a PO intemucleoside linkage and all of the other intemucleoside linkages adjacent to X nucleosides are PN.

[0085] In certain embodiments, the intemucleoside linkages adjacent to the X and Z nucleotides are PS and the oligonucleotides contains 1-4 stabilizing intemucleoside linkages adjacent to any one to five Y nucleosides.

[0086] In certain embodiments. 1-3 intemucleoside linkages adjacent any one to three X nucleosides are stabilizing intemucleoside linkages, and 1-3 intemucleoside linkages adjacent to any one to three Z nucleosides are stabilizing intemucleoside linkages. In certain embodiments, 1-3 intemucleoside linkages adjacent any one to three X nucleosides are PS2, MsPA, PN. or OiPS, and 1 -3 intemucleoside linkages adj acent to any one to three Z nucleosides are PS2, MsPA, PN. or OiPS. In certain embodiments. 1-3 intemucleoside linkages adjacent any one to three X nucleosides are PN, and 1-3 intemucleoside linkages adjacent to any one to three Z nucleosides are PN.

[0087] In certain embodiments, at least one intemucleoside linkage adjacent any one to two X nucleosides is a stabilizing intemucleoside linkage, and at least one intemucleoside linkage adjacent to any one to two Z nucleosides is a stabilizing intemucleoside linkage. In certain embodiments, the at least one intemucleoside linkage adjacent any one to two X nucleosides is a PS2, MsPA, PN, or OiPS, and the at least one intemucleoside linkage adjacent to any one to two Z nucleosides is a PS2, MsPA, PN, or OiPS. In certain embodiments, the at least one intemucleoside linkage adjacent any one to two X nucleosides is a PN. and the at least one intemucleoside linkage adjacent to any one to two Z nucleosides is a PN.

[0088] In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Xs are each independently a stabilizing intemucleoside linkage, and at least two intemucleoside linkages adjacent to any two or three Zs are each independently a stabilizing intemucleoside linkage. In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Xs are each independently PS2, MsPA, PN, or OiPS, and at least two intemucleoside linkages adjacent to any two or three Zs are each independently PS2, MsPA, PN, or OiPS. In certain embodiments, at least two intemucleoside linkages adjacent to any two or three Xs are each PN. and at least two intemucleoside linkages adjacent to any two or three Zs are each PN.

[0089] In certain embodiments, two intemucleoside linkages adjacent to any two or three Xs are stabilizing intemucleoside linkages, and two intemucleoside linkages adjacent to any two or three Zs are stabilizing intemucleoside linkages. In certain embodiments, two intemucleoside linkages adjacent to any two or three Xs are each independently PS2, MsPA, PN, or OiPS, and two intemucleoside linkages adjacent to any two or three Zs are each independently PS2, MsPA. PN, or OiPS. In certain embodiments, two intemucleoside linkages adjacent to any two or three Xs are each PN, and two intemucleoside linkages adjacent to any two or three Zs are each PN. In some embodiments, the stabilizing intemucleoside linkages adjacent to X nucleosides are between nucleosides 1 and 2 and between nucleosides 2 and 3. In some embodiments, the stabilizing intemucleoside linkages adjacent to X nucleosides are between nucleosides 2 and 3 and between nucleosides 3 and 4. In some embodiments, the stabilizing intemucleoside linkages adjacent to X nucleosides are between nucleosides 1 and 2 and between nucleosides 3 and 4. In some embodiments, the stabilizing intemucleoside linkages adjacent to Z nucleosides are between nucleosides n and n-1 and between nucleosides n-1 and n-2. In some embodiments, the stabilizing intemucleoside linkages adjacent to Z nucleosides are between nucleosides n and n-1 and between nucleosides n-2 and n-3. In some embodiments, the stabilizing intemucleoside linkages adjacent to Z nucleosides are between nucleosides n-1 and n-2 and between nucleosides n-2 and n-3.

[0090] In certain embodiments, the two 3' terminal intemucleoside linkages are stabilizing intemucleoside linkages and the intemucleoside linkages adjacent to the X nucleosides are PS. In certain embodiments, the two 3' terminal intemucleoside linkages are independently PS2, MsPA, PN, or OiPS, and the intemucleoside linkages adjacent to the X nucleosides are PS. In certain embodiments, the two 3' terminal intemucleoside linkages are PN and the intemucleoside linkages adjacent to the X nucleosides are PS.

[0091] In certain embodiments, the two 5' terminal intemucleoside linkages are stabilizing intemucleoside linkages and the intemucleoside linkages adjacent to the Z nucleosides are PS. In certain embodiments, the two 5' terminal intemucleoside linkages are independently PS2, MsPA, PN, or OiPS. and the intemucleoside linkages adjacent to the Z nucleosides are PS. In certain embodiments, the two 5' terminal intemucleoside linkages are PN and the intemucleoside linkages adjacent to the Z nucleosides are PS.

[0092] In certain embodiments, the two 3' terminal intemucleoside linkages and the 5’ terminal intemucleoside linkages are stabilizing intemucleoside linkages. In certain embodiments, the two 3' terminal intemucleoside linkages and the 5' terminal intemucleoside linkages are each independently PS2, MsPA, PN, or OiPS. In certain embodiments, the two 3' terminal intemucleoside linkages and the 5' terminal intemucleoside linkages are each PN.

[0093] In certain embodiments, the two 3' terminal intemucleoside linkages and the two 5' terminal intemucleoside linkages are stabilizing intemucleoside linkages. In certain embodiments, the two 3' terminal intemucleoside linkages and the two 5' terminal intemucleoside linkages are each independently PS2, MsPA, PN, or OiPS. In certain embodiments, the two 3' terminal intemucleoside linkages and the two 5' terminal intemucleoside linkages are each PN.

[0094] In certain embodiments, the three 3' terminal intemucleoside linkages and the three 5' terminal intemucleoside linkages are stabilizing intemucleoside linkages. In certain embodiments, the three 3' terminal intemucleoside linkages and the three 5' terminal intemucleoside linkages are each independently PS2. MsPA, PN. or OiPS. In certain embodiments, the three 3' terminal intemucleoside linkages and the three 5' terminal intemucleoside linkages are each PN.

[0095] In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Xs are each independently a stabilizing intemucleoside linkage, and at least three intemucleoside linkages adjacent to any three or four Zs are each independently a stabilizing intemucleoside linkage. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Xs are each independently PS2, MsPA, PN, or OiPS, and at least three intemucleoside linkages adjacent to any three or four Zs are each independently PS2, MsPA. PN, or OiPS. In certain embodiments, at least three intemucleoside linkages adjacent to any three or four Xs are each PN, and at least three intemucleoside linkages adjacent to any three or four Zs are each PN.

[0096] In certain embodiments, the three 5’ terminal intemucleoside linkages are stabilizing intemucleoside linkages and one intemucleoside linkage adjacent to one to two Z nucleosides is a PO. In certain embodiments, the three 5' terminal intemucleoside linkages are independently PS2, MsPA, PN, or OiPS and one intemucleoside linkage adjacent to one to two Z nucleosides is a PO. In certain embodiments, the three 5' terminal intemucleoside linkages are PN and one intemucleoside linkage adjacent to one to two Z nucleosides is a PO. The linkages adjacent to Z nucleotides that are not PO are stabilizing intemucleoside linkages. In certain embodiments, the linkages adjacent to Z nucleotides that are not PO are independently PS2, MsPA, PN, or OiPS. In certain embodiments, the linkages adjacent to Z nucleotides that are not PO are PN.

[0097] In certain embodiments, the three 3’ terminal intemucleoside linkages are stabilizing intemucleoside linkages and one intemucleoside linkage adjacent to one to two X nucleosides is a PO. In certain embodiments, the three 3' terminal intemucleoside linkages are independently PS2, MsPA, PN, or OiPS and one intemucleoside linkage adjacent to one to two X nucleosides is a PO. In certain embodiments, the three 3’ terminal intemucleoside linkages are PN and one intemucleoside linkage adjacent to one to two X nucleosides is a PO. The linkages adjacent to X nucleotides that are not PO are stabilizing intemucleoside linkages. In certain embodiments, the linkages adjacent to X nucleotides that are not PO are independently PS2, MsPA, PN, or OiPS. In certain embodiments, the linkages adjacent to X nucleotides that are not PO are PN.

[0098] In certain embodiments, each X and each Z is LNA. In some embodiments, each X and each Z is an MOE. In some embodiments, each X and each Z is an cEt.

[0099] In certain embodiments, 1 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently a stabilizing intemucleoside linkage. In certain embodiments, 2 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently a stabilizing intemucleoside linkage. In certain embodiments, 1 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently a stabilizing intemucleoside linkage. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently a stabilizing intemucleoside linkage. In certain embodiments, 1 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS. In certain embodiments, 2 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS. In certain embodiments, 1 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2. MsPA. PN. and OiPS. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides together link three or four contiguous Y nucleotides. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are PS2. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are MsPA. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are PN. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are OiPS.

[0100] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0101] In certain embodiments, u is an integer from 10 to 14. In certain embodiments, u is an integer from 10 to 12. In certain embodiments, u is 10, 11, 12, 13, 14, 15 or 16.

[0102] In certain embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group. The 5' terminal group can be. but is not limited to, a PO group, a PS group, a PS2 group, a MsPA group, a PN group, or an OiPS group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of PO, PS, or PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains aPN 5' terminal group.

[0103] In certain embodiments, t is 3; each X nucleoside is a LNA, MOR or cEt; 0-3 intemucleoside linkages adjacent to the X nucleosides are stabilizing intemucleoside linkages; u is an integer from 10 to 16; 0 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each stabilizing intemucleoside linkages; v is 3; each Z is a LNA, MOR or cEt; 0-3 intemucleoside linkages adjacent to the Z nucleosides are stabilizing intemucleoside linkages; and, each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a PS or PO. In certain embodiments, the stabilizing intemucleoside linkages are independently selected from the group consisting of PS2, MsPA, PN, or OiPS. In certain embodiments, 1 intemucleoside linkages that each links two contiguous Y nucleotides is a stabilizing intemucleoside linkage. In certain embodiments, 2 intemucleoside linkages that each links two contiguous Y nucleotides is a stabilizing intemucleoside linkage. In certain embodiments, 3 intemucleoside linkages that each links two contiguous Y nucleotides is a stabilizing intemucleoside linkage. In certain embodiments. 4 intemucleoside linkages that each links two contiguous Y nucleotides is a stabilizing intemucleoside linkage.

[0104] In certain embodiments, t is 3; each X is a LNA that is adjacent to a PN intemucleoside linkage; u is an integer from 10 to 16; 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, and OiPS, and together link three or four contiguous Y nucleotides; v is 3; each Z is a LNA that is adjacent to a PN intemucleoside linkage; and, each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0105] In certain embodiments, the stabilized oligonucleotide is conjugated to a polypeptide. The polypeptide can be, but is not limited to, a means for targeted delivery.

[0106] In certain embodiments, the subject matter described herein is directed to oligonucleotide polypeptide conjugates that possess desirable properties, such as plasma stability, plasma clearance, target knockdown, and target sequence specificity, and are of Formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; t is an integer from 2 to 5; each Y is independently a ribonucleoside or a deoxyribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X. Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; wherein each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate or a phosphodiester; and wherein the sum of t, u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of: PS2, MsPA, PN, and OiPS.

[0107] In certain embodiments, the subject matter described herein is directed to oligonucleotide polypeptide conjugates that possess desirable properties, such as plasma stability , slow plasma clearance, target knockdown, and target sequence specificity, and are of Formula I: #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LNA. MOE and cEt; t is an integer from 2 to 5; each Y is independently a ribonucleoside or a deoxyribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X, Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) there are adjacent to a Z nucleoside or an X nucleoside, at least one intemucleoside linkage means for stabilizing the oligonucleotide; and (b) there are 1 to 5 intemucleoside linkage means for stabilizing the oligonucleotide; wherein each intemucleoside linkage that is not an intemucleoside linkage means for stabilizing the oligonucleotide is a phosphorothioate or a phosphodiester; and wherein the sum of t, u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of: PS2, MsPA, PN, and OiPS.

[0108] In certain embodiments, an intemucleoside linkage means for stabilizing is determined by an oligonucleotide catabolism assay. In certain embodiments, an intemucleoside linkage means for stabilizing is a modified intemucleoside linkage.

[0109] In certain embodiments, the subject matter described herein is directed to an improved oligonucleotide polypeptide conjugate, wherein the oligonucleotide is conjugated to a polypeptide, the improvement comprising an oligonucleotide of formula (I): #-(X)t-(Y)u-(Z)v          (I) wherein: each X is a nucleoside independently selected from the group consisting of LNA. MOE and cEt; t is an integer from 2 to 5; each Y is independently a ribonucleoside or a deoxyribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; # is the point of attachment of an X nucleoside to the polypeptide by a linking group; and, each X, Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; and wherein the sum of t, u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage.

[0110] In certain embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group. The 5' terminal group can be. but is not limited to. a PO group, a PS group, a PS2 group, a MsPA group, a PN group, or an OiPS group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of: PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PN 5' terminal group.

[0111] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0112] As set forth above, oligonucleotide polypeptide conjugates are comprised of stabilized oligonucleotides as described herein. Once an oligonucleotide is stabilized, it can be covalently linked using standard techniques to any known polypeptide using any known linking groups. i. Stabilized Oligonucleotides

[0113] The oligonucleotides described herein may be synthesized using standard solid or solution phase synthesis techniques that are known in the art. In certain embodiments, the oligonucleotides are synthesized using solid-phase phosphoramidite chemistry (U.S. Patent No. 6,773,885) with automated synthesizers. Chemical synthesis of nucleic acids allows for the production of various forms of the nucleic acids with modified linkages, chimeric compositions, and nonstandard bases or modifying groups attached in chosen places through the nucleic acid’s entire length.

[0114] Each oligonucleotide is an ASO. The term “antisense oligonucleotide (ASO)” refers to single strands of DNA-like or RNA-like molecules (e.g., modified nucleotides such as those described herein) that are complementary or partially complementary to a chosen target polynucleotide sequence, e.g., an mRNA. By binding to a complementary target sequence ASOs can alter or modulate gene expression through a number of mechanisms, including, e.g., by altering splicing (exon exclusion or exon inclusion); by recruiting RNase H leading to target degradation; through translation inhibition; and by small RNA inhibition.

[0115] As described herein, ASOs range from about 16 to 22 base pairs (bp) in length. For example, in certain embodiments, the ASO is about 16 to about 22 nucleotides in length, or about 17 to about 22 nucleotides in length, or about 18 to about 22 nucleotides in length, or about 19 to about 22 nucleotides in length, or about 20 to about 22 nucleotides in length, or about 21 to about 22 nucleotides in length. In certain embodiments, the ASO is about 16, 17, 18. 19, 20, 21 or 22 nucleotides in length.

[0116] Selection of antisense oligonucleotide sequences specific for a given target sequence is based upon analysis of the chosen target sequence and determination of a number of factors, including secondary structure, Tm, binding energy, and relative stability. Additionally, antisense oligonucleotides may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Target regions of the mRNA include, but are not limited to, regions at or near the AUG translation initiation codon. Secondary structure analyses and target site selection considerations can be performed using software and algorithms known in the art, for example, using v.4 of the OLIGO primer analysis software (Molecular Biology Insights) and / or the BLASTN 2.0.5 algorithm software (Altschul et al, Nucleic Acids Res. 1997, 25(17):3389-402).

[0117] In certain embodiments, the nucleic acid modifications within the oligonucleotide are included in a pattern. In certain embodiments, the oligonucleotide is a gapmer. The modification pattern of a gapmer oligonucleotide generally has the formula 5'-Xt-Yu-Zv-3', with Xt and Zv as flanking regions around a gap region Yu. In certain embodiments, the Yu region is a contiguous stretch of nucleotides, e.g., a region of 10 to 16 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAse H. In certain embodiments, the Yu region is 12 DNA nucleosides in length. In certain embodiments, the gapmer binds to the target nucleic acid, at which point an RNAse is recruited resulting in cleavage the target nucleic acid. The Yu region is flanked both 5' and 3' by regions Xt and Zv, which comprise modified nucleosides, e.g., two to five modified nucleosides in each of Xt and Zv. In certain embodiments, the modified nucleosides are present in the 5' and 3' regions of the oligonucleotide, while certain modified nucleosides and / or modified linkages may or may not present in the central portion of the molecule. In certain embodiments, the modified nucleosides are present in the 5' and 3' regions of the oligonucleotide and certain modified nucleotides are not present in the central portion of the molecule (e.g., LNA residues are not present in the central portion; however, the central region will contain modified linkages, such as PS linkages, and one to five modified linkages selected from the group consisting of PS2, MsPA, PN, and OiPS.) In certain embodiments, Xt and Zv each comprise 3 modified nucleosides. In certain embodiments, the 3 modified nucleosides are arranged in tandem in each of Xt and Zv.

[0118] In certain embodiments, the oligonucleotide comprises one or more modifications to the intemucleosidebackbone (i.e., the natural phosphodiester (PO) linkage is modified). In certain embodiments, such modifications are made to. e.g., reduce nuclease degradation of the ASO. Thus, in certain embodiments, an oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified intemucleoside^inkages. In some embodiments, 100% of the intemucleoside%linkages present in the oligonucleotide are modified.

[0119] While backbone modifications are known in the art and include, but are not limited, to, e.g., phosphorothioate linkages, phosphoramidate linkages, and phosphorodiamidate linkages, as described herein, it is now possible to specifically pattern backbone modifications at targeted loci to increase desirable properties of the oligonucleotide, and an oligonucleotide polypeptide conjugate.

[0120] In certain embodiments, the intemucleoside linkages are stereorandom with regard to the chiral centers (Rp and Sp). In certain other embodiments, the Rp and Sp configurations in the oligonucleotide are optimized in particular configurations.

[0121] In certain embodiments, the oligonucleotide is a gapmer comprising LNA and PS modifications and 2 to 9 PS2. MsPA, PN. or OiPS intemucleoside linkages. In certain embodiments, the oligonucleotide is a gapmer comprising LNA and PS modifications and 2 to 6 PS2, MsPA, PN, or OiPS intemucleoside linkages. For example, in certain embodiments, the oligonucleotide is a gapmer having a modification pattern of the formula 5'-Xi-Yu-Zv-3', with Xt and Zv as flanking regions around a gap region Yu, wherein Xt and Zv each comprise 3 LNA modified nucleotides (e.g., 3 consecutive LNA modified nucleotides), and wherein the gap region Yu comprises PS linkages and 1-2 PS2, MsPA, PN, or OiPS intemucleoside linkages. In certain embodiments, the oligonucleotide is a gapmer having a modification pattern of the formula 5'-Xt-Yu-Zv-3', with Xt and Zv as flanking regions around a gap region Yu, wherein Xt and Zv each comprise 3 LNA modified nucleotides (e.g.. 3 consecutive LNA modified nucleotides), and wherein the gap region Yu comprises PS linkages and 1-4 PS2, MsPA, PN, or OiPS intemucleoside linkages. In certain embodiments, the oligonucleotide is a gapmer having a modification pattern of the formula 5'-Xt-Y«-Zv-3', with Xt and Zv as flanking regions around a gap region Yu, wherein Xt and Zv each comprise 3 LNA modified nucleotides (e.g., 3 consecutive LNA modified nucleotides), and wherein the gap region Yu comprises PS linkages and 1, 2, 3, or 4 PS2, MsPA, PN, or OiPS intemucleoside linkages. In certain embodiments, the oligonucleotide further comprises one or more 5-methylcytidine residues. In certain embodiments, the gap region Yu does not comprise LNA residues.

[0122] In some embodiments, the subject matter described herein is directed to an oligonucleotide of Formula (IA), (X)t-(Y)u-(Z)v            (IA) wherein: each X is a nucleoside independently selected from the group consisting of LNA. MOE and cEt; t is an integer from 2 to 5; each Y is independently a ribonucleoside or a deoxyribonucleoside; u is an integer 10 to 16; each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt; v is an integer from 2 to 5; and, each X. Y, Z nucleoside is covalently bound by an intemucleoside linkage; provided that: (a) at least one intemucleoside linkage(s) adjacent to a Z or an X is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and / or (b)    1 to 5 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; wherein each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate or a natural phosphodiester; and wherein the sum of t, u and v is an integer from 16 to 22. In certain embodiments, the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleoside are each independently selected from the group consisting of: PS2, MsPA, PN, and OiPS. In some embodiments, the oligonucleotide contains a 5' terminal group, wherein the 5' terminal group can be, but is not limited to, a phosphodiester (PO) group, a phosphorothioate (PS) group, a phosphorodithioate (PS2) group, a mesyl-phosphoramidate (MsPA) group, a cyclic phosphory l guanidine (PN) group, or an O-isopropyl phosphorothioate (OiPS) group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a 5' terminal group selected from the group consisting of: PO, PS, and PN. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PO 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PS 5' terminal group. In some embodiments, the oligonucleotide (or stabilized oligonucleotide) contains a PN 5' terminal group.

[0123] In some embodiments, the oligonucleotide comprises at least two different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides. The at least two stabilizing intemucleoside linkages that each link two contiguous Y nucleosides can be the same or different stabilizing intemucleoside linkages. In some embodiments, the oligonucleotide comprises 2-5 intemucleoside linkages that each link two contiguous Y, wherein the 2-5 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

[0124] In certain embodiments, each intemucleoside linkage that is not a PS2, MsPA, PN or OiPS is a phosphorothioate or phosphodiester.

[0125] In certain embodiments, the sum of t and v is an integer from 4 to 8.

[0126] In certain embodiments, t is 2, 3 or 4. In certain embodiments, t is 3.

[0127] In certain embodiments, v is 2. 3 or 4. In certain embodiments, v is 3.

[0128] In certain embodiments, t is 2, 3. or 4 and v is 2. 3, or 4. In certain embodiments, t is 3 and v is 3.

[0129] In certain embodiments, at least two intemucleoside linkages adjacent to a Z nucleoside are each PN. In certain embodiments, at least three intemucleoside linkages adjacent to a Z nucleoside are each PN.

[0130] In certain embodiments, at least two intemucleoside linkages adjacent to an X nucleoside are each PN. In certain embodiments, at least three intemucleoside linkages adjacent to an X nucleoside are each PN.

[0131] In certain embodiments, at least two intemucleoside linkages adjacent to an X nucleoside are each PN, and at least two intemucleoside linkages adjacent to a Z nucleoside are each PN.

[0132] In certain embodiments, at least three intemucleoside linkages adjacent to an X nucleoside are each PN, and at least three intemucleoside linkages adjacent to a Z nucleoside are each PN.

[0133] In certain embodiments, each X nucleoside and each Z nucleoside is LNA.

[0134] In certain embodiments, 1 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS. In certain embodiments, 2 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA. PN, and OiPS. In certain embodiments, 1 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2. MsPA, PN. and OiPS. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides together link three or four contiguous Y nucleotides. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are PS2. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are MsPA. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are PN. In certain embodiments, 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are OiPS.

[0135] In certain embodiments, u is an integer from 10 to 14. In certain embodiments, u is an integer from 10 to 12. In certain embodiments, uis 10, 11, 12, 13, 14, 15 or 16.

[0136] In certain embodiments, t is 3; each X is a LNA that is adjacent to a PN intemucleoside linkage; u is an integer from 10 to 16; 0 to 4 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, and OiPS, and together link three or four contiguous Y nucleotides; v is 3; each Z is a LNA that is adjacent to a PN intemucleoside linkage; and. each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0137] In certain embodiments, t is 3; each X is a LNA that is adjacent to a PN intemucleoside linkage; u is an integer from 10 to 16; 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, and OiPS, and together link three or four contiguous Y nucleotides; v is 3; each Z is a LNA that is adjacent to a PN intemucleoside linkage; and, each intemucleoside linkage that is not a PS2, MsPA, PN. or OiPS is a phosphorothioate.

[0138] In certain embodiments, t is 3; v is 3; 1 to 4 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS; 2 to 3 intemucleoside linkages adjacent to aZ is / are each independently selected from the group consisting of PS2. MsPA. PN, and OiPS; 2-3 intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0139] In certain embodiments, t is 3; v is 3; 1 to 4 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA. PN, and OiPS; zero intemucleoside linkages adjacent to a Z is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2-3 intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0140] In certain embodiments, t is 3; v is 3; 1 to 4 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS; 2 to 3 intemucleoside linkages adjacent to aZ is / are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS; zero intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0141] In certain embodiments, t is 3; v is 3; 1 to 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA. PN. and OiPS; 2 to 3 intemucleoside linkages adjacent to aZ is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; zero intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0142] In certain embodiments, t is 3; v is 3; 1 intemucleoside linkage that links two contiguous Y nucleotides is selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA. PN, and OiPS; and each intemucleoside linkage that is not a PS2. MsPA. PN, or OiPS is a phosphorothioate.

[0143] In certain embodiments, t is 3; v is 3; 1 intemucleoside linkage that links two contiguous Y nucleotides is selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z is / are each independently selected from the group consisting of PS2. MsPA. PN, and OiPS; zero intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0144] In certain embodiments, t is 3; v is 3; 2 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to aZ is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; zero intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0145] In certain embodiments, t is 3; v is 3; 2 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; zero intemucleoside linkages adjacent to aZ is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a X are selected from the group consisting of PS2. MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0146] In certain embodiments, t is 3; v is 3; 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to aZ is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; zero intemucleoside linkages adjacent to a X are selected from the group consisting of PS2, MsPA, PN, and OiPS; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0147] In certain embodiments, t is 3; v is 3; 1 to 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z are each PN; 2 to 3 intemucleoside linkages adjacent to aX are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0148] In certain embodiments, t is 3; v is 3; 1 intemucleoside linkage that links two contiguous Y nucleotides is selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkage(s) adjacent to aZ are each PN; 2 to 3 intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN. or OiPS is a phosphorothioate.

[0149] In certain embodiments, t is 3; v is 3; 2 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z are each PN; 2 to 3 intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0150] In certain embodiments, t is 3; v is 3; 2 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z are each PN; 1 to 2 intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0151] In certain embodiments, t is 3; v is 3; 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA. PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z are each PN; 2 to 3 intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0152] In certain embodiments, t is 3; v is 3; 2 to 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; zero intemucleoside linkages adjacent to a Z are each PN; zero intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0153] In certain embodiments, t is 3; v is 3; zero intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z are each PN; 2 to 3 intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0154] In certain embodiments, t is 3; v is 3; 1 to 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS; 2 intemucleoside linkages adjacent to aZ are each PN; 1 intemucleoside linkage adjacent to a Z is a PO; 2 to 3 intemucleoside linkages adjacent to a X are each PN; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0155] In certain embodiments, t is 3; v is 3; 1 to 3 intemucleoside linkages that each link two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; 2 to 3 intemucleoside linkages adjacent to a Z are each PN; 2 intemucleoside linkages adjacent to a X are each PN; 1 intemucleoside linkage adjacent to a X is a PO; and each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

[0156] In certain embodiments, the location of one or more of the PS2, MsPA, PN, and OiPS present in the oligonucleotide was first identified in an oligonucleotide catabolism assay. In certain embodiments, the location of two or more of the PS2, MsPA, PN, and OiPS present in the oligonucleotide was first identified in an oligonucleotide catabolism assay. In certain embodiments, the location of each PS2, MsPA, PN, and OiPS present in the oligonucleotide was first identified in an oligonucleotide catabolism assay. ii. Polypeptides

[0157] A stabilized oligonucleotide as described herein can be covalently linked to any known polypeptide that is capable of covalently linking to a linking group. In some embodiments, the polypeptide specifically binds to a target molecule or target protein on a target cell. Thus, some embodiments are directed to an oligonucleotide conjugated to a polypeptide means for delivery. The target cell can be in a tissue. The target cell can be, but is not limited to, a cell of the central nervous system (CNS), a cell in the brain, or a cell of the blood brain barrier (BBB). The target molecule or target protein can be, but is not limited to, a cell surface receptor. In some embodiments, the target molecule or target protein comprises a protein present on a cell of the BBB. In some embodiments, the target protein comprises a BBB transport protein, a protein expressed on the luminal surface of the BBB, or a brain retention protein. The polypeptide can be monovalent or multivalent for binding to the target molecule or target protein.

[0158] In some embodiments, the polypeptide specifically binds a protein selected from the group consisting of: transferrin receptor (TfR), CD98 heavy chain (CD98hc), Large neutral amino acids transporter small subunit 1 (CD98 light chain), Glucose transporter 1 (GLUT1), Low density lipoprotein receptor, Insulin-like growth factor 1 receptor, Insulin-like growth factor 2 receptor, IgG receptor FcRn large subunit p51, Low density lipoprotein receptor-related protein 1, Low density lipoprotein receptor-related protein 2, Insulin receptor, Cell cycle control protein 50A, Transmembrane protein 50A, Basigin, Leptin Receptor, Claudin-5, P-selectin, Lactoferrin receptor, Folate receptor, Sodium-dependent lysophosphatidylcholine symporter L Solute carrier organic anion transporter family member ICT, Sodium-coupled neutral amino acid transporter 5, LDL receptor-related protein 8, High affinity cationic ammo acid transporter I, Sodium- and chloride-dependent taurine transporter, Insulin-like growth factor-binding protein 7, Solute carrier family 40 member 1, Zinc transporter 6, heparin-binding epidermal growth factor-like growth factor, and Myelin-oligodendrocyte glycoprotein (MOG). Additional BBB transport proteins include those expressed by brain endothelial cell genes associated with either receptor-mediated transcytosis or small molecule transport (see Zhang et al. Fluids and Barriers or the CNS (2020) 17:47 and Yang AC et al. Nature (2022) 603:885-892). In some embodiments, the polypeptide specifically binds TfR. In some embodiments, the polypeptide specifically binds CD98hc.

[0159] The polypeptide can be, but is not limited to, (a) a peptide that specifically binds a target protein (e.g, a BBB transport protein), (b) an antibody antigen binding domain that specifically binds a target protein (e.g., a BBB transport protein), (c) an Fc polypeptide modified to specifically bind a target protein (e.g., a BBB transport protein), (d) a CH3 peptide modified to specifically bind a target protein (e.g., a BBB transport protein), (e) a first fibronectin type III domain peptide modified to specifically bind a target protein (e.g., a BBB transport protein), or (!) a bicyclic peptide that specifically binds a target protein (e.g., a BBB transport protein).

[0160] An antibody antigen binding domain can be. but is not limited to, a Fab, a single chain Fab (scFab), a heavy chain only antibody variable domain (nanobody, e.g., a VHH or a vNAR), a Fv fragment, or a single chain variable fragment (scFv).

[0161] In some embodiments, the peptide can be, but is not limited to, (a) a peptide that specifically binds TfR or CD98hc, (b) an antibody antigen binding domain that specifically binds TfR or CD98hc. (c) a Fc polypeptide modified to specifically bind TfR or CD98hc, (d) a CH3 peptide modified to specifically bind TfR or CD98hc, (e) a fibronectin type III domain peptide modified to specifically bind TfR or CD98hc, or (f) a bicyclic peptide that specifically binds TfR or CD98hc.

[0162] An antibody antigen binding domain can be, but is not limited to, a Fab, a single chain Fab (scFab), a heavy chain only antibody variable domain (nanobody, e.g., a VHH or a vNAR), a Fv fragment, or a single chain variable fragment (scFv).

[0163] An antibody antigen binding domain comprises the antigen binding domain of an immunoglobulin or a peptide having a structure similar to the antigen binding domain of an immunoglobulin. The immunoglobulin can be, but is not limited to, an IgG, IgM, IgE, IgA, IgD, or a heavy chain antibody. An antibody antigen binding domain can be, but is not limited to, a Fab, a scFab, a Fv fragment, a scFv, or a heavy chain only antibody variable domain (nanobody, e.g., a VHH or a vNAR).

[0164] The term ‘Tab” refers to an antigen-binding fragment consisting of a light chain variable region (Vt)and a light chain constant region (together the antibody light chain), and a heavy chain variable region (Vh) and a heavy chain CHI constant region (together an antibody Fd fragment).

[0165] The term “single-chain Fab” or “scFab” refers to an antigen-binding fragment consisting of a Fab wherein the Fd fragment and the light chain linked together via a peptide linker. The linker can connect the N-terminus of the Fd fragment with the C-terminus of the light chain or the N-terminus of the light chain with the C-terminus of the Fd fragment.

[0166] The term “Fv fragment” refers to an antigen-binding fragment consisting of a Vh and a Vl that together form a binding site for an antigen.

[0167] The term “single-chain variable fragment” or “scFv” refers to an antigen-binding fragment consisting of a heavy chain variable region and a light chain variable region linked together via a peptide linker. The linker can connect the N-terminus of the Vh with the C- terminus of the Vl or the N-terminus of the Vl with the C-terminus of the Vh. An scFv lacks constant regions. Modified scFv and methods of modifying a scFv to bind to a target protein are described in WO2022258841 (incorporated herein by reference).

[0168] The term “nanobody” refers to an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies derived from camelid heavy chain antibodies can be referred to as “VHH” fragments. Nanobodies derived from cartilaginous fish heavy chain antibodies can be referred to as “vNARs.” Modified VHH fragments and methods of modifying a VHH fragment to bind to a target protein, including TfR and CD98hc, are described in WO2019246288, WO2021205361, WO2020056327, WO2022103769, and WO2023023166, and WO2023224956 (each of which is incorporated herein by reference).

[0169] In some embodiments, a modified CH3 peptide comprises a CH3 peptide that is modified to bind a target protein, such as a BBB transport protein (e.g., TfR. or CD98hc), a BBB cell surface protein, or a brain retention protein. Modified CH3 peptide and methods of modifying a CH3 peptide to bind to a target protein are described in US20180237496, US20200223935, and WO2023114499 (each of which is incorporated herein by reference). Exemplary modified CH3 peptides that bind TfR and CD98 are described in WO2018152326, WO2019140050, WO2019032955, WO2023279099, WO2023114499, and WO2023114510 (each of which is incorporated herein by reference).

[0170] In some embodiments, a peptide that specifically binds a target protein (e.g., a BBB transport protein, a BBB cell surface protein or brain retention protein) comprises a heavy chain complementary determining region 3 (CDRH3) peptide that specifically binds the target protein (e.g., the BBB transport protein, the BBB cell surface protein, or the brain retention protein). The CDRH3 can be inserted into a constant domain of an antibody, such as a CH3 peptide, a CH2 peptide, a CHI peptide, or a CL peptide as described in described in WO2023087017 (incorporated herein by reference).

[0171] An Fc polypeptide or CH3 peptide used herein can be derived from an IgG, e.g., a human IgGl, IgG2, IgG3, or IgG4.

[0172] A modified fibronectin type III (FN3) domain comprises aFN3 domain that is modified to bind a target protein, such as a BBB transport protein (e.g.. TfR or CD98hc), a BBB cell surface protein, or a brain retention protein. A FN3 domain is a consensus sequence of multiple FN3 domains from human Tenascin-C. Modified FN3 domains and methods of modifying a FN3 domain to bind to a target protein are described in US20100216708 (incorporated herein by reference). Specific examples of modified FN3 domains that bind TfR (CD71) are described in WO2021076546 and WO2022221505 (each of which is incorporated herein by reference). Additional examples of modified FN3 domains the specifically bind target proteins are described in WO2021076574, WO 2022221550, and WO 2023003874 (each of which is incorporated herein by reference).

[0173] A bicyclic peptide comprises a synthetic short peptide constrained by a chemical linker to form two loops. The chemical linker can form linkages to 3 different amino acids in the peptide. Bicyclic peptides can be made that specifically bind to target proteins, such as a BBB transport protein (e.g., TfR or CD98hc), a BBB cell surface protein, or a brain retention protein. Bicyclic peptides and methods of making bicyclic peptides that bind to a target protein are described in US Patents 8680022, 8685890, 8778844 (each of which is incorporated herein by reference). Specific bicyclic peptides that bind TfR. are described in WO2022101633, hereby incorporated by reference.

[0174] In some embodiments, the polypeptide comprises a multi-specific antibody or multispecific antibody-like molecule. Each antibody antigen binding domain of a multi-specific antibody or multi-specific antibody-like molecule can be, but is not limited to, a Fab, a scFab, a Fv fragment, a scFv, or a nanobody. In some embodiments, the polypeptide comprises a bispecific antibody or bispecific antibody-like molecule. A bispecific antibody can have two antigen binding domains of the same type (e.g., two Fabs, two scFabs, two Fv fragments, two scFvs, or two nanobodies) or a bispecific antibody can have two antigen binding domains of two different types (e.g., a Fab and an scFab, Fv fragment, a scFv, or a nanobody; a scFab and a Fab, a Fv fragment, a scFv or a nanobody; an Fv fragment and a Fab, a scFab, a scFv, or a nanobody; a scFv and a Fab, a scFab, a Fv fragment, or a nanobody; or a nanobody and a Fab, a scFab, a Fv fragment, or a scFv).

[0175] In some embodiments, the polypeptide comprises a modified antibody heavy chain constant domain or Fc polypeptide (e.g, modified CH3 peptide) that has been engineered to specifically bind to a target protein. In some embodiments, the domain that is modified is a human Ig CH3 domain. The CH3 domain can be of any IgG subtype, i.e., from IgGl, IgG2, IgG3, or IgG4. In the context of IgG antibodies, a CH3 domain refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme. a. CH3 transferrin receptor-binding proteins

[0176] In some embodiments, the polypeptide comprises a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) that specifically binds TfR. In some embodiments, the polypeptide comprises a modified CH3 domain protein that specifically binds the apical domain of TfR. The modified CH3 domain protein may bind to the transferrin receptor without blocking or otherwise inhibiting binding of transferrin to the receptor. In some embodiments, binding of transferrin to TfR is not substantially inhibited. In some embodiments, binding of transferrin to TfR. is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, binding of transferrin to TfR. is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%. 16%. 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%. 6%, 5%, 4%, 3%, 2%, or 1%). Exemplary modified CH3 peptides that bind TfR. are described in WO2018152326. WO2019140050, WO2019032955, WO2023279099, WO2023114499, and WO2023114510 (each of which is incorporated herein by reference).

[0177] In some embodiments, a polypeptide that specifically binds TfR. comprises an anti-TfR antibody antigen binding domain. An anti-TfR antibody antigen binding domain can comprise an antibody, a Fab (including a F(ab')2), a scFab, a Fv fragment, an scFv, or a nanobody. Anti-TfR antibody antigen binding domains include, but are not limited to: a 17H10 anti-TfR Fab or scFv; a 17H10.1 anti-TfR Fab or scFv;aJC-141 anti-TfR antibody; a JC-141 anti-TfR Fab; a JC-141 anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the JR-141 antibody (WO2016208695); a JC-171 anti-TfR antibody; a JC-171 anti-TfR Fab; a JC-171 anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, andCDR3 sequences of the JR-171 antibody (WO2018124121); a“Brain shuttle’7 (BS) anti-TfR Fab; an anti-TfR antibody, Fab, scFab. Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the BS anti-TfR Fab (WO2018210898, WO2015101588, WO2014033074, and Bioconjugate Chemi stiy 2023 34 (11), 2096-2111. each of which is incorporated herein by reference); a 13E4v2ii anti-TfR antibody: a 13E4v2ii anti-TfR Fab; a 13E4v2ii anti-TfR scFv; an anti-TfR antibody. Fab, scFab, Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the 13E4v2ii antibody (WO2020132584); a TfR12 anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the TfR12 anti-TfR scFv (WO2021205358); a TfR13 anti-TfR scFv; or an anti-TfR antibody, Fab. scFab, Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the Tfrl3 anti-TfR scFv (WO2021205358). Additional anti-TfR antibodies are described in WO2015098989, WO2023090409, and WO2019151539 (each of which is incorporated herein by reference).

[0178] Additional anti-TfR antibodies are described in WO2021205358 (incorporated herein by reference), and the polypeptide of the invention can include any antibody antigen binding domain with the CDRs or variable regions of any one of TfRl, TfR2, TfR3, TfR4. TfR5, TfR6, TfR7, TfR8, TfR9, TfRIO, TfRll, TfR12, TfR13, TfR14, TfR15. TfR16, TfR17, TfR18, TfRl 9, TfR20, T1R21, TfR22, TfR23, TfR24, TfR25, TfR26, TfR27, TfR28, T1R29, TfR30, TfR31, TfR32, TfR33, TfR34, TfR35, TfR36, TfR37, and TfR38 described therein.

[0179] Additional anti-TfR antibodies are known in the art and are available from various commercial sources. In some embodiments, the anti-TfR antibody or the TfR-binding fragment of an anti-TfR antibody binds to an apical domain of the TfR. In some embodiments, binding of the anti-TfR antibody or the TfR-binding fragment of an anti-TfR antibody to the TfR does not inhibit binding of transferrin to the TfR. Exemplary anti-TfR antibodies include, but are not limited to, B3 / 25, RBC4, 7579, E2.3, A27.15, D65.30, D2C, chl28.1Av, chl28. l / IgG3, ch!28.1 / IgGl, hu!28.1, (Candelaria et al. Front. Immunol. 12 (17 March 2021), 2021), Ri7, 8D3 (Weber et al. Cell Reports 22:149-162, 2018). Exemplary anti-TfR antibodies are also descnbed in U.S. patent publications: US2018282408Al, US2020071413A1, US20210138083A1, US20190092870A1, and US20130028891 (each of which is incorporated herein by reference). Exemplary anti-TfR antibodies / polypeptide suitable for conjugation to an oligonucleotide are also described in WO2020028840. WO2021142234, WO2021150382. WO2022020107, WO2022020108, WO2022020109 (each of which is incorporated herein by reference).

[0180] Exemplary anti-TfR vNARs are described in WO2022103769.

[0181] Brain shuttles containing anti-TfR or other anti-BBB protein binding domains are described in WO2014033074 and WO2015101588 and Bioconjugate Chemistry 2023 34 (11), 2096-2111 (each of which is incorporated herein by reference).

[0182] Antibody antigen binding domains that specifically bind BBB proteins can be identified using methods available in the art for generating and / or identifying antibodies that specifically bind to a target protein. Such methods include, but are not limited to, immunization, phage display, and ribosome display.

[0183] In some embodiments, a TfR-binding region comprises a fibronectin ty pe III domain peptide modified to specifically bind TfR, for example, any of the domains described in WO2021076546 and WO2022221505 (each of which is incorporated herein by reference).

[0184] In some embodiments, a peptide that specifically binds TfR includes a peptide as descnbed in WO2023022234, WO2023027125, or WO2021167107 (each of which is incorporated herein by reference).

[0185] In some embodiments, a TfR-binding region comprises a bicyclic peptide that specifically binds TfR. for example, any of the bicyclic peptides described in WO2022101633 (incorporated herein by reference).

[0186] A TfR-binding region can be derived from a protein know n to bind the TfR, such as, P. vivax reticulocyte-binding protein 2b (PvRBP2b) or a viral protein such as an arenavirus protein (e.g, Machupo, Sabia, Junin, Guanarito or Chapare virus) known to bind TfR.

[0187] In some embodiments, the TfR-binding region comprises an engineered polypeptide. An engineered polypeptide can be a polypeptide (e.g., an antibody Fc polypeptide) or antigenbinding region of an anti-TfR antibody modified to alter affinity of the polypeptide or the antigen-binding region of the anti-TfR antibody for the Tfr. Engineered peptides can be identified or generated using methods available in the art for identifying or generating a peptide having affinity for a known target (e.g., TfR). Such methods include, but are not limited to, phage display, yeast display (e.g., yest surface display), and directed evolution, and combinations thereof. b. CD98hc binding polypeptides

[0188] In some embodiments, the polypeptide comprises a modified antibody heavy chain constant domain or Fc polypeptide (e.g., modified CH3 peptide) that specifically binds CD98hc. Exemplary modified CH3 peptides that bind CD98hc are described in WO2023114499 and WO2023114510 (each of which is incorporated herein by reference).

[0189] In some embodiments, a CD98hc-binding region comprises an anti-CD98hc antibody antigen binding domain. An anti-CD98hc antibody antigen binding domain can comprise an antibody, a Fab (including an F(ab')2), a scFab, a Fv fragment, an scFv, or a nanobody. Anti-CD98hc antibody antigen binding domains include, but are not limited to: a 1C03 anti-CD98hc VHH (e.g.. 1C03-4 or 1C03-5 anti-CD98hc VHH); a CD98hc2 anti-CD98hc nanobody; an anti-CD98hc heavy chain antibody or nanobody having the CDR1, CDR2, and CDR3 sequences of the CD98hc2 anti-CD98hc nanobody; a CD98hc4 anti-CD98hc scFv; an anti-CD98hc antibody, Fab, scFab, Fv fragment, or scFv having the heavy' chain and light chain CDR1, CDR2, and CDR3 sequences of the CD98hc4 anti-CD98hc scFv; a CD98hc5 anti-CD98hc scFv; or an anti-CD98hc antibody, Fab. scFab. Fv fragment, or scFv having the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the CD98hc5 anti-CD98hc scFv as disclosed in WO2021205361 (incorporated herein by reference). The oligonucleotide polypeptide conjugates of the present invention can include polypeptides comprising antigen binding domains that have the CDRs or variable chains of any of antibodies CD98hcl, CD98hc2, CD98hc3, CD98hc4, CD98hc5, CD98hc6, CD98hc7, CD98hc8, CD98hc9, CD98hclO. CD98hcll, CD98hcl2, CD98hcl3, CD98hcl4, and CD98hcl5 as described in WO2021205361 (incorporated herein by reference).

[0190] Additional anti-CD98hc antibodies are known in the art and are available from various commercial sources. Exemplary anti-CD98hc antibodies include, but are not limited to, IGN523 (Hayes GM et al. Int J Cancer 2015 137(3):710-720). Exemplary anti-CD98hc antibodies are also described in patent publications: WO2008017828. WO2013078377, WO2021205361, WO2022252167, WO2015146132, US20130052197. and US7943745 (each of which is incorporated herein by reference).

[0191] Brain shuttles containing anti-CD98hc antibodies are described in Pomnoppadol G et al. “Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs’? bioRxiv April 29, 2023 (incorporated herein by reference).

[0192] Anti-CD98hc vNARs are described in WO2021205361, WO2019246288, and WO2023023166 (each of which is incorporated herein by reference).

[0193] Antibody antigen binding domains that specifically bind BBB proteins can be identified using methods available in the art for generating and / or identifying antibodies that specifically bind to a target protein. Such methods include, but are not limited to, immunization, phage display, and ribosome display.

[0194] In some embodiments, a CD98hc-binding region comprises a heavy chain complementary determining region 3 (CDRH3) engineered into an antibody constant domain as described in WO2023087017 (incorporated herein by reference).

[0195] In some embodiments, a CD98hc-binding region comprises a fibronectin type III domain peptide modified to specifically bind CD98hc.

[0196] In some embodiments, a CD98hc-binding region comprises a bicyclic peptide that specifically binds CD98hc. c. Additional Polypeptide Modifications

[0197] Any of the antibodies or Fc polypeptides described herein, including those comprising a modified CH3 peptide, may further comprise additional mutations, e.g., to promote heterodimer formation, to modulate effector function, to extend serum half-life and / or stability, to influence glycosylation, and / or to reduce immunogenicity in humans. 1. Polypeptide Modifications for Heterodimerization

[0198] Any of the antibodies or Fc polypeptides described herein, including those comprising a modified CH3 peptide, may further comprise one or more mutations that promote heterodimer formation and hinder homodimer formation. These modifications are useful, for example, where it is desired to from a bi-specific antibody or to form a heteromeric heavy chain dimer or heteromeric Fc dimer. Formation of a heterodimeric Fc dimer can be used to from, e.g., an Fc dimer having one Fc polypeptide that binds a target protein (e.g, TfR. or CD98hc) and a second Fc polypeptide that binds a different target protein, or to form an Fc dimer that is monovalent for binding to the target protein.

[0199] Exemplary mutations that promote Fc heterodimer formation include knob and hole mutations. The knobs-into-holes approach generally involves introducing a protuberance (‘‘knob”) at the interface of a polypeptide (e.g., an Fc polypeptide) and a corresponding cavity (‘‘hole”) in the interface of a second polypeptide (e.g, an Fc polypeptide), such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing amino acid having smaller side chains from the interface of the first polypeptide (e.g.. an Fc polypeptide) with amino acids having larger side chains (e.g., Tyr or Trp). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide (e.g., an Fc polypeptide) by replacing amino acids having larger side chains with amino acids having smaller side chains (e.g, Ala or Thr). In some embodiments, such additional mutations are at a position in the polypeptide (e.g. an Fc polypeptide) that does not have a negative effect on binding of the polypeptide to target protein.

[0200] By way of example, a knob and hole approach for dimerization can comprise replacing a native Thr at position 366 of one of the polypeptides of a dimer (e.g., an Fc polypeptide) with a Trp to form a knob (i.e., a T366W knob mutation), and replacing a native Tyr at position 407 of the other polypeptide of the dimer (e.g., an Fc polypeptide) with a Vai to form a hole (i.e., Y407V hole mutation). The other polypeptide (e.g., Fc polypeptide) may further comprise substitution of a native Thr at position 366 with Ser and substitution of a native Leu at position 368 with an Ala (i.e., T366S and L368A hole mutations). In some embodiments, one of the polypeptides of a dimer (e.g., an Fc polypeptide) has the T366W knob mutation and the other polypeptide (e.g., an Fc polypeptide) has the Y407V hole mutation, which is typically accompanied by the T366S and L368A hole mutations. All positions are numbered per EU numbering.

[0201] The knobs-into-holes approach, e.g., a T366W knob substitution on one polypeptide (e.g., an Fc polypeptide) with T366S, L368A, and Y407V hole substitutions on the other polypeptide (e.g., an Fc polypeptide) can be used with antibodies, heavy chain dimers, or Fc dimers, including any of the described CD98hc-binding Fc polypeptides and / or TfR.-binding Fc polypeptides. 2. Polypeptide Modifications for Modulating Effector Function

[0202] Any of the antibodies or Fc polypeptides described herein, including those comprising a modified CH3 peptide, may further comprise one or more mutations that reduce or eliminate effector function. Reducing or eliminating effector function includes reducing or eliminating the ability’ of the Fc polypeptide or Fc dimer to induce certain biological functions upon binding of the Fc polypeptide or dimer to an Fc receptor expressed on an effector cell that mediates the effector function. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and cytotoxic T cells. Examples of antibody effector functions include, but are not limited to, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptor), and B-cell activation.

[0203] In some embodiments, any of the antibodies or Fc polypeptides described herein may comprise a CH2 peptide having one or more mutations that reduce effector function. Mutations that reduce effector function can be, but are not limited to, mutations at positions 234, 235, and 390, according to EU numbering. The mutations may be present in a single heavy chain of an antibody, a single Fc polypeptide of an Fc dimer, in both heavy chains of an antibody, or in both Fc polypeptides of an Fc dimer.

[0204] In some embodiments, any of the antibodies or Fc polypeptides described herein may comprise a CH2 peptide having an A at position 234, an A at position 235; a G at position 329 (“P329G” or “PG” mutation); a S at position 329 (“P329S” or “PS” mutation); an A at position 234 and an A at position 235 (“LALA” mutation). A at position 234, an A at position 235, and a G at position 329 (“LALA PG” mutation), or an A at position 234, an A at position 235, and a S at position 329 (“LALA PS” herein).

[0205] In some embodiments, any of the antibodies or Fc dimers described can comprise an A at position 234, an A at position 235; a G at position 329 (“P329G” or “PG” mutation); a S at position 329 (“P329S” or “PS” mutation); an A at position 234 and an A at position 235 (“LALA” mutation), A at position 234, an A at position 235, and a G at position 329 (“LALA PG” mutation), or an A at position 234, an A at position 235, and a S at position 329 (“LALA PS” herein) in both CH2 peptides.

[0206] In some embodiments, any of the antibodies or Fc dimers described herein can comprise an A at position 234, an A at position 235; a G at position 329 (“P329G” or “PG” mutation); a S at position 329 ("P329S" or “PS” mutation); an A at position 234 and an A at position 235 (“LALA” mutation). A at position 234, an A at position 235, and a G at position 329 (“LALA PG” mutation), or an A at position 234, an A at position 235, and a S at position 329 (“LALA PS” herein) in a single CH2 peptide.

[0207] Additional mutations that modulate an effector function include, but are not limited to: (a) substitution of the native P at position 329 for a G, A, S, R, or an amino acid residue large enough to inhibit formation of the Fc / Fcy receptor interface that is formed between P329 of the Fc and Trp87 and Trpl 10 of FcyRIIL and / or (b) one or more of S228P, E233P, L235E, N297A, N297D, or P331S substitutions, according to the EU numbering scheme.

[0208] Combination of substitutions that modulate an effector function include, but are not limited to: L234A. L235A, and P329G of human IgGl; S228P and L235E of human IgG4; L234A and G237A of human IgGl; L234A, L235A, and G237A of human IgGl; V234A and G237A of human IgG2; L235A, G237A, and E318A of human IgG4; and S228P and L236E of human IgG4, according to the EU numbering scheme. 3. Polypeptide Modifications for Extending Serum HalfLife

[0209] Any of the antibodies or Fc polypeptides described herein, including those comprising a modified CH3 peptide, may further comprise one or more mutations that alter serum halflife. In some embodiments, any of the antibodies or Fc polypeptides described herein, including those comprising a modified CH3 peptide, may further comprise one or more mutations that enhance or prolong serum half-life.

[0210] Exemplary mutations that enhance serum half-life include, but are not limited to: a M428L substitution, a N434S substitution, a M428L and a N434S substitutions (“LS” substitutions), a N434S substitution, a N434A substitution, a M428L substitution, a M252Y substitution, a S254T substitution, a T256E substitution, or combination of M252Y, S254T, and T256E substitutions, as numbered according to the EU numbering scheme.

[0211] The one or more mutations that enhance serum half-life may be present in a single heavy chain of an antibody, a single Fc polypeptide of an Fc dimer, in both heavy chains of an antibody, or in both Fc polypeptides of an Fc dimer. iii. Linking Groups

[0212] A stabilized oligonucleotide as described herein can be covalently attached to any known linking group that is a bifunctional linker capable of also covalently linking to a polypeptide. Thus, in certain embodiments, the linking group covalently joins an oligonucleotide to the polypeptide. The linking group may be any group suitable for joining an oligonucleotide to the polypeptide.

[0213] Oligonucleotide polypeptide conjugates can be generated using well-known chemical cross-linking reagents and protocols that covalently link the oligonucleotide and the polypeptide through a linking group. For example, there are a large number of chemical crosslinking agents that are known to those skilled in the art and useful for cross-linking a protein with an agent of interest. For example, the cross-linking agents are heterobifunctional crosslinkers, which can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers. A wide variety of heterobifunctional cross-linkers are known in the art. including N-hydroxysuccinimide (NHS) or its water soluble analog N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), l-ethyl-3-(3-dimethylarmnopropyljcarbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Those cross-linking agents having N-hydroxysuccinimide moieties can be obtained as the N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional cross-linkers, there exist a number of other cross-linking agents including homobifunctional and photoreactive cross-linkers. Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate.2HCl (DMP) are examples of useful homobifunctional cross-linking agents, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers.

[0214] Non-limiting examples of conjugate linkers include those described in WO 2023 / 279099 and WO2023 / 056388, each of which is incorporated by reference in its entirety. In certain embodiments, the linking group comprises a Val-cit linker as described in U.S. 6,214,345, which is incorporated herein by reference. In certain embodiments, the linking group comprises those described in WO 2020 / 028840, WO 2022 / 212886 and US 2023 / 0287108, each of which is incorporated by reference in its entirety. Other linkers can include those described in Bioconjugate Chemistry 2023 34 (11), 2096-2111.

[0215] The linking group may be attached to any region of the polypeptide, (e.g, to the N-terminal region, to the C-terminal region, or to an amino acid within the protein, such as a cysteine residue or a glutamine residue), so long as the oligonucleotide does not prevent binding of the polypeptide to its target. Similarly, the linking group may be attached to any region of the oligonucleotide (e.g. the 5' end, the 3' end or to a nucleic acid residue within the molecule), so long as the polypeptide does not interfere with the functionality of the oligonucleotide (e.g., complementary binding to a target nucleic acid). For example, the linker may be attached to the oligonucleotide through any number of synthetically feasible points located throughout the oligo, such as at the 3' or 5' terminal residues of the oligo; at a sugar moiety; at a base moiety; or at a residue located within the backbone.

[0216] In certain embodiments, the linker is attached to the oligonucleotide at the 5' terminal residue of the oligonucleotide. In certain embodiments, the linker is attached to the oligonucleotide at the 3' terminal residue of the oligonucleotide. In certain embodiments, the linking group comprises a Val-cit linker as described in U.S. 6.214.345, which is incorporated herein by reference. In certain embodiments, the linking group comprises those described in WO 2020 / 028840, WO 2022 / 212886, US 2023 / 0287108, and Bioconjugate Chemistry 2023 34 (11), 2096-2111, each of which is incorporated by reference in its entirety.

[0217] In certain embodiments, the linking group comprises spacers. In certain embodiments, the spacers are hydrophilic spacers. In certain embodiments, the hydrophilic spacers are polyethylene glycol (PEG).

[0218] In certain embodiments, the linking group is a homobifuctional linker or a heterobifunctional linker.

[0219] In some embodiments, the linking group is cleavable (e.g, a nuclease-cleavable linker, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020). In certain embodiments, the linking group comprises one or more nucleotides (e.g, 1, 2, 3. or more) or one or more nucleosides (e.g, 1. 2, 3, or more). In certain embodiments, the one or more nucleotides or one or more nucleosides are unmodified. In certain embodiments, the linking group comprises one or more nucleotides having unmodified bases, unmodified sugar groups and / or unmodified phosphate groups. In certain embodiments, the linking group comprises one or more nucleosides having unmodified bases and / or unmodified sugar groups. In certain embodiments, the linking group comprises TCA, a nuclease linker. In certain embodiments, TCA is modified with a C6 amine at T position. In certain embodiments, the linking group does not comprise TCA. In certain embodiments, the linking group is enzymatically cleavable. In certain embodiments, the linking group is cleavable by an enzyme present in the central nervous system or muscle. In certain embodiments, a cleavable linking group is selected for conjugates comprising ASOs (e.g, to enable the ASO to dissociate from the remainder of the conjugate for transport into the nucleus). In certain embodiments, the cleavable linking group is a cleavable dipeptide linker. In certain embodiments, the cleavable dipeptide linker is a val-cit cleavable linking group or val-ala cleavable linker. In certain embodiments, the cleavable linking group is an acid cleavable linker. In certain embodiments, the acid cleavable linker is a carbonate linker or a hydrazone linker. In certain embodiments, the cleavable linking group comprises PEG spacers. In certain embodiments, the cleavable linking group is a disulfide such as SPDP (succinimidyl 3-(2-pyridyldithio)propionate) or lys-conjugated acid-cleavable hydrazide.

[0220] In certain embodiments, the linking group is a non-cleavable linking group. In certain embodiments, the linking group is a covalent linking group. In certain embodiments, the covalent linking group is derivable from an APN or an acrylamide. In certain embodiments, the covalent linking group comprises a group CH2CH2C(=O). In certain embodiments, the covalent linking group comprises a group:

[0221] In certain embodiments, the covalent linking group is derivable from a haloacetamide, e.g., bromoacetamide, chloroacetamide, iodoacetamide.

[0222] In certain embodiments, the linking group comprises a Ce amine group having the formula -(CEbje-NH-.

[0223] In certain embodiments, the linking group is derivable from a maleimide. For example, in certain embodiments, the linking group comprises a group: O KO .

[0224] In certain embodiments, the linking group may be attached to the polypeptide (P) at the valence marked * (e.g., to a sulfur atom of a modified site within P).

[0225] In certain embodiments, the maleimide is a modified maleimide. In certain embodiments, the modified maleimide is an alkyl-, aryl-, cycloalkyl-, or exocyclic- maleimide. In certain embodiments, the linking group comprises a protected maleimide. For example, in certain embodiments, the linking group comprises a protected maleimide of formula: O .

[0226] In certain embodiments, the protected maleimide is removed after bioconjugation.

[0227] In certain embodiments, the linking group is a self-hydrolyzing linking group.

[0228] Certain specific, non-limiting embodiments (abbreviated as Linker Embodiments LE1-LE42) are described below.

[0229] In Linker Embodiments LEI, the linking group has a molecular weight of from about 20 daltons to about 5,000 daltons. In In Linker Embodiments LE2, the linking group has a molecular weight of from about 20 daltons to about 1,000 daltons. In Linker Embodiments LE3, the linking group has a molecular weight of from about 20 daltons to about 200 daltons. In Linker Embodiments LE4, the linking group has a length of about 5 angstroms to about 60 angstroms.

[0230] In Linker Embodiments LE5, the linking group separates the peptide from the remainder of the conjugate of formula I by about 5 angstroms to about 40 angstroms, inclusive, in length.

[0231] In Linker Embodiments LE6, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein one or more (e.g., 1, 2, 3. or 4) of the carbon atoms is optionally replaced by (-O-), (-NH-), (S), an amino acid, a hydrazone (C(R?)=N=N(R’)). a nucleotide, or a 3-12 membered di-valent heterocycle, wherein the chain and any 3-12 membered di-valent heterocycle is optionally substituted with one or more (e.g, 1, 2, 3, or 4) substituents independently selected from the group consisting of (Ci-Ce)alkoxy, (Cs-Cejcycloalkyl, (Ci-Ce)alkanoyl, (Ci-Ce)alkanoyloxy, (Ci-Cejalkoxycarbonyl, (Ci-Cejalkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), a hydrazone (=N=N(R’)) carboxy, aryl, aryloxy, heteroaryl, and heteroaryl oxy; wherein each R’ is independently H or (Ci-Ce)alkyl.

[0232] In Linker Embodiments LE7, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein one or more (e.g., 1, 2. 3. or 4) of the carbon atoms is optionally replaced by (-O-). (-NH-). or a 3- 12 membered di-valent heterocycle, wherein the chain and any 3-12 membered di-valent heterocycle is optionally substituted with one or more (e.g, 1, 2. 3, or 4) substituents independently selected from the group consisting of (Ci-C6)alkoxy, (C3-C6)cycloalkyl, (Ci-Ce)alkanoyl, (Ci-C6)alkanoyloxy, (Ci-Ce)alkoxy carbonyl, (Ci-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0233] In Linker Embodiments LE8, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein one or more (e.g, 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (-0-), (-NH-), (S), an amino acid, a hydrazone (C(R’)=N=N(R’)), a nucleotide, or a 3-12 membered di-valent heterocycle, wherein the chain and any 3-12 membered di-valent heterocycle is optionally substituted with one or more (e.g., 1, 2. 3, or 4) substituents independently selected from the group consisting of (Ci-C6)alkoxy, (C3-C6)cycloalkyl, (Ci-C6)alkanoyl, (Ci-C6)alkanoyloxy, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), a hydrazone (=N=N(R’)) carboxy, aryl, ary loxy, heteroaiyl, and heteroaryloxy; wherein each R’ is independently H or (Ci-Ce)alkyl.

[0234] In Linker Embodiments LE9, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (-0-), (-NH-), or a 312 membered di-valent heterocycle, wherein the chain and any 3-12 membered di-valent heterocycle is optionally substituted with one or more (e.g, 1, 2. 3, or 4) substituents independently selected from the group consisting of (Ci-C6)alkoxy, (C3-C6)cycloalkyl, (Ci-Ce)alkanoyl, (Ci-C6)alkanoyloxy, (Ci-Ce)alkoxycarbonyl, (Ci-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroary l, and heteroaryloxy.

[0235] In Linker Embodiments LE10, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (Ci-Ce)alkoxy, (C3-C6)cycloalkyl, (Ci-C6)alkanoyl, (Ci-C6)alkanoyloxy, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy. heteroaryl. and heteroaryloxy.

[0236] In Linker Embodiments LEI 1, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (Ci-Cs)alkoxy, (C3-C6)cycloalkyl, (Ci-C6)alkanoyl. (Ci-Ce)alkanoyloxy. (Ci- C6)alkoxycarbonyl, (Ci-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0237] In Linker Embodiments LE12, the linking group is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms.

[0238] In Linker Embodiments LE13, the linking group is a divalent, branched or unbranched, saturated hydrocarbon chain, having from 2 to 10 carbon atoms.

[0239] In Linker Embodiments LE14, the linking group is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 10 carbon atoms.

[0240] In Linker Embodiments LEI5, the linking group is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more disulfide linkages.

[0241] In Linker Embodiments LEI6, the linking group is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more hydrazone groups in the chain or appended to a carbon atom of the chain.

[0242] In Linker Embodiments LEI7. the linking group is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more amino acids in the chain.

[0243] In Linker Embodiments LEI 8, the linking group is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises a dipeptide in the chain.

[0244] In Linker Embodiments LEI9, the linking group is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises the dipeptide val-cit in the chain.

[0245] In Linker Embodiments LE20, the linking group comprises one or more nucleotides in the chain.

[0246] In Linker Embodiments LE21, the linking group comprises two or more nucleotides in the chain.

[0247] In Linker Embodiments LE22. the linking group comprises a tri-nucleotide group in the chain.

[0248] In Linker Embodiments LE23, a linking group is attached to two or more oligonucleotides (e.g., for a compound of formula (I) at least one “y’‘ is greater than 1).

[0249] In Linker Embodiments LE24, only one linking group is attached to two or more oligonucleotides (e.g., for a compound of formula (I) one '‘y” is greater than 1).

[0250] In Linker Embodiments LE25, at least two linking groups are attached to two or more oligonucleotides (e.g., for a compound of formula (I) at least two “y” are greater than 1).

[0251] In Linker Embodiments LE26, at least two linking groups are attached to two oligonucleotides (e.g., for a compound of formula (I), “y” is 2 and n is greater than 1).

[0252] In Linker Embodiments LE27, the linking group is attached to the oligonucleotide through a phosphate of the oligonucleotide (e.g., associated with the 5' terminal residue).

[0253] In Linker Embodiments LE28, the linking group is attached to the oligonucleotide through a phosphorothioate of the oligonucleotide (e.g, associated with the 5' terminal residue).

[0254] In Linker Embodiments LE29, the linking group comprises a polyethyleneoxy chain. In another embodiment of the invention the polyethyleneoxy chain comprises 2, 3, 4, 5. 6, 7, 8, 9, or 10 repeating ethyleneoxy units.

[0255] In Linker Embodiments LE30, the linking group comprises a 5-membered di-valent heterocycle.

[0256] In Linker Embodiments LE31, the linking group has the following structure: wherein L’ is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (-O-). (-NH-), (-S-), an amino acid, a hydrazone (-C(R')=N=N(R')-). a nucleotide, or a 3-12 membered divalent heterocycle, wherein the chain and any 3-12 membered divalent heterocycle is optionally substituted with one or more (e.g, 1, 2, 3, or 4) substituents independently selected from the group consisting of (Ci-C6)alkoxy, (C3-C6)cycloalkyl, (Ci-C6)alkanoyl, (Ci-C6)alkanoyloxy, (Ci-C6)alkoxy carbonyl. (Ci-C6)alkylthio. azido, cyano, nitro, halo, hydroxy, oxo (=0), a hydrazone (=N=N(R')-) carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy; wherein each R' is independently H or (Ci-Ce)alkyl; and wherein the valence marked * is attached to the polypeptide and the valence marked ** is attached to X in formula (I) (or a 5' terminal group of X). In another embodiment. L’ is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more disulfide linkages. In another embodiment, L' is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more hydrazone groups in the chain or appended to a carbon atom of the chain. In another embodiment, L’ is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises one or more amino acids in the chain. In another embodiment. L’ is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises a dipeptide in the chain. In another embodiment, L’ is a divalent, branched or unbranched, saturated or unsaturated, chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain comprises the dipeptide val-cit in the chain. In another embodiment, L’ comprises one or more nucleotides. In another embodiment, L’ comprises two or more nucleotides. In another embodiment, L’ comprises a tri-nucleotide group. In another embodiment, L‘ comprises one or more nucleotides having unmodified bases, unmodified sugar groups and / or unmodified phosphate groups.

[0257] In certain embodiments, a stabilized oligonucleotide polypeptide conjugate comprises: o-I .0—P—O—Oligonucleotide wherein P is the polypeptide, and wherein the phosphodiester (PO) group is a 5' terminal group of the oligonucleotide.

[0258] In certain embodiments, a stabilized oligonucleotide polypeptide conjugate comprises: wherein P is the polypeptide, and wherein the phosphorothioate (PS) group is a 5' terminal group of the oligonucleotide.

[0259] In certain embodiments, a stabilized oligonucleotide polypeptide conjugate comprises: wherein P is the polypeptide, and wherein the cyclic phosphory l guanidine (PN) group is a 5' terminal group of the oligonucleotide.

[0260] In Linker Embodiments LE32. L' has the following structure: wherein t is 1, 2, 3. 4, 5, 6, 7, or 8; z is 0, 1, 2, 3, 4, 5, 6. 7, or 8; and each of Ri, R2, and R3 is independently a nucleotide.

[0261] In Linker Embodiments LE33, L’ has the following structure: 10

[0262] In Linker Embodiments LE34, the linking group has the following structure: O wherein t is 1, 2, 3, 4, 5, 6, 7, or 8; and z is 0, 1,2, 3, 4, 5, 6, 7, or 8.

[0263] In Linker Embodiments LE35, the linking group has the following structure: o wherein t is 1, 2, 3, 4, 5, 6, 7, or 8; and z is 0, 1,2, 3, 4, 5, 6, 7, or 8, wherein the valence marked * is attached to the polypeptide and the valence marked ** is attached to X in formula (I). In certain embodiments, the valence marked ** is attached to X through a 5   phosphate of the oligonucleotide (e.g., associated with the 5' terminal residue).

[0264] In Linker Embodiments LE36. the linking group has the following structure:

[0265] In Linker Embodiments LE37, the linking group has the following structure: 10 wherein the valence marked * is attached to the polypeptide and the valence marked ** is attached to X in formula (I). In certain embodiments, the valence marked ** is attached to X through a phosphate of the oligonucleotide (e.g., associated with the 5' terminal residue).

[0266] In Linker Embodiments LE38, the linking group has the following structure:

[0267] In Linker Embodiments LE39, the linker is a peptide linker or formed from a protein, peptide, or amino acid. For example, in certain embodiments, the linking group is a divalent radical formed from a protein. In another embodiment, the linking group is a divalent radical formed from a peptide. In another embodiment, the linking group is a divalent radical formed 5 from an amino acid.

[0268] In Linker Embodiments LE40, the linking group may be configured such that it allows for the rotation of the oligonucleotide and the TfR binding protein relative to each other; and / or is resistant to digestion by proteases. In some embodiments, the linking group may be a flexible linker, e.g., containing amino acids such as Gly, Asn, Ser, Thr, Ala, and the like. Such linking 10 groups are designed using known parameters. For example, the linking groups may have repeats, such as Gly-Ser repeats.

[0269] In Linker Embodiments LE41, the linking group has or comprises a formula selected O 0 wherein 5           each A is independently (Ci-Ci5)alkyl; each D is -(CH2-CH2-O)m-; and each mis 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0270] In Linker Embodiments LE42, the linking group has or comprises a formula selected 10 from the group consisting of: 5

[0271] The term “halo” is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.

[0272] The term "alkyl", by itself or as part of another substituent, means, unless otherwise 10 stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., Ci-6 means one to six carbons). Examples include (Ci-C6)alkyl, (C2-Ce)alkyl and (C3-Ce)alkyl. Examples of alky l groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and higher homologs and isomers. 15

[0273] The term "alkoxy" refers to an alkyl groups attached to the remainder of the molecule via an oxygen atom (“oxy”).

[0274] The term “alkylthio” refers to an alkyd groups attached to the remainder of the molecule via a thio group.

[0275] The term “alkoxycarbonyl'’ as used herein refers to a group (alkyl)-O-C(=O)-, wherein the term alkyl has the meaning defined herein.

[0276] The term “alkanoyloxy” as used herein refers to a group (alkyl)-C(=O)-O, wherein the term alkyl has the meaning defined herein.

[0277] The term "aryloxy" refers to an aryl group attached to the remainder of the molecule via an oxygen atom (Aryl-O-).

[0278] The term "heteroaryloxy" refers to a heteroaryl group attached to the remainder of the molecule via an oxygen atom (Heteroaryl-O-).

[0279] As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0280] The term “cycloalkyl” refers to a saturated or partially unsaturated (non-aromatic) all carbon ring having 3 to 6 carbon atoms (i.e., (C3-C6)carbocycle). Non-limiting examples of cycloalkyds include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0281] The term “and” as used herein refers to a single all carbon aromatic ring or a multiple condensed all carbon ring system wherein at least one of the rings is aromatic. For example, in certain embodiments, an and group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes a phenyl radical. Aryl also includes multiple condensed carbon ring systems (e.g., ring systems comprising 2, 3 or 4 rings) having about 9 to 20 carbon atoms in which at least one ring is aromatic and wherein the other rings may be aromatic or not aromatic (z.e., cycloalkyl. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the point of attachment of a multiple condensed ring system, as defined above, can be at any position of the ring system including an aromatic or a carbocycle portion of the ring. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1, 2, 3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0282] The term “heterocycle” refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. The sulfur and nitrogen atoms may also be present in their oxidized forms. Exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl. The term “heterocycle” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from cycloalkyl, aryl, and heterocycle to form the multiple condensed ring system. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heterocycle) can be at any position of the multiple condensed ring system including a heterocycle, aryl and carbocycle portion of the ring. In one embodiment the term heterocycle includes a 3-12 membered heterocycle. In one embodiment the term heterocycle includes a 37 membered heterocycle. In one embodiment the term heterocycle includes a 3-6 membered heterocycle. In one embodiment the term heterocycle includes a 4-6 membered heterocycle. In one embodiment the term heterocycle includes a 3-12 membered monocyclic or bicyclic heterocycle heterocycle comprising 1 to 3 heteroatoms. In one embodiment the term heterocycle includes a 3-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. In one embodiment the term heterocycle includes a 4-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. Exemplary' heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl. morpholinyl, thiomorpholinyl. piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro [cyclopropane-1,1 '-isoindoliny 1] -3 '-one,           isoindoliny 1-1 -one,           2-oxa-6- azaspiro[3.3]heptanyl, imidazolidin-2-one imidazolidine. pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, 1,4-di oxane and

[0283] In one embodiment the heterocycle can be di-valent, i.e., attached to the remainder of the molecule or the linking group at two positions of the heterocycle (heterocycle). In one embodiment, the heterocycle is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of (Ci-C6)alkoxy. (C3-Ce)cycloalkyl, (Ci- Ce)alkanoyl, (Ci-Ce)alkanoyloxy, (Ci-Cs)alkoxy carbonyl. (Ci-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), and carboxy. As used herein a wavy line ” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wary bond intersects in the chemical structure to the remainder of a molecule. IV. Methods

[0284] In certain embodiments, the subject matter described herein is directed to preparing stabilized oligonucleotides and oligonucleotide polypeptide conjugate comprising a stabilized oligonucleotide.

[0285] In certain embodiments, the subject matter described herein is directed to a method for preparing a stabilized oligonucleotide polypeptide conjugate, the method comprising: (i) determining the in vivo plasma clearance of a first oligonucleotide polypeptide in a first oligonucleotide conjugate; (ii) determining a soft spot in the first oligonucleotide of the first oligonucleotide polypeptide conjugate in an oligonucleotide catabolism assay; (iii) providing a second oligonucleotide polypeptide conjugate comprising a stabilized second oligonucleotide and determining the in vivo plasma clearance of the second oligonucleotide in the second oligonucleotide polypeptide conjugate, wherein the stabilized second oligonucleotide comprises: (a) the same nucleotide sequence as the first oligonucleotide; (b) 1 to 10 stabilizing intemucleoside linkages that are not present in the first oligonucleotide, wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the oligonucleotide as determined by an oligonucleotide catabolism assay; and (c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; and, wherein at least one of the 1 to 10 stabilizing intemucleoside linkages not present in the first oligonucleotide is located at or adjacent to the soft spot identified in step (ii); wherein the in vivo plasma clearance of the second oligonucleotide polypeptide conjugate is determined to be slower than that of the first oligonucleotide polypeptide conjugate.

[0286] In certain embodiments, the method further comprises: (iv) providing a third oligonucleotide polypeptide conjugate comprising a further stabilized oligonucleotide having (a) one or more additional stabilizing intemucleoside linkages not present in the first or second oligonucleotides, and (b) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodi ester linkages.

[0287] In certain embodiments, the subject matter described herein is directed to methods of preparing a stabilized oligonucleotide polypeptide conjugate, the methods comprise: (i) determining the in vivo plasma clearance of a first oligonucleotide in a first oligonucleotide polypeptide conjugate; (ii) determining a soft spot in the first oligonucleotide of the first oligonucleotide polypeptide conjugate in an oligonucleotide catabolism assay; (iii) providing a one or more stabilized second oligonucleotides comprising: (a) the same nucleotide sequence as the first oligonucleotide; (b) 1 to 10 stabilizing intemucleoside linkages that are not present in the first oligonucleotide, wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the oligonucleotide as determined by an oligonucleotide catabolism assay: and (c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; wherein at least one of the 1 to 10 stabilizing intemucleoside linkages not present in the first oligonucleotide is located at or adjacent to the soft spot identified in step (ii); (iv) providing one or more second oligonucleotide polypeptide conjugates comprising the one or more stabilized second oligonucleotides; and (v) determining the in vivo plasma clearances of the one or more second oligonucleotides in the one or more second oligonucleotide polypeptide conjugates; and (vi) selecting a second oligonucleotide polypeptide conjugate having slower clearance relative to the first oligonucleotide polypeptide conjugate.

[0288] In certain embodiments, the method further comprises: (vi) providing a one or more stabilized third oligonucleotides comprising: (a) the same nucleotide sequence as the first oligonucleotide; (b) 1 to 10 stabilizing intemucleoside linkages that are not present in the stabilized second oligonucleotide of the second oligonucleotide polypeptide conjugate selected in step (v), wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the oligonucleotide as determined by an oligonucleotide catabolism assay; and (c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; wherein at least one of the 1 to 10 stabilizing intemucleoside linkages is not present in the selected stabilized second oligonucleotide; (vii) providing one or more third oligonucleotide polypeptide conjugates comprising the one or more stabilized third oligonucleotides; and (viii) determining the in vivo plasma clearances of the one or more third oligonucleotides in the one or more third oligonucleotide polypeptide conjugates; and (ix) selecting a third oligonucleotide polypeptide conjugate having slower clearance relative to the second oligonucleotide polypeptide conjugate select in step (v).

[0289] In certain embodiments, one or more additional rounds of stabilization are performed by repeating steps (vi)-(ix).

[0290] In certain embodiments, the 1 to 10 stabilizing intemucleoside linkages of step (iii) or step (vi) that are not present in the first or second oligonucleotide comprise a substitution of a phosphorothioate or phosphodi ester for a stabilizing intemucleoside (e.g., an PS2, PN, MsPA, or OiPS). In some embodiments, the 1 to 10 stabilizing intemucleoside linkages of step (iii) or step (vi) that are not present in the first or second oligonucleotide comprises a substitution of one stabilizing intemucleoside linkage for a different stabilizing intemucleoside linkage.

[0291] The selected second or third (or subsequent) oligonucleotide polypeptide conjugate can have enhanced pharmacokinetic and pharmacodynamic properties relative to the prior (first or second, respectively) oligonucleotide polypeptide conjugate. Thus, the stabilized oligonucleotides are particularly useful for conjugation to a polypeptide to form an oligonucleotide polypeptide conjugate having enhanced therapeutic potential.

[0292] In certain embodiments, the subject matter described herein is directed to methods of preparing a stabilized oligonucleotide polypeptide conjugate, the methods comprise: (i) determining the in vivo plasma clearance of a first oligonucleotide (e.g., gapmer; e.g, a gapmer having three nucleotides each in the wing segments) in a first oligonucleotide polypeptide conjugate; (ii) identifying a soft spot in the first oligonucleotide of the first oligonucleotide polypeptide conjugate in an oligonucleotide catabolism assay; (iii) substituting one or more intemucleoside linkages in the first oligonucleotide for one or more stabilizing intemucleoside linkages to form one or more stabilized second oligonucleotides, wherein the one or more substituted intemucleoside linkages are (a) at and / or adjacent to the soft spot identified in step (ii); and / or (b) within 3-4 nucleotides of the 5' or 3' terminus of the first oligonucleotide; (iv) providing one or more second oligonucleotide polypeptide conjugates comprising the one or more stabilized second oligonucleotides; and (v) determining the in vivo plasma clearances of the one or more second oligonucleotides in the one or more second oligonucleotide polypeptide conjugates; and (vi) selecting a second oligonucleotide polypeptide conjugate having slower clearance relative to the first oligonucleotide polypeptide conjugate. In certain embodiments, substituting an intemucleoside linkage in the first oligonucleotide for stabilizing intemucleoside linkage comprises substituting a PS or PO for a stabilizing intemucleoside linkage (e.g.. aPS2, MsPA, PN. or OiPS). In certain embodiments, substituting an intemucleoside linkage in the first oligonucleotide for stabilizing intemucleoside linkage comprises substituting one stabilizing intemucleoside linkage for a different stabilizing intemucleoside linkage (e.g., substituting a PS2 for a MsPA or PN). In certain embodiments, substituting one or more intemucleoside linkages at and / or adjacent to a soft spot comprises: substituting the intemucleoside linkage identified as the soft spot, substituting the intemucleoside linkage adjacent (5' and / or 3') to the soft spot, or substituting the intemucleoside linkage identified as the soft spot and one or two intemucleoside linkages adjacent (5' and / or 3') to the soft spot. In certain embodiments, substituting one or more intemucleoside linkages within the 3-4 nucleotides of the 5' or 3' terminus comprises substituting on or more of the intemucleoside linkages between nucleosides: 1 and 2, 2, and 3, 3, and 4, n and n-1, n-1 and n-2, and n-2 and n-3 (wherein n is the number of nucleosides in the first oligonucleotide). In certain embodiments, a stabilized second oligonucleotide contains 1 to 10 stabilizing intemucleoside linkages that were not present in the first oligonucleotide. In certain embodiments, a stabilized second oligonucleotide contains 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages. In certain embodiments, a stabilized second oligonucleotide contains 1 to 10 stabilizing intemucleoside linkages that were not present in the first oligonucleotide and 8 to 15 phosphorothioate and / or natural phosphodiester intemucleoside linkages.

[0293] In certain embodiments, steps can be performed iteratively any number of times. In such embodiments, the method comprises determining the in vivo plasma clearance of a prior oligonucleotide polypeptide conjugate; and, providing a subsequent oligonucleotide polypeptide conjugate having the same sequence as a prior oligonucleotide polypeptide conjugate and comprising a further stabilized oligonucleotide having (a) one or more additional stabilizing intemucleoside linkages not present in a prior oligonucleotide, and (b) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages.

[0294] In certain embodiments, the order of performance of certain steps can be performed in any order that is practicable.

[0295] In certain embodiments, in vivo plasma clearance is measured in a rodent or non-human primate model. In certain embodiments, the rodent is a rat. In certain embodiments, the nonhuman primate is a cynomolgus monkey.

[0296] In certain embodiments, in vivo plasma clearance of an oligonucleotide polypeptide conjugate is measured by plasma concentration taken at one or more timepoints from zero to about 24, about 48, about 72, about 168, or about 336 hours after injection of the oligonucleotide polypeptide conjugate. In certain embodiments, plasma concentration is measured at about 0.25, about 4, 24, about 48, and / or about 72 hours post-injection. In certain embodiments, additional measurements are taken daily up to about two weeks post-injection.

[0297] In certain embodiments, in vivo plasma clearance of an oligonucleotide polypeptide conjugate is measured by (i) injecting (e.g.. via intravenous injection) a rodent or non-human primate with about 1 to about 300 mg / kg of an oligonucleotide polypeptide conjugate, (ii) collecting plasma from the rodent or non-human primate at one or more times (e.g.. about 0.25, about 4, about 24, about 48, and / or about 72 hours after injection), and (iii) measuring plasma concentration of the oligonucleotide or oligonucleotide polypeptide conjugate at each timepoint of (ii). Measuring plasma concentration of the oligonucleotide or oligonucleotide polypeptide conjugate can be done using methods available in the art for measuring the concentration of an oligonucleotide or oligonucleotide polypeptide conjugate. In certain embodiments, about 1,2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.21,22,23, 24. 25. 26. 27. 28. 29 or 30 mg / kg of an oligonucleotide polypeptide conjugate is injected in the rodent or non-human primate. In certain embodiments, about 10 mg / kg of an oligonucleotide polypeptide conjugate is injected in the rodent or non-human primate. In certain embodiments, about 25 mg / kg of an oligonucleotide polypeptide conjugate is injected in the rodent or non-human primate.

[0298] In certain embodiments, when the method is performed iteratively the in vivo plasma clearance of a subsequent oligonucleotide polypeptide conjugate is determined to be slower than that of a prior oligonucleotide polypeptide conjugate when the plasma concentration of the subsequent oligonucleotide polypeptide conjugate is greater than that of a prior oligonucleotide polypeptide conjugate at a given timepoint. In certain embodiments, the in vivo plasma clearance of the second oligonucleotide polypeptide conjugate is determined to be slower than that of the first oligonucleotide polypeptide conjugate when the plasma concentration of the second oligonucleotide polypeptide conjugate is greater than that of the first oligonucleotide polypeptide conjugate at a given timepoint. In certain embodiments, the timepoint is about 0.25 to about 72 hours (e.g., about 0.25, about 4, about 24. about 48, and / or about 72 hours) post injection of the oligonucleotide polypeptide conjugate in the rodent or non-human primate. In certain embodiments, additional measurements beyond 72 hours post injection, e.g., at about 96 to about 168 hours (e.g., at about 96, about 120, about 144, and / or about 168 hours), are taken to determine the plasma concentration of an oligonucleotide polypeptide conjugate.

[0299] In certain embodiments, the in vivo plasma clearance of the second oligonucleotide polypeptide conjugate is determined to be slower than that of the first oligonucleotide polypeptide conjugate when the plasma concentration of the second oligonucleotide polypeptide conjugate is greater than that of the first oligonucleotide polypeptide conjugate at a given timepoint by at least about 1%. 2%, 3%. 4%, 5%, 6%, 7%, 8%, 9%, 10, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more; or about 1-10%, 5-15%, 10-20%, 20-50%, or above about 50%.

[0300] In certain embodiments, the in vivo plasma clearance of an oligonucleotide polypeptide conjugate is measured according to Example 2 below.

[0301] In certain embodiments, each oligonucleotide is a gapmer. In certain embodiments, the gapmer comprises 16 to 22 nucleotides. In some embodiments, the gapmer comprises wing segments of 3 nucleotides each.

[0302] In certain embodiments, the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of phosphorodithioate, phosphoramidates, phosphonates, phosphotriesters, and phosphoryl guanidines. In certain embodiments, the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of phosphorodithioate, phosphoramidates, phosphoryl guanidines, and O-isopropyl phosphorothioate. In certain embodiments, the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of PS2, MsPA, PN, and OiPS.

[0303] In certain embodiments, the stabilized oligonucleotide comprises 3 to 8 stabilizing intemucleoside linkages not present in the first (or prior) oligonucleotide. In certain embodiments, the stabilized oligonucleotide comprises 3 to 8 PS2, MsPA, PN, or OiPS intemucleoside linkages not present in the first (or prior) oligonucleotide.

[0304] In certain embodiments, each intemucleoside linkage of the first oligonucleotide is a phosphorothioate linkage.

[0305] In certain embodiments, at least one location of a stabilizing intemucleoside linkage in the stabilized oligonucleotide is at or is adjacent to a soft spot identified in an oligonucleotide catabolism assay. In certain embodiments, at least one location of a PS2. MsPA. PN, or OiPS intemucleoside linkage in the stabilized oligonucleotide is at or is adjacent to a soft spot identified in an oligonucleotide catabolism assay.

[0306] In certain embodiments, the intemucleoside linkages in step (iii)(b) are selected from the group consisting of PS2, MsPA, PN, and OiPS. In certain embodiments, the intemucleoside linkages in step (iii)(b) comprise 3 to 8 of PS2, MsPA, PN, and OiPS. In certain embodiments, the intemucleoside linkages in step (iii)(b) comprise 2 to 6 PN. In certain embodiments, the intemucleoside linkages in step (iii)(b) comprise 2 to 4 MsPA. In certain embodiments, the intemucleoside linkages in step (iii)(b) comprise 1 to 3 PS2. In certain embodiments, the intemucleoside linkages in step (iii)(b) comprise 1 to 3 OiPS.

[0307] In certain embodiments, the gapmer of the stabilized oligonucleotide polypeptide conjugate comprises a gap, wherein 1 to 4 PS2, MsPA, or OiPS are present in the gap. In certain embodiments, the gapmer of the stabilized oligonucleotide polypeptide conjugate comprises a first wing, wherein 1 to 3 PN are present in the first wing. In certain embodiments, the first wing is at the 3' end of the gapmer. In certain embodiments, the gapmer of the stabilized oligonucleotide polypeptide conjugate further comprises a second wing. In certain embodiments, 1 to 3 PN are present in the second wing.

[0308] As used herein, a “soft spot” is a site of catabolism (i.e.. the cleavage or clipping of an intemucleoside linkage, for example by a nuclease) in an oligonucleotide. A site of catabolism of an oligonucleotide may be identified, for example, by in vitro or in vivo methods which expose the oligonucleotide to liver microsomes, liver tissue, or the liver of an animal for a given period of time followed by analysis of a sample to determine catabolites of the oligonucleotide. In certain embodiments, a soft spot is more sensitive to catabolism than other sites in the oligonucleotide (e.g, a soft spot intemucleoside linkage is cleaved more rapidly than other intemucleoside linkages in the oligonucleotide). In certain embodiments, the identified soft spot is more resistant to cleavage following substitution with a stabilizing intemucleoside linkage. In certain embodiments, after the soft spot is identified in an oligonucleotide catabolism assay, one or more intemucleoside linkages relevant to the soft spot is determined to be a locus for modification as described herein to prepare a stabilized oligonucleotide. In certain embodiments, an oligonucleotide may be determined to have one or more soft spots. Once one or more soft spot is identified, one or more relevant intemucleoside linkages can be replaced by a stabilizing intemucleoside linkage, e g., a PS2, MsPA, PN, or OiPS group. More than one intemucleoside linkage adjacent to the soft spot may be replaced. The process may be repeated as necessary after stability testing in order to improve the stability of an ASO. In certain embodiments, the location of the modification is at the soft spot, or is adjacent to the soft spot. In certain embodiments, the stabilizing intemucleoside linkage provides resistance to cleavage or clipping by a nuclease.

[0309] In certain embodiments, the site of catabolism of an oligonucleotide is identified by exposure of the oligonucleotide to liver tissue homogenate of a rodent for about 24 to about 72 hours, followed by analysis of a sample by mass spectrometry (MS) in order to determine catabolites of the oligonucleotide. In certain embodiments, liquid chromatography is used to separate oligonucleotide from other components of the sample prior to MS analysis.

[0310] In certain embodiments, the site of catabolism of an oligonucleotide is identified by (i) exposure of the oligonucleotide to mouse liver tissue homogenate at about 37 °C for about 48 hours in pH 7.4 buffer containing 1% NP-40, (ii) separation of the oligonucleotide from other components of the sample by liquid chromatography, and (iii) analysis of a sample by mass spectrometry (MS) in order to determine catabolites of the oligonucleotide.

[0311] The oligonucleotide polypeptide conjugates as described herein may be used for a variety of purposes, including therapeutic indications.

[0312] In some embodiments, the oligonucleotide polypeptide conjugates are used to deliver an oligonucleotide (e.g, an ASO) to a target cell or target tissue in a subject.

[0313] In some embodiments, the described oligonucleotide polypeptide conjugates can be used to deliver the oligonucleotide to a target cell or target tissue to modulate the expression of a target gene or sequence. V. Formulations

[0314] An advantage of the oligonucleotide polypeptide conjugates and methods thereof described herein is that an otherwise non-stable oligonucleotide with limited use can be stabilized and made into a useful composition for pharmaceutical uses. Another advantage of the oligonucleotide polypeptide conjugates and methods thereof described herein is that a less-stable oligonucleotide can be stabilized without substantially sacrificing efficacy, thus improving is usefulness for pharmaceutical uses. Any of the described oligonucleotide polypeptide conjugates can be prepared, provided, or formulated as a salt, mixed salt, or a free acid. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts.

[0315] Any of the described oligonucleotide polypeptide conjugates can be provided in or formulated in a pharmaceutical composition or medicament. A pharmaceutical composition or medicament includes a pharmaceutically effective amount of at least one of the described oligonucleotide polypeptide conjugates and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product (e.g., dual transporter)) that are intentionally included in the pharmaceutical composition. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to (a) aid in processing of the API during manufacture, (b) protect, support, or enhance stability, bioavailability, or patient acceptability of the API, (c) assist in product identification, and / or (d) enhance any other attribute of the overall safety, effectiveness, or delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0316] Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.

[0317] The carrier can be, but is not limited to, a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, Ringer’s solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. A carrier may also contain adjuvants or additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. A carrier may also contain isotonic agents, such as sugars, polyalcohols, sodium chloride, and the like.

[0318] The pharmaceutical compositions can contain other additional components commonly found in pharmaceutical compositions. Such additional components can include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g, antihistamine, diphenhydramine, etc.).

[0319] Pharmaceutically acceptable refers to those properties and / or substances which are acceptable to the subject from a pharmacological / toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a subject. In some embodiments, a pharmaceutically acceptable compound is approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0320] In some embodiments, the pharmaceutical compositions further comprise one or more additional active ingredients. The additional active pharmaceutical ingredient can be, but is not limited to, a small molecule drug.

[0321] In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

[0322] An oligonucleotide polypeptide conjugate or a pharmaceutical composition containing an oligonucleotide polypeptide conjugate can be formulated for administration to a subject. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0323] The term “subject,’7 “individual.” and “patient,” as used interchangeably herein, refer to a mammal, including but not limited to humans, non-human primates, rodents (e.g, rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0324] The terms “treatment,” “treating,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of an injury, disease, or condition, including any objective or subjective parameter such as abatement, remission, improvement in patient survival, increase in survival time or rate, diminishing of symptoms or making the injury, disease, or condition more tolerable to the patient, slowing in the rate of degeneration or decline, or improving a patient’s physical or mental well-being. Additionally, “treating” or “treatment” may refer to the modulation of the target gene expression such as gene knockdown or gene knockout. For instance, the expression of the target gene or sequence is inhibited or reduced, e.g.. by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, as compared to the expression in a control. The treatment or 77 amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.

[0325] The term '‘pharmaceutically acceptable excipient” refers to anon-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as but not limited to a buffer, carrier, or preservative.

[0326] As used herein, a “therapeutic amount” or “therapeutically effective amount” of an agent is an amount of the agent that treats, alleviates, abates, or reduces the severity of symptoms of a disease in a subject. A “therapeutic amount” or “therapeutically effective amount” of an agent may improve patient survival, increase survival time or rate, diminish symptoms, make an injury, disease, or condition more tolerable, slow the rate of degeneration or decline, or improve a patient’s physical or mental well-being.

[0327] The term “administer” refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical delivery’, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the proteins described herein are administered intravenously.

[0328] Pharmacokinetic (PK) studies are used to measure the concentration of amount of a drug in plasma versus time. PK can be measured by collecting a blood or plasma sample at any time after drug administration and measuring the amount of a drug in a given volume of blood or plasma of the sample. A PK profile is generally the result of absorption, distribution, metabolism, and excretion.

[0329] Gene knockdown is the level of decrease in expression of a gene following administration of a drug, such as an ASO relative to a control ASO or negative control treatment. Knockdown can be determined by measuring protein activity, measuring protein levels, or by measuring RNA levels using methods available in the art (e.g., ELISA, qPCR, etc.). EXAMPLES

[0330] The present subject matter will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g, amounts, temperatures, etc.), but some experimental error and deviation may be present. The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature.

[0331] The oligonucleotide polypeptide conjugates described herein can be prepared using known techniques, such as those described in WO2023 / 279099. As an example, a Fab-Fc dimer fusion was prepared, containing a first Fc polypeptide having a S239C cysteine substitution, hole and LALAPS mutations (SEQ ID NO: 1). a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide and comprises a modified constant domain that specifically binds to a transferrin receptor (35.23.2), knob and LALAPS mutations (SEQ ID NO: 2), and wherein the first and second Fc polypeptide are each fused to a non-targeting Fab (SEQ ID NO: 3 and 4). Therefore, the first Fab-Fc polypeptide fusion has a sequence of SEQ ID NO: 5 and the second Fab-Fc polypeptide fusion has a sequence of SEQ ID NO: 6. Additionally, the C-terminal lysine residue of an Fc polypeptide may be fully or partially removed by the cellular machinery during protein production. Therefore, a first Fc polypeptide may comprise SEQ ID NO: 7 and a second Fc may comprise SEQ ID NO: 8. Similarly, the first Fab-Fc polypeptide fusion may have a sequence of SEQ ID NO: 9 and the second Fab-Fc polypeptide fusion may have a sequence of SEQ ID NO: 10 Constructs were cloned by gene synthesis and Gibson assembly into a mammalian expression vector pRK5.

[0332] Fab-Fc dimer fusions protein expression, and purification. Vectors were co-transfected to Expi293 cells along with the corresponding light chain vector in the ratio knob:hole:light chain of 1:1:2. The expressed protein was purified from conditioned media by loading the supernatant over a Protein A column. The column was washed with 10 column volumes of PBS, pH 7.4. The proteins were eluted with 50 mM sodium citrate, pH 3.0 containing 150 mM NaCl, and immediately neutralized with 200 mM arginine, 137 mM succinic acid, pH 5.0. The proteins were further purified by size-exclusion chromatography (SEC) (GE Superdex200) using 200 mM arginine, 137 mM succinic acid, pH 5.0 as running buffer. The purified proteins were confirmed by intact mass LC / MS, and purity of > 95% was confirmed by SDS-PAGE and analytical HPLC-SEC.

[0333] ASO Synthesis. Oligonucleotide synthesis was performed on a MerMade 12 (LGC) or Oligo Pilot Plus (Akta) DNA / RNA synthesizer at 100 pmol scale following standard solidphase oligonucleotide synthesis protocols. All locked nucleic acid (LNA) and deoxyribonucleic acid (DNA) phosphoramidites were purchased from Hongene Biotech Corporation, including LNA-A(Bz), LNA-5MeC(Bz), LNA-T, LNA-G(dmf). and dA(Bz), dC(Ac). dT, dG(dmf). Phosphorodithioate (PS2) monomers dA(Bz) ThioPhosphamidite and dG(iBu) ThioPhosphamidite were purchased from Wuxi TIDES. LNA-5MeC(Bz) was dissolved in a mixed solvent of DCM / acetonitrile (1:1, v / v), PS2 amidites were dissolved in DCE, and all other phosphoramidites were dissolved in acetonitrile and molecular sieves (3 A) were added. The parent antisense oligonucleotide (ASO) sequences were first assembled on UnyLinker 5 CPG solid support, followed by attachment of amino linker on 5’-end using 6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (CAS: 133975-85-6; Glen Research Cat. # 10-1916). The synthesis cycle for adding one nucleotide (or non-nucleic acid) unit consists of four individual steps, detritylation, coupling, oxidation (or sulfurization), and capping. 5-Ethylthio-lH-tetrazole (ETT, 0.25 M in acetonitrile) was 10 used as activator solution. A 0.2 M solution of PADS (phenylacetyl disulfide) in 50% pyridine / 50% acetonitrile or 0.2 M ADTT in pyridine was employed to introduce phosphorothioate linkages. Detailed protocols for 100 pmol scale ASO synthesis are summarized in Tables 1-4. 15 Table 1. Generic 100 pmol scale ASO synthesis parameters Process Reagents Parameters Solid support UnyLinker CPG, 500 A, 80 pmol / g 100 pmol scale Wash Anhydrous acetonitrile (moisture content <10 ppm) 10 mL, 20 s Detritylation 3% Trichloroacetic acid (TCA) in DCM 10 mL, 45 s Coupling 0.1 M phosphoramidites in ACN or ACN / DCM or DCE (1:1, v / v) 2.5 mL amidite, 0.25 M ETT in acetonitrile 5 mL ETT. 6 min Oxidation 0.02 MI2 in THF / Pyridine / H2O (7:2:1) 10 mL, 2 min Sulfurization 0.2 M PADS in 50% pyridine / 50% acetonitrile or ADTT (0.2 M, 120 eq.) in pyridine, 10 min 10 mL, 4 min Capping Cap Mix A: THF / Pyridine / Ac2O (8:1:1) 2.5 mL Cap A. 2.5 Cap Mix B: 16% Methyl imidazole in THF mL Cap B, 40 s Table 2. Example synthesis of oligo containing MsPA backbone modifications Synthesizer AKTA oligopilot plus 100 (CV=6.3 ml) CPG Universal CPG (1000 A, 40 pmol / g) Scale (pmol) 100 pmol Coupling 1 (recycle) 0.2 M amidite, 6.0 eq.; 1.0 M DCI / 0.1 M NMI, 45 eq., 17 min (cycled twice for the 1st monomer; one time for all other amidites) Oxidation for PS bonds 0.02 M I2 in MeCN / pyridine / EEO. 75 / 20 / 5 (v / v / v), 5 eq., 5 min Oxidation for MsPA bonds methanesulfonyl azide (0.5 M, 75 eq.) in MeCN. 30 min; repeated 2 times, 60 min in total Sulfurization ADTT (0.2 M, in pyridine), 10 CV, 126 eq., 10 min Table 3. Example synthesis of oligo containing PN backbone modifications Synthesizer AKTA oligopilot plus 100 (CV=6.3 ml) CPG Universal CPG (1000 A, 40 pmol / g) Scale (pmol) 100 pmol Coupling 1 (recycle) 0.2 M amidite. 6.0 eq.; 1.0 M DCI / 0.1 M NMI, 45 eq., 17 min (cycled twice for the 1st monomer; one time for all other amidites) Oxidation for PS bonds 0.02 M U in MeCN / pyridine / H2O, 75 / 20 / 5 (v / v / v), 5 eq.. 5 min Oxidation for PN bonds 2-Azido-l,3-dimethylimidazolinium Hexafluorophosphate (0.5 M, in MeCN). 10 mL, 50 eq., 120 min Sulfurization ADTT (0.2 M, in pyridine), 10 CV, 126 eq.. 10 min Table 4a. Example synthesis of oligo containing PS2 and PN backbone modifications Synthesizer AKTA oligopilot plus 100 (CV=6.3 ml) CPG Universal CPG (500 A, 24 pmol / g) Scale 100 pmol Coupling for PS and PN LNA-C in 4:1 MeCN / DCM, other amidites in MeCN at 0.2 M, 6.0 eq.; 10.6 M ETT. T1 eq., 19 min (cycled twice for the 1st monomer; one time for all other amidites) Oxidation for PS bonds 0.02 M E in MeCN / pyridine / H2O, 75 / 20 / 5 (v / v / v), 5 eq., 5 min Oxidation for PN bonds 2-Azido-l,3-dimethylimidazolinium Hexafluorophosphate (0.5 M, in MeCN). 10 mL, 50 eq., 120 min Coupling for PS2 monomers PS2 amidite 0.2M in DCE. 6 eq.; 0.6 M ETT. 27 eq., 19 min; one time Sulfurization ADTT (0.2 M. in pyridine). 10 CV, 126 eq., 10 min Table 4b. Example synthesis of oligo containing OiPS and PN backbone modifications Synthesizer AKTA oligopilot plus 100 (CV=6.3 ml) CPG Universal CPG (500 A, 12 pmol / g) Scale 12 pmol Coupling for PS and PN LNA-C in 4:1 MeCN / DCM. other amidites in MeCN at 0.05 M, 2.0 eq.; 0.25 M ETT, 20 eq., 2.1 min (cycled twice for the 1st monomer; one time for all other amidites) Oxidation for PS bonds 0.02 M E in MeCN / pyridine / H2O, 75 / 20 / 5 (v / v / v), 5 eq.. 5 min Oxidation for PN bonds 2-Azido-l,3-dimethylimidazolinium Hexafluorophosphate (0.2 M, in MeCN), 10 mL, 50 eq., 60 min Sulfurization for PS and OiPS bonds ADTT (0.2 M. in pyridine), 10 CV, 126 eq., 10 min

[0334] Preparation of 5 '-maleimide modified ASO. To a solution of ASO amine TEA salt (30 mg, by OD) in PBS buffer (TEKNOVA lOx PBS stock solution, pH 6.0, 2.5 ml) was added a solution of 3-maleimidopropionic acid N-hydroxysuccinimide ester (MCOSu, 10 eq.) in DMF (2.5 ml) at room temperature. The resulting solution was shaken at room temperature for 18 h. 10 Upon completion, the solution was desalted by passing through a sephadex G25 column (2.5x40 cm) on AKTA pure 25 M, eluting with Milli Q water at 3 mL / min, monitored by UV 260 / 280 and conductivity. The appropriate fractions were pooled and lyophilized to give desired 5'-maleimide ASO. The 5'-maleimide ASO may have as its terminal phosphate linkage either a PO, PS, or PN modified phosphate, for example: (5'-maleimide ASO with PO 5' terminal group), (5'-maleimide ASO with PS 5' terminal group), or (5'-maleimide ASO with PN 5' terminal group). —P—O—ASO

[0335] ASO-Polypeptide Conjugation. The Fab-Fc dimer fusions generated above containing the S239C cysteine modification for conjugation was first reduced using 30 molar equivalents of TCEP and 2 mM EDTA, 37 °C for 1 hr. Reduction was confirmed by LC / MS. Post reduction, remaining TCEP was removed by dialysis using lx PBS, pH 6.8 with 2 mM EDTA (punfication by e.g.. dialysis) and the Fab-Fc dimer fusions were reoxidized with 50 molar equivalents of dHAA at room temperature for 3 hr. Oxidation was confirmed by LC / MS of dHAA. For the bioconjugation, 1.2 molar equivalents of the 5'-maleimide modified ASOs, generated above were added to the oxidized Fab-Fc dimer fusions at room temperature for 1 hr. The resulting conjugates were purified by anion exchange chromatography using Resouce Q column (equilibration buffer: 50 mM Tris, pH 7.5, elution buffer: 50 mM Tris, pH 7.5 + 2 M NaCl) to remove unwanted and unconjugated products. Purity of the conjugates were determined by LC / MS and SEC.

[0336] For sequence comparison of oligonucleotides (e.g., to determine identity or complementarity), typically one nucleotide sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. Example 1. Soft Spot Identification Assay

[0337] In order to determine the catabolism and biotransformation of oligonucleotides (ASOs) and to further characterize the oligonucleotides and oligonucleotide polypeptide conjugates described herein, a stability assay to identify likely sites of catabolism (clipping sites) of ASOs (referred to herein as "soft spots") had to be developed. The catabolism assay, described in detail below, was used to identify soft spots for potential modification in order to design and provide more stable molecules. Previous assay methods for in vitro of oligonucleotides are described, for example, by Basiri et al. in Molecular Therapy: Nucleic Acids; 21: 725-736 (2020).

[0338] Tissue Homogenization and Incubation—Frozen mouse livers were homogenized at 200mg / ml tissue concentration in pbs buffer (pH 7.4) containing 1% NP-40. 200 ^.L of the homogenate was spiked with ASOs at l-2mM concentration and incubated at 37° C for 48 h (at 300 g shaking).

[0339] Sample Preparation—200 mL of 10% phosphoric acid w as added to 200 mL incubated tissue homogenates and were vortexed for 5 min. 600 mL of the Clarity OTX Lysis-Loading Buffer (Phenomenex, PN ALO-8579) was added to each tube and were vortexed for 5 min followed by centrifugation at 3200 rpm at 4° C for 10 min. Clarify OTX SPE plates were used. Table 5 shows the detailed procedure. Table 5. Sample Preparation SPE Step Solvent Condition 1 mL Methanol Equilibrate 1 mL 50mM NaH2PO4, 2 mM NaN3. 10 mM K2EDTA in water (pH = 5.5) Load 0.4 mL pretreated sample Equilibrate 1 mL 50mM NaH2PO4, 2 mM NaN3, 10 mM K2EDTA in water (pH = 5.5) Wash 1 1 mL 50 mM NaH2PO4 in 50:50 (v:v) water: acetonitrile (pH = 5.5) Wash 2 1 mL 50 mM NH4HCOs in 50:50 (v:v) water: acetonitrile (pH = 5.5) Elute 2x0.5 mL 100 mM NH4HCO3. 10 mM TCEP in 50:40:10 (v:v:v) water:acetonitrile:tetrahydofuran (pH = 9.8) Evaporation Under nitrogen dryer (60° C) until dry Reconstitute Reconstitute with 100 mL 5% MeOH in water Analysis 10-20 mL samples injected into the LC-HRMS for analysis

[0340] Sample Analyses were conducted by liquid chromatography and mass spectrometry.

[0341] Liquid chromatography (LC): The ASO separation was carried out by ion pairing chromatography at 70C with a Waters BEH oligonucleotide 2.1 X50 mm column. Mobile phase buffer A and B are water and method with 100 mM hexafluoroisopropanol (HFIP) and 15 mM N,N-diisopropylethylamine (DIEA). Chromatography was performed at 0.3 mL / min under the following gradient condition (min-%B) 0-5, 1-5, 6-50, 6.1-95, 7.1-95. 7.2-5, 10-5. The total run time was 10 min and the LC output was diverted to waste from 0-1 min and 7-10min.

[0342] Mass spectrometry (MS): A high resolution MS with IDA method was used to identify catabolites. Identified Soft Spots—The table below shows soft spots that were identified according to the methods provided herein. Once one or more soft spot is identified, the relevant intemucleoside linkage is replaced by a PS2, MsPA. PN, or OiPS group. More than one intemucleoside linkage adjacent to the soft spot may be replaced. The process may be repeated as necessary after stability testing in order to improve the stability of an ASO. Table 6A. Soft Spots Identified in Exemplary ASO Sequences. ASOs ASO Sequences: listed from 5' to 3' (“+’’ indicates a LNA, “[%C]” indicates 5-methylC, indicate a PS linkage, “n" indicates a PN linkage, indicates a soft spot) SEQ ID NO. 1 +[%C]*+G*+T*G*T*T*T*G*A*T*A*T*T$*A*T*+[%C]*+[%C]*+T 12 2 +A* +[%C]*+[%C]*T*T*A*$A*$G*T*A*T*T*A*C*T*+T*+G*+[%C] 11 3 +T*+[%C]*+A*A*C*T*C*T*T*A*G*C*$A*T*C*C*+A*+[%C]*+T* 13 4 + [%C]*+T+T*A*T*C*A*T*T*T*G*C*A*$T*T*+G*+G*+T 14 5 +[%C]*+T*+G*T*T*A*G*A*C*A*T*T*C*A*$T*T*+[%C]*+T*+[%C] 15 6 +A* +[%C]* +[%C]*T*T*A*AnGnT*A*T*T*$A*C*T*+nT+nG+[%C] 11 Example 2. In Vivo Plasma Pharmacokinetics

[0343] To assess the pharmacokinetics of oligonucleotide polypeptide conjugates, OTVM1, 0TVM2, 0TVM3, 0TVM4, 0TVM5, and OTVM_unmod (see Table 7) were administered to Sprague Dawley rats at a concentration of lOmg / kg by intravenous injection. All oligonucleotide polypeptide conjugates were administered at a dose volume of 2mL / kg. Plasma was collected at 0.25, 4, 24, 48, and 72 hr after injection. huIgG (FIG. 10) and ASO (FIG. 11) concentrations were measured in plasma using the methods described below.

[0344] Experimental Protocols for the in vivo pharmacokinetic experiments are described below.

[0345] Animal handling and tissue collection—Animals were peripherally administered therapeutic treatment via intravenous (IV) tail vein injection (-200 pL total volume). For in life plasma collection, blood was collected via submental puncture and transferred to EDTA coated tubes, then spun down at 12,700 rpm for 7 min at 4 °C before collecting the top plasma layer. For tissue collection, animals were anesthetized with tribromoethanol and whole blood was collected via cardiac puncture into EDTA coated tubes for plasma drug concentration assessment. Following transfer to EDTA coated tubes, whole blood was spun down at 12,700 rpm for 7 min at 4 °C before collecting the top plasma layer. Animals were then perfused with ice-cold PBS transcardially at a rate of 5 mL / min for 5 min. For biochemical analysis, tissues were collected, weighed, snap frozen on dry ice, and then stored at -80 °C.

[0346] Tissue homogenization for drug concentration measurement—Weighed frozen tissue samples were processed for biochemical assays by adding 10x volume chilled 1% NP40 + PBS homogenization buffer with added complete Protease Inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) phosphatase inhibitors. Samples were homogenized using 3 mm tungsten carbide beads in 1.5 mL Eppendorf tubes, shaken using the Qiagen TIssueLyzer II (Cat No. / ID: 85300) (2x3 min at 27 Hz).

[0347] Tissue homogenization for RNA measurements—Weighed frozen tissue samples were processed for RNA assays by adding 10x volume Qiazol reagent. Samples were homogenized using 5 mm tungsten carbide beads in 2 mL Eppendorf tubes, shaken using the Qiagen TIssueLyzer II (Cat No.TD: 85300) (2x3 min at 27 Hz). After lysis, samples were incubated for 5 min at room temperature, then chloroform was added. Samples were vortexed, incubated at room temperature for 3 min, then centrifuged for 15 min at 12000xg at 4 °C. The aqueous phase was then isolated. RNA was then isolated by adding isopropanol, vortexing, incubating for 10 min at room temperature, then centrifuging for lOmin at 12000xg at 4 °C. The resulting pellet was then resuspended in 75% ethanol, vortexed and centrifuged for 5min at 7500xg at 4 °C. The final pellet was resuspended in water.

[0348] huIgG Assay—Quantification of humanized antibodies in plasma and tissue lysates were measured using a generic electrochemiluminescence immunoassay (ECLIA). Briefly, to the w'dls of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discover}', Rockville, MD), a working concentration of biotinylated goat anti-human IgG polyclonal primary antibody (Southern Biotech, Birmingham, AL) prepared in assay diluent was incubated for approximately 1 hr. Following this incubation and a plate wash step, prepared test samples (with sample pre-dilution, where appropriate) and relevant standards were added to the assay plate and allowed to incubate for approximately 1 hr. Following test sample incubation and a plate wash step, secondary ruihenylated (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, Rockville, MD) at a working concentration in assay diluent was added to the assay plate and incubated for approximately 1 hr. Following a plate wash, a lx MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate the electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). AH of the assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); and all test samples were pre-diluted at the assay MRD of 1:20 prior to analyzing in the assay plate. Sample ECLU signals generated in the assay subsequently were processed into concentrations by back-calculating off the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating concentrations for unknown / test samples.

[0349] Total ASO Assay—Quantification of total ASO (in conjugated and free forms) in animal plasma and tissue homogenates were measured using a hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, custom biotinylated and digoxigenin-conjugated antisense probes (synthesized by Integrated DNA Technologies, Coralville, IA) at working concentrations were combined with prepared test samples (with sample pre-dilution, where appropriate) and relevant standards in TE Buffer (1 OmM Tris-HCL containing 1 mM EDTA). Prepared samples in TE buffer were added, in a 1:1 mix, into lx SSC Buffer (Sigma-Aldrich, St. Louis, MO) containing a working concentration of recombinant proteinase K enzyme (ThermoFisher, Waltham, MA). Hybridization / Enzyme mixture was then digested, denatured, annealed, and cooled in a thermal cycler instrument. Following hybrid product incubation, samples were added to the wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery. Rockville, MD) and incubated for approximately 30 nuns. Following incubation and a plate wash step, secondary ruthenylated (SULFO-TAG) sheep anti-digoxigenin antibody (Novus Biologicals, Littleton, CO) at a working concentration in assay diluent was added to the plate and incubated for approximately 30 mms. Following a plate wash, a 1 x MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate the electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All of the assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); and all test samples were prediluted at the assay MRD of 1:20 prior to analyzing in the assay plate. Sample ECLU signals generated in the assay subsequently were processed into concentrations by back-calculating off the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating concentrations for unknown / test samples.

[0350] qPCR analysis—To evaluate target mRNA levels, qRT-PCR was run on RNA extracted from tissue lysates. Target mRNA levels were evaluated using Taqman probes (hMAPT, mGapdh) and the Express One-Step Kit. For each sample, hMAPT mRNA levels were normalized to the housekeeping gene Gapdh. qRT-PCR was performed using a QuantStudio 6 Flex system (Applied Biosystems) and average CT values were measured for each probe using technical duplicates. Next, the delta delta CT values were calculated relative to the non-ASO 5 treated group and plotted as relative expression levels.

[0351] Results—The plasma pharmacokinetic profile: huIgG is depicted in FIG. 10. The plasma pharmacokinetic profile: ASO is depicted in FIG. 11. These results demonstrate slower clearance profiles with modified oligonucleotide polypeptide conjugates as compared to the unmodified analog. Lower clearance profiles as provided by modifying the oligonucleotides of 10 oligonucleotide polypeptide conjugates can enhance residence time and uptake into relevant tissues, which is desirable to enhance the therapeutic index and uses of the oligonucleotides. Example 3. In Vivo Potency and Stability Assessment of MAPT ASOs

[0352] ASO SEQ ID NO: 11 was further modified as shown in Table 7A below: 15 Table 7A. Stabilized ASO sequences. ASO ID ASO Sequence Conjugated Molecule ID SEQ ID NO. ASON unmod Ll+A*+[%C]*+[%C]*dT*dT*dA*dA*dG*dT*dA*dT*dT*dA *dC*dT*+T*+G*+[%C] or\ ,m unmod 11 ASOM1 Ll+A*+[%C]*+[%C]*dT*dT*dA*dAndGndT*dA*dT*dT*dA *dC*dT*+Tn+Gn+[%C] OTVM1 11 ASOM2 Ll+An+[%C]n+[%C]*dT*dT*dAudAudG*dT*dA*dT*dT*dA *dC*dT*+Tn+Gn+[%C] 0TVM2 11 ASOM3 Ll+An+[%C]n+[%C]*dT*dT*dAtdAtdG*dT*dA*dT*dT*dA *dC*dT*+Tn+Gn+[%C] 0TVM3 11 ASOM4 Ll+A*+[%C]*+[%C]*dT*dT*dAudAudGudT*dA*dT*dT*dA *dC*dTn+Tn+Gn+[%C] 0TVM4 11 ASOM5 Ll+An+[%C]n+[%C]ndT*dT*dAtdAtdG*dT*dA*dT*dT*dA *dC*dTn+Tn+Gn+[%C] 0TVM5 11 Control ASO 1 LlmG*inG*mT*mT*mG*dA*d[%C]*dA*dT*d[%C]*dG*dT *d[%Cl*dT*dG*m[%Cl*m[%Cl*mT*mG*mT 16 Control ASO 2 Llm[%C]*m[%C]PmG*mT*mT*dT*dT*d[%C]*dT*dT*dA *d[%C]*d[%C]*mA*m[%C]Pm[%C]*m[%C]*mT 17 “+” indicates LNA nucleosides, all others are DNA nucleosides; [%C] = 5-methylcytosine indicate a PS linkage; “n” indicates a PN linkage; “t” indicates a phosphorodithioate (PS2) linkage; “P” indicates a PO linkage; u indicates a MsPA linkage; LI indicates a 5' tenninal phosphodiester (PO) group

[0353] ASOs were diluted in sterile saline and administered to hTau mice (mTau ) via intracerebroventricular (ICV) injection at a single dose of either 10 or 25 pg (according to Table 6 below) in 10 pL total volume per mouse. 10-14 days post dose (according to Table 8 below), brain tissues were harvested to measure relative MAPT RNA levels (according to the tissue homogenization and qPCR procedures below) and total ASO concentration (according to the tissue homogenization and total ASO assay procedures below). Table 8. ASO Dose (pg) Takedown (days post dose) ASOMunmod 10 14 ASOMunmod 25 14 Control ASO 1 10 14 Control ASO 1 25 14 Control ASO 2 10 14 Control ASO 2 25 14 ASOMunmod 10 10 ASOMunmod 10 28 ASOM4 10 10 ASOM4 10 28 ASOM5 10 10 ASOM5 10 28

[0354] Results are shown in FIG. 8 (relative MAPT RNA levels) and FIG. 9 (total ASO). These results suggest enhanced potency of the ASO base sequence compared to existing control MAPT ASOs, and enhanced tissue stability of the modified ASOs (e.g., ASOM4 and ASOM5) compared to the unmodified version (ASOM_unmod), which should extend the duration of action of the modified ASOs in vivo. Example 4. In Vivo OTV Brain Uptake and MAPT Knockdown after modified OTV:MAPT

[0355] Peripheral Dosing. The five modified ASOs above selected for potency were synthesized and bioconjugated to TV proteins according to the procedures described above to make five MAPT OTV molecules (0TVM1, 0TVM2, 0TVM3, 0TVM4, OTVM5) for assessing tau knockdown in the brain following peripheral intravenous administration.

[0356] TfRmu / hu knock-in mice (see, US Patent No. 10,143,187 herein incorporated by reference) were dosed once at 25mpk with each of the OTVs. The MAPT OTVs were diluted in sterile saline and administered intravenously. Mice were weighed prior to each dose to determine appropriate dosage. Plasma was collected 4, 24, and 72 hr post dose, while terminal plasma and tissue samples (z.e., brain and liver) were collected one week post dose to determine the level of total full length ASO deposited to the brain (FIG. 1 and FIG. 2) according to the procedures below.

[0357] Additionally, TfRmu / hu knock-in mice (see, US Patent No. 10,143,187 herein incorporated by reference) were crossed to mice transgenic forhTau+ / " (human Tau) and mTau" ' (mouse Tau knockout) and resulting mice were administered either 4 doses (Day 0, 1 week, 2 week, 3 week) or 8 doses (Day 0, Day 3, Day 7, Day 10. Day 14, Day 17, Day 21, Day 24) of each of the MAPT OTVs. The MAPT OTVs were diluted in sterile saline and administered to the mice intravenously. Anti-CD4 was administered prior to the initial and 3rd week of dosing to prevent anti-drug antibody responses in the mice. Mice were weighed prior to each dose to determine appropriate dosage. Brain samples were collected one week after the final dose to determine ASO concentration in the brain using the total ASO assay described below (FIG. 3), and the level of human Tau knockdown relative to Gapdh and saline-dosed mice (FIG. 4 and FIG. 5) according to the procedures below.

[0358] These values were then compared to a separate study in which the same mouse line was used to assess brain uptake and target knockdown an OTV molecule conjugated to an unmodified ASO. In this study, mice were administered either 1 (Day 0) or 4 doses (Day 0, 1 week, 2 week, 3 week) of the unmodified MAPT OTV. The MAPT OTV was diluted in sterile saline and administered to the mice intravenously. Anti-CD4 was administered prior to the initial and 3rd week of dosing to prevent anti-drug antibody responses in the mice. Mice were weighed prior to each dose to determine appropriate dosage. Brain samples were collected one week after the final dose to determine an ASO concentration in the brain using the total ASO assay (FIG. 6), and the level of human Tau knockdown relative to Gapdh and saline-dosed mice (FIG. 7) according to the procedures below.

[0359] Taken together, these results demonstrate superior ASO brain uptake and target knockdown with modified MAPT OTVs compared to unmodified MAPT OTV. We observed 20-27nM ASO brain uptake (modified sequences) one week after a 25mpk IV dose (FIG. 1 and FIG. 2) compared to 18nM (unmodified) (FIG. 6), 33-53nM ASO brain uptake (modified sequences) one week after four 25mpk IV doses (FIG. 3) compared to 18nM (unmodified) (FIG. 6), 53-75% MAPT knockdown in the brain (modified sequences) one week after four 25mpk IV doses (FIG. 4) compared to 21% (unmodified) (FIG. 7), and 137nM ASO brain uptake along with 84% MAPT knockdown in the brain one week after eight 25mpk IV doses (FIG. 3 and FIG. 5).

[0360] Mouse handling and tissue collection. Mice were peripherally administered therapeutic treatment via intravenous (IV) tail vein injection (-200 pL total volume). For in-life plasma collection, blood was collected via submental puncture and transferred to EDTA coated tubes, then spun down at 12,700 rpm for 7 min at 4C before collecting the top plasma layer. For tissue collection, animals were anesthetized with tribromoethanol and whole blood was collected via cardiac puncture into EDTA coated tubes for plasma drug concentration assessment. Following transfer to EDTA coated tubes, whole blood was spun down at 12,700 rpm for 7 min at 4C before collecting the top plasma layer. Mice were then perfused with ice-cold PBS transcardially at a rate of 5 mL / min for 5 min. For biochemical analysis, tissues were collected, weighed, snap frozen on dry ice, and then stored at -80°C.

[0361] Intracerebroventricular Bolus (ICV) surgery. Followed procedures from DeVos, J Vis Exp (2013) herein incorporates by reference and summarized in brief as follows: in preparation for the surgery, the surgical area was sterilized with 70% ethanol. Mice were brought under anesthesia with 4% isoflurane. Hair was shaved between from the shoulder region to between the eyes prior to placing mouse on stereotax surface. With a maintenance level of 2% isoflurane, an incision was made from the base of the neck up to between the eyes. Following cleaning with hydrogen peroxide, a needle was slowly driven through the skull at a rate of 1 mm / s. After a 2-3-minute period to allow7 for brain sealing around the needle, a dose of 10 pL ASO at 1 pL per second was administered. With a cotton swab held against the skull at the base of the needle, the needle was raised at a rate of 1 mm per second. The cotton swab was held at the site of injection for 1 minute to limit drug leakage. Following ICV bolus, the incision was sutured and treated with an antibiotic ointment. The mouse was transferred to a headed recovery pad and observed for full recovery. Mice were monitored daily after surgery to check for pain, discomfort, or infections.

[0362] Tissue homogenization for drug concentration measurement. Weighed frozen tissue samples were processed for biochemical assays by adding 10X volume chilled 1% NP40 + PBS homogenization buffer with added cOmplete Protease Inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) phosphatase inhibitors. Samples were homogenized using 3 mm tungsten carbide beads in 1,5mL Eppendorf tubes, shaken using the Qiagen TIssueLyzer II (Cat No. / ID: 85300) (2x3 min at 27 Hz).

[0363] Tissue homogenization for RNA measurements. Weighed frozen tissue samples w ere processed for RNA assays by adding 10x volume Qiazol reagent. Samples were homogenized using 5 mm tungsten carbide beads in 2mL Eppendorf tubes, shaken using the Qiagen TIssueLyzer II (Cat No. / ID: 85300) (2x3 min at 27 Hz). After lysis, samples were incubated for 5min at room temperature, then chloroform was added. Samples were vortexed, incubated at room temperature for 3 min, then centrifuged for 15min at 12000xg at 4°C. The aqueous phase was then isolated. RNA was then isolated by adding isopropanol, vortexing, incubating for lOminutes at room temperature, then centrifuging for lOmin at 12000xg at 4°C. The resulting pellet was then resuspended in 75% ethanol, vortexed and centrifuged for 5min at 7500xg at 4°C. The final pellet was resuspended in water.

[0364] huIgG Assay. Quantification of humanized antibodies in mouse plasma and tissue lysates were measured using a generic electrochemiluminescence immunoassay (ECL1A). Briefly, to the wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery', Rockville, MD), a working concentration of biotinylated goat anti-human IgG polyclonal primary antibody (Southern Biotech, Birmingham, AL) prepared in assay diluent was incubated for approximately 1 hr. Following this incubation and a plate wash step, prepared test samples (with sample pre-dilution, where appropriate) and relevant standards were added to the assay plate and allowed to incubate for approximately 1 hr. Following test sample incubation and a plate wash step, secondary' ruthenylated (SULFO-TAG) goat antihuman IgG antibody (Meso Scale Discovery. Rockville. MD) at a working concentration in assay diluent was added to the assay plate and incubated for approximately 1 hr. Following a plate wash, a lx MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate the electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All of the assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate): and all test samples were pre-diluted at the assay MRD of 1:20 prior to analyzing in the assay plate. Sample ECLU signals generated in the assay subsequently were processed into concentrations by back-calculating off the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating concentrations for unknown / test samples.

[0365] Total ASO Assay. Quantification of total ASO (in conjugated and free forms) in mouse plasma and tissue homogenates were measured using a hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, custom biotinylated and digoxigenin-conjugated antisense probes (synthesized by Integrated DNA Technologies. Coralville, IA) at working concentrations were combined with prepared test samples (with sample pre-dilution, where appropriate) and relevant standards in TE Buffer (lOmM Tris-HCL containing ImM EDTA). Prepared samples in TE buffer were added, in a 1:1 mix, into lx SSC Buffer (Sigma-Aldrich, St. Louis. MO) containing a working concentration of recombinant proteinase K enzyme (ThermoFisher, Waltham, MA). Hybridization / Enzy me mixture was then digested, denatured, annealed, and cooled in a thermal cycler instrument. Following hybrid product incubation, samples were added to the wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) and incubated for approximately 30 mins. Following incubation and a plate wash step, secondary ruthenylated (SULFO-TAG) sheep anti-digoxigenin antibody (Novus Biologicals, Littleton, CO) at a working concentration in assay diluent was added to the plate and incubated for approximately 30 mins. Following a plate wash, a lx MSD Read Buffer I (Meso Scale Discovery’. Rockville. MD) was then added to generate the electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All of the assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate); and all test samples were prediluted at the assay MRD of 1:20 prior to analyzing in the assay plate. Sample ECLU signals generated in the assay subsequently were processed into concentrations by back-calculating off the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating concentrations for unknown / test samples.

[0366] qPCR analysis. To evaluate target mRNA levels, qRT-PCR was run on RNA extracted from tissue lysates. Target mRNA levels were evaluated using Taqman probes (hMAPT, mGapdh) and the Express One-Step Kit. For each sample, hMAPT mRNA levels were normalized to the housekeeping gene Gapdh. qRT-PCR w as performed using a QuantStudio 6 Flex system (Applied Biosystems) and average CT values were measured for each probe using technical duplicates. Next, the delta delta CT values were calculated relative to the non-ASO treated group and plotted as relative expression levels. Example 5. In Vivo Plasma Pharmacokinetics

[0367] ASO SEQ ID NOs: 11 and 15 were further modified as shown in Table 9 below: Table 9. Stabilized ASO sequences. ASO ID ASO Sequence Conjugated Molecule ID SEQ ID NO. ASOM6 Ll+A*+[%C]n+[%C]ndT*dT*dAndAndG*dT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] OTVM6 11 ASOM7 Ll+A*+[%C]*+[%C]*dT*dT*dAndAndGndT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] OTVM7 11 ASOM8 Ll+A*+[%C]*+[%C]*dT*dT*dA*dAndGndT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] OTVM8 11 ASOM9 L2+An+[%C]n+[%C]*dT*dT*dA*dAndGndT*dA*dT*dT *dA*dC*dT*+T*+G*+[%C] OTVM9 11 ASOM10 Ll+An+[%C]n+[%C]ndT*dT*dA*dAtdG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] OTVM10 11 AS0M11 Ll+An+[%C]n+[%C]ndT*dT*dAtdA*dG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] 0TVM11 11 AS0M12 Ll+An+[%C]n+[%C]ndT*dT*dA*dA*dG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] 0TVM12 11 ASOM13 Ll+An+[%C]n+[%C]*dT*dT*dA*dAndG*dT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] 0TVM13 11 ASOM14 Ll+An+[%C]n+[%C]*dT*dT*dA*dAndGndT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] 0TVM14a 11 ASOM14 L2+An+[%C]n+[%C]*dT*dT*dA*dAndGndT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] 0TVM14b 11 AS0M15 Ll+An+[%C]n+[%C]ndT*dT*dAodAodG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] 0TVM15 11 ASOM16 L3+An+[%C]*+[%C]*dT*dT*dAndAndG*dT*dA*dT*dT *dA*dC*dT*+Tn+Gn+[%C] 0TVM16 11 ASOM17 Ll+A*+[%C]*+[%C]*dT*dT*dAndAndGndT*dA*dT*dT *dA*dC*dT*+T*+G*+[%C] 0TVM17 11 ASOM18 Ll+An+[%C]n+[%C]*dT*dT*dAndAndG*dT*dA*dT*dT *dA*dC*dT*+T*+G*+[%C] 0TVM18 11 AS0M19 Ll+An+[%C]n+[%C]ndT*dT*dAtdAtdG*dT*dA*dT*dT *dA*dC*dTn+Tn+GP+[%C] 0TVM19 11 ASOM20 Ll+An+[%C]n+[%C]ndT*dT*dAtdAtdG*dT*dA*dT*dT *dA*dC*dTn+TP+Gn+[%C] OTVM20 11 ASOM21 Ll+An+[%C]n+[%C]ndT*dT*dAtdAtdG*dT*dA*dT*dT *dA*dC*dTP+Tn+Gn+[%C] 0TVM21 11 ASOM22 Ll+An+[%C]n+[%C]PdT*dT*dAtdAtdG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] OTVM22 11 ASOM23 Ll+An+[%C]P+[%C]ndT*dT*dAtdAtdG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] OTVM23 11 ASOM24 Ll+AP+[%C]n+[%C]ndT*dT*dAtdAtdG*dT*dA*dT*dT *dA*dC*dTn+Tn+Gn+[%C] OTVM24 11 ASOM25 Ll+[%C]n+Tn+G*dT*dTndAndG*dA*dC*dA*dT*dT*dC ndAndT*dT*+[%C] *+T*+[%C] OTVM25 15 ASOM26 Ll+[%C]*+T*+G*dT*dTndAndG*dA*dC*dA*dT*dT*dC ndAndT*dT*+[%C]n+Tn+[%C] OTVM26 15 ASOM27 Ll+[%C]n+Tn+GndTndT*dAtdG*dA*dC*dA*dT*dT*dC *dAtdT*dTn+[%C]n+Tn+[%C] OTVM27 15 ASOM28 Ll+[%C]n+Tn+G*dT*dT*dAtdGtdA*dC*dA*dT*dT*dC ndAndT*dT*+l%Cln+Tn+l%Cl OTVM28 15 +A, +[%C], +T, +G indicate adenine 5-methylcytosine, thymine, and guanine locked nucleosides, respectively. dA, dC, dT, dG indicate deoxyadenosine, deoxycytidine, deoxythymidine, and deoxy guanosine, respectively. * indicates phosphorothioate intemucleoside linkage n indicates cyclic phosphoryl guanidine (PN) internucleoside linkage t indicates phosphorodithioate (PS2) internucleoside linkage P indicates phosphodiester intemucleoside linkage o indicates OiPS (O-(isopropy1) phosphorothioate LI indicates a 5' terminal phosphodiester (PO) group L2 indicates a 5' terminal phosphorothioate (PS) group L3 indicates a 5' terminal cyclic phosphoryl guanidine (PN) group

[0368] To assess the pharmacokinetics of additional oligonucleotide polypeptide conjugates (see Table 7B), the additional conjugates were administered to Sprague Dawley rats at a concentration of 10 mg / kg by intravenous injection as described above. All oligonucleotide polypeptide conjugates were administered at a dose volume of 2 mL / kg. Plasma was collected 5 at 0.25, 4, 24, 48, and 72 h after injection. huIgG and ASO concentrations were measured in plasma as described above.

[0369] Results are shown in Table 10. These results demonstrate that adding stabilizing modifications in various placements and formats consistently improves huIgG clearance, and modifying the region at or near the soft spot with backbone modifications can adjust the ASO 10 clearance by protecting or shifting the clipping site. Table 10. Rat PK data including ASO CL and IgG CL (all conjugated to S239C site), all MAPT). Conj. Mol. ID huIgG clearance (mL / d / kg) ASO clearance (mL / d / kg) OTVM6 9.2 70.8 OTVM7 15 86.9 OTVM9 13 89.1 OTVM8 21.3 85.8 OTVM13 18.1 76.3 OTVM12 13.6 72.2 OTVM19 22.7 43.9 OTVM20 28 50 OTVM21 20.6 42.4 OTVM22 28.2 56.6 OTVM23 30.3 68.6 OTVM24 33.7 82.7 OTVM14a 17.3 70.7 OTVM17 26.2 98.0 OTVM18 17.8 OTVM15 13.4 49.5 OTVM16 12.3 58.3 OTVM14b 8.8 55.1 OTVM25 17.1 56.8 OTVM26 16.2 27.9 OTVM27 16.5 51.8 OTVM28 44.7 67.2

[0370] ASOs (unconjugated) were administered to hTau mice (mTau ) via intracerebroventricular (ICV) injection at a single dose in 10 pL total volume per mouse. Brain tissues were harvested to measure relative MAPT RNA levels. The results, which demonstrate significant knockdown of MAPT expression across the tested ASOs, are shown in FIG. 12. INFORMAL SEQUENCE LISTING SEQ ID NO. Sequence Description 1 APEAAGGPCVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKT KPRE EQYNS T YRWS VL TVLHQDWLNGKE YKCKVS NKAL SAPIE KT IS KAK GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc sequence with hole, LALAPS, and S239C mutations 2 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKT KPRE EQ YNS T YRWS VL TVLHQDWLNGKE YKCKVS NKAL SAP IE KT IS KAK GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWANYKTTP PVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLSLSPGK Clone CH3C.35.23.2 with knob and LALAPS mutations 3 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNLA TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPITFGQGTKVEIKRT VAAP SVFIFPPSDEQLKS GTASWC LLNNFY PREAKVQWKVDNALQS GNSQE SVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NTF light chain 4 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWGWIRQPPGKGLEWIGSMSYS GSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK TH NTF heavy chain 5 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWGWIRQPPGKGLEWIGSMSYS GSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPCVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK Fab-Fc polypeptide LALAPS S239C Hole 6 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWGWIRQPPGKGLEWIGSMSYS GSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTE WANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LSPGK Fab-Fc polypeptide 35.23.2 LALAPS Knob 7 APEAAGGPCVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKT KPRE EQ YNS T YRWS VL TVLHQDWLNGKE YKCKVS NKAL SAP IE KT IS KAK GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc sequence with hole, LALAPS, and S239C mutations 8 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKT KPRE EQ YNS T YRWS VL TVLHQDWLNGKE YKCKVS NKAL SAP IE KT IS KAK GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTEWANYKTTP PVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLSLSPG Clone CH3C.35.23.2 with knob and LALAPS mutations 9 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWGWIRQPPGKGLEWIGSMSYS GSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV Fab-Fc polypeptide LALAPS S239C HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPCVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG Hole w / lysine truncation 10 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWGWIRQPPGKGLEWIGSMSYS GSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGWPLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALSAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESYGTE WANYKTTPPVLDSDGSFFLYSKLTVTKEEWQQGFVFSCSVMHEALHNHYTQKSLS LSPG Fab-Fc polypeptide 35.23.2 LALAPS Knob w / lysine truncation 11 ACCT TAAGTAT T AC T T GC ASO 12 CGTGTTTGATATTATCCT ASO 13 T C AAC T C T TAGCAT C C AC T ASO 14 C T TAT CAT TT GCAT T GGT ASO 15 CTGTTAGACATTCATTCTC ASO 16 GGTTGACATCGTCTGCCTGT Control ASO 1 17 CCGTTTTCTTACCACCCT Control ASO 2

Claims

1. An oligonucleotide polypeptide conjugate, comprising an oligonucleotide conjugated to a polypeptide, wherein the oligonucleotide is of formula (I):#-(X)t-(Y)u-(Z)v          (I)wherein:each X is a nucleoside independently selected from the group consisting of locked nucleoside (LNA), 2' methoxyethyl ribosyl nucleoside (MOE), or P-D ribosyl bicyclic sugar nucleoside (cEt);t is an integer from 2 to 5;each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside;u is an integer 10 to 16;each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt;v is an integer from 2 to 5;# is the point of attachment of an X nucleoside to the polypeptide by a linking group; and,each X, Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that:(a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; and / or(c)    1 to 5 intemucleoside linkages that each links two contiguous Ynucleoside are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS;wherein each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; andwherein the sum of t, u and v is an integer from 16 to 22.

2. The oligonucleotide polypeptide conjugate of claim 1 wherein the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and the 1 to 5intemucleoside linkages that each links two contiguous Y nucleosides are each independently-selected from the group consisting of a stabilizing intemucleoside linkage3. The oligonucleotide polypeptide conjugate of claim 1 or 2, wherein the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of phosphorodithioate (PS2), phosphoramidates, phosphonates, phosphotriesters, phosphoryl guanidines, and (9-isopropyl phosphorothioate (OiPS).

4. The oligonucleotide polypeptide conjugate of claim 3, wherein the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of PS2, phosphoramidates, phosphoryl guanidines, and OiPS.

5. The oligonucleotide polypeptide conjugate of claim 1, wherein the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of PS2, mesyl-phosphoramidate (MsPA). cyclic phosphoryl guanidine (PN), and OiPS.

6. The oligonucleotide polypeptide conjugate of any one of claims 1-5, wherein the oligonucleotide contains a single phosphodiester intemucleoside linkage adjacent to an X nucleoside or a Z nucleoside.

7. The oligonucleotide polypeptide conjugate of any one of claims 1-6, wherein each intemucleoside linkage that is not a stabilizing intemucleoside linkage or a phosphodiester intemucleoside linkage is a phosphorothioate.

8. The oligonucleotide polypeptide conjugate of any one of claim 1-7, wherein the sum of t and v is an integer from 4 to 8.

9. The oligonucleotide polypeptide conjugate of claim 8, wherein(a) t is 2, 3 or 4;(b) v is 2. 3, or 4;(c) u is 10, 11, 12, 13, 14, 15, or 16;(d)   u is 10, 11, or 12(d)   t is 2, 3 or 4 and v is 2, 3, or 4;(e)   t is 2, 3 or 4, v is 2, 3, or 4, and u is 10, 11.

12.

13. 14, 15, or 16;(1)   t is 3. v is 3, and u is 10, 11, 12, 13, 14, 15, or 16; or(g) t is 3, v is 3, and u is 10, 11, or 12.

10. The oligonucleotide polypeptide conjugate of claim 8 or 9, wherein v is 2, 3 or 4 and at least two stabilizing intemucleoside linkages adjacent to a Z nucleoside are each PN.

11. The oligonucleotide polypeptide conjugate of claim 8 or 9, wherein v is 3 and:(a) one intemucleoside linkage adjacent to a Z nucleoside is PN;(b) two intemucleoside linkages adjacent to Z nucleosides are each PN;(c) three intemucleoside linkages adjacent to Z nucleosides are each PN; or(d) two intemucleoside linkages adjacent to Z nucleosides are each PN and one intemucleoside linkage adjacent to a Z nucleoside is a phosphodiester intemucleoside linkage.

12. The oligonucleotide polypeptide conjugate of claim 8. 9, or 11, wherein t is 3 and:(a) one intemucleoside linkage adjacent to an X nucleoside is PN;(b) two intemucleoside linkages adjacent to X nucleosides are each PN;(c) three intemucleoside linkages adjacent to X nucleosides are each PN; or(d) two intemucleoside linkages adjacent to X nucleosides are each PN and one intemucleoside linkage adjacent to a X nucleoside is a phosphodiester intemucleoside linkage.

13. The oligonucleotide polypeptide conjugate of any one of claims 8-12. wherein(a) one stabilizing intemucleoside linkages adjacent to an X nucleoside is PN and two intemucleoside linkages adjacent to Z nucleosides are each PN;(b) two intemucleoside linkages adjacent to X nucleosides are each PN and two intemucleoside linkages adjacent to Z nucleosides are each PN;(c) three intemucleoside linkages adjacent to X nucleosides are each PN and three intemucleoside linkages adjacent to Z nucleosides are each PN;(d) three intemucleoside linkages adjacent to X nucleosides are each PN and two intemucleoside linkages adjacent to Z nucleosides are each PN;(e) two intemucleoside linkages adjacent to X nucleosides are each PN and three intemucleoside linkages adjacent to Z nucleosides are each PN;14. The oligonucleotide polypeptide conjugate of claim 8 or 9, wherein(a) two stabilizing intemucleoside linkages adjacent to an X nucleoside is PN and zero intemucleoside linkages adjacent to Z nucleosides are each stabilizing intemucleoside linkages; or(b) zero intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages and two intemucleoside linkages adjacent to Z nucleosides are each PN;(c) zero intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages and zero intemucleoside linkages adjacent to Z nucleosides are each stabilizing intemucleoside linkages15. The oligonucleotide polypeptide conjugate of any one of claims 1-14. wherein each X nucleoside and each Z nucleoside is an LNA.

16. The oligonucleotide polypeptide conjugate of any one of claims 1-15. wherein 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of PS2, phosphorami dates, phosphonates, phosphotriesters, phosphoryl guanidines, and OiPS.

17. The oligonucleotide polypeptide conjugate of claim 16, wherein the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of: PS2, MsPA, PN, and OiPS.

18. The oligonucleotide polypeptide conjugate of any one of claims 1-15. wherein 2 to 3 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, phosphorami dates, phosphonates, phosphotriesters, phosphoryl guanidines, and O-isopropyl phosphorothioates.

19. The oligonucleotide polypeptide conjugate of claim 18, wherein the 2 to 3 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS.

20. The oligonucleotide polypeptide conjugate of claim 18 or 19, wherein the 2 to 3 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides together link three or four contiguous Y nucleotides.

21. The oligonucleotide polypeptide conjugate of claim 17. wherein(a) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each MsPA;(b) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each OiPS;(c) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each PS2; or(d) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each PN.

22. The oligonucleotide polypeptide conjugate of any of claims 1-21, wherein the oligonucleotide contains at least two different stabilizing intemucleoside linkages.

23. The oligonucleotide polypeptide conjugate of claim 22, wherein the oligonucleotide comprises 2 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides, wherein the 2 to 4 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

24. The oligonucleotide polypeptide conjugate of claim 1. wherein t is 3;each X nucleoside is an LNA;u is an integer from 10 to 16;v is 3;each Z nucleoside is an LNA;each stabilizing intemucleoside linkage is a PS2, MsPA, PN, or OiPS; andeach intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

25. The oligonucleotide polypeptide conjugate of claim 24, wherein 2 to 3 intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages; 2 to 3 intemucleoside linkages adjacent to Z nucleosides are stabilizing intemucleoside linkages; and 1 to 4 intemucleoside linkages adjacent to Y nucleosides are stabilizing intemucleoside linkages.

26. An oligonucleotide polypeptide conjugate, comprising an oligonucleotide conjugated to a means for targeted delivery, wherein the oligonucleotide is of formula (I):#-(X)t-(Y)u-(Z)v          (I)wherein:each X is a nucleoside independently selected from the group consisting of LNA, MOE, and cEt;t is an integer from 2 to 5;each Y nucleoside is independently a ribonucleoside or a deoxy ribonucleoside;u is an integer 10 to 16;each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt;v is an integer from 2 to 5;# is the point of attachment of an X to the means for targeted delivery by a linking group; and,each X, Y and Z nucleoside is covalently bound by an intemucleoside linkage; provided that:(a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside is / are each independently selected from the group consisting of PS2, MsPA. PN, and OiPS;(b) at least one intemucleoside linkage(s) adjacent to an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS; and / or(c)    1 to 5 intemucleoside linkages that each links two contiguous Ynucleosides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS;wherein each intemucleoside linkage that is not a PS2, MsPA. PN, or OiPS is a phosphorothioate or a phosphodiester; andwherein sum of t, u and v is an integer from 16 to 22.

27. An improved oligonucleotide polypeptide conjugate, wherein the oligonucleotide is conjugated to a polypeptide, the improvement comprising an oligonucleotide of formula (I):#-(X)t-(Y)u-(Z)v          (I)wherein:each X is a nucleoside independently selected from the group consisting of LN A, MOE, and cEt;t is an integer from 2 to 5;each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside;u is an integer 10 to 16;each Z is a nucleoside independently selected from the group consisting of LNA, MOE and cEt;v is an integer from 2 to 5;# is the point of attachment of an X to the polypeptide by a linking group; and, each X, Y and Z is covalently bound by an intemucleoside linkage;provided that:(a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay; and / or(b)    1 to 5 intemucleoside linkages that each links two contiguous Ynucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage that stabilizes the oligonucleotide as determined by an oligonucleotide catabolism assay;wherein each intemucleoside linkage that is not a stabilizing intemucleoside linkage is a phosphorothioate or a natural phosphodiester; andwherein the sum of t, u and v is an integer from 16 to 22.

28. A method for preparing a stabilized oligonucleotide polypeptide conjugate, the method comprising:(i) determining the in vivo plasma clearance of a first oligonucleotide polypeptide conjugate comprising a first oligonucleotide;(ii) determining a soft spot in the first oligonucleotide of the first oligonucleotide polypeptide conjugate in an oligonucleotide catabolism assay;(iii) providing a second oligonucleotide polypeptide conjugate comprising a stabilized second oligonucleotide and determining the in vivo plasma clearance of the second oligonucleotide polypeptide conjugate, wherein the stabilized second oligonucleotide comprises:(a) the same nucleotide sequence as the first oligonucleotide;(b) 1 to 10 stabilizing intemucleoside linkages that that are not present in the oligonucleotide, wherein the 1 to 10 stabilizing intemucleoside linkages stabilize the second oligonucleotide relative to the first oligonucleotide as determined by an oligonucleotide catabolism assay that are not present in the first oligonucleotide;(c) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodiester linkages; and, wherein at least one of the 1 to 10 stabilizing intemucleoside linkages not present in the first oligonucleotide is located at or adjacent to the soft spot identified in step (ii);wherein, the in vivo plasma clearance of the second oligonucleotide polypeptide conjugate is determined to be slower than that of the first oligonucleotide polypeptide conjugate.

29. The method of claim 28, further comprising:(iv) providing a third oligonucleotide polypeptide conjugate comprising a further stabilized oligonucleotide having (a) one or more additional stabilizing intemucleoside linkages not present in the first or second oligonucleotides, and (b) 8 to 15 intemucleoside linkages in total that are phosphorothioate or natural phosphodi ester linkages.

30. The method of claim 28 or 29, wherein the oligonucleotide in each instance is a gapmer.

31. The method of claim 30, wherein the gapmer in each instance comprises 16 to 22 nucleosides.

32. The method of any one of claims 28-31. wherein the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of: PS2, phosphoramidates, phosphonates, phosphotriesters, phosphoryl guanidines, and O-isopropyl phosphorothioate.

33. The method of claim 32, wherein the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of: PS2, phosphoramidates, phosphoryl guanidines, and O-isopropyl phosphorothioate.

34. The method of claim 33, wherein the stabilizing intemucleoside linkages are in each instance independently selected from the group consisting of PS2, MsPA, PN, and OiPS.

35. The method of claim 28, wherein the stabilized oligonucleotide comprises 1 to 9 stabilizing intemucleoside linkages not present in the first oligonucleotide.

36. The method of claim 35, wherein the stabilized oligonucleotide comprises 3 to 9 stabilizing intemucleoside linkages not present in the first oligonucleotide.

37. The method of claim 28, wherein each intemucleoside linkage of the first oligonucleotide is a phosphorothioate linkage.

38. The method of claim 35, wherein the stabilizing intemucleoside linkages comprise:(a) 2 to 6 PN;(b) 1-2 PS2;(c) 1-2 MsPA;(d) 1-2 OiPS(e) 2 to 6 PN and 1-2 PS2, MsPA, or OiPS.

39. The method of claim 30, wherein the gapmer of the stabilized oligonucleotide polypeptide conjugate comprises 1 to 4 PS2, MsPA, PN, or OiPS intemucleoside linkages adj acent to a nucleoside present in the gap region of the gapmer.

40. The method of claim 30 or 31, wherein the gapmer of the second oligonucleotide polypeptide conjugate comprises at least one wing segment comprising 1 to 3 stabilizing intemucleoside linkages, optionally wherein the stabilizing intemucleoside linkages are PN.

41. The method of claim 40, wherein the at least one wing comprises a wing at the 3' end of the gapmer, a wing at the 5' end of the gapmer, or a 3' end of the gapmer and a wing at the 5' end of the gapmer.

42. The method of any one of claims 28-41, wherein the plasma clearance is measured by plasma concentration.

43. The method of claim 42, wherein the plasma concentration is determined at one or more timepoint(s)(a) from about zero to about 336 hours after injection of the oligonucleotide polypeptide conjugate being assayed;(b) from about zero to about 168 hours after injection of the oligonucleotide polypeptide conjugate being assayed; or(c) from about zero to 72 hours after injection of the oligonucleotide polypeptide conjugate being assayed.

44. The method of claim 43, wherein the plasma concentration is determined at one or more timepoints at about 0.25, about 4, about 24, about 48, and / or about 72 hours.

45. The method of any one of claims 28-44, wherein the plasma clearance of the second oligonucleotide polypeptide conjugate is determined to be slower when the plasma concentration of the second oligonucleotide polypeptide is greater as compared to that of the first oligonucleotide polypeptide conjugate of the timepoint(s).

46. The method of claim 45, wherein the plasma concentration of the second oligonucleotide polypeptide is greater by about 1-10%, 5-15%, 10-20%, 20-50%, or above 50%.

47. An oligonucleotide of formula (IA):(X)t-(Y)u-(Z)v            (IA)wherein:each X is a nucleoside independently selected from the group consisting of LNA, MOE, and cEt;t is an integer from 2 to 5;each Y nucleoside is independently a ribonucleoside or a deoxyribonucleoside;u is an integer 10 to 16;each Z is a nucleoside independently selected from the group consisting of LNA, MOE, and cEt;v is an integer from 2 to 5;and,each X, Y and Z nucleoside is covalently bound by an intemucleoside linkage;provided that:(a) at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS; and / or(b)    1 to 5 intemucleoside linkages that each links two contiguous Ynucleosides are each independently selected from the group consisting of PS2, MsPA, PN, and OiPS;wherein each intemucleoside linkage that is not a PS2, MsPA. PN, or OiPS is a phosphorothioate or a natural phosphodiester; andwherein the sum of t, u and v is an integer from 16 to 22.

48. The oligonucleotide of claim 47 wherein the at least one intemucleoside linkage(s) adjacent to a Z nucleoside or an X nucleoside is / are each independently selected from a stabilizing intemucleoside linkage; and the 1 to 5 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of a stabilizing intemucleoside linkage49. The oligonucleotide of claim 47 or 48, wherein each intemucleoside linkage that is not a PS2, MsPA, PN, or OiPS is a phosphorothioate.

50. The oligonucleotide of any one of claims 47-49, wherein the sum of t and v is an integer from 4 to 8.

51. The oligonucleotide of claim 50. wherein(a) t is 2, 3 or 4;(b) v is 2, 3, or 4;(c) u is 10, 11, 12, 13, 14, 15, or 16;(d) uis 10, 11, or 12(d)   t is 2. 3 or 4 and v is 2.

3. or 4;(e)   t is 2, 3 or 4, v is 2, 3, or 4, and u is 10, 11, 12, 13, 14, 15, or 16;(1)   t is 3, v is 3, and u is 10, 11, 12, 13, 14, 15, or 16; or(g)   t is 3, v is 3, and u is 10, 11, or 12.

52. The oligonucleotide conjugate of claim 50 or 51, wherein v is 2, 3 or 4 and at least two intemucleoside linkages adjacent to aZ are each PN.

53. The oligonucleotide polypeptide conjugate of claim 50 or 51, wherein v is 3 and:(a) one intemucleoside linkage adjacent to a Z nucleoside is PN;(b) two intemucleoside linkages adjacent to Z nucleosides are each PN;(c) three intemucleoside linkages adjacent to Z nucleosides are each PN(d) two intemucleoside linkages adjacent to Z nucleosides are each PN and one intemucleoside linkage adjacent to a Z nucleoside is a phosphodiester intemucleoside linkage.

54. The oligonucleotide polypeptide conjugate of claim 50, 51, or 53, wherein t is 3 and:(a) one intemucleoside linkage adjacent to an X nucleoside is PN;(b) two intemucleoside linkages adjacent to X nucleosides are each PN;(c) three intemucleoside linkages adjacent to X nucleosides are each PN(d) two intemucleoside linkages adjacent to X nucleosides are each PN and one intemucleoside linkage adjacent to a X nucleoside is a phosphodiester intemucleoside linkage.

55. The oligonucleotide of any one of claims 50-54, wherein(a) one stabilizing intemucleoside linkages adjacent to an X nucleoside is PN and two intemucleoside linkages adjacent to Z nucleosides are each PN;(b) two intemucleoside linkages adjacent to X nucleosides are each PN and two intemucleoside linkages adjacent to Z nucleosides are each PN;(c) three intemucleoside linkages adjacent to X nucleosides are each PN and three intemucleoside linkages adjacent to Z nucleosides are each PN;(d) three intemucleoside linkages adjacent to X nucleosides are each PN and two intemucleoside linkages adjacent to Z nucleosides are each PN;(e) two intemucleoside linkages adjacent to X nucleosides are each PN and three intemucleoside linkages adjacent to Z nucleosides are each PN;56. The oligonucleotide polypeptide conjugate of claim 50 or 51, wherein(a) two stabilizing intemucleoside linkages adjacent to an X nucleoside is PN and zero intemucleoside linkages adjacent to Z nucleosides are each stabilizing intemucleoside linkages; or(b) zero intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages and two intemucleoside linkages adjacent to Z nucleosides are each PN;(c) zero intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages and zero intemucleoside linkages adjacent to Z nucleosides are each stabilizing intemucleoside linkages57. The oligonucleotide of any one of claims 47-56, wherein each X nucleoside and each Z nucleoside is an LNA.

58. The oligonucleotide of any one of claims 47-57, wherein 1 to 4 intemucleoside linkages that each links two contiguous Y nucleosides are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS.

59. The oligonucleotide of any one of claims 47-57, wherein 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides are each independently selected from the group consisting of PS2, MsPA, PN. and OiPS.

60. The oligonucleotide of claim 59, wherein 2 to 3 intemucleoside linkages that each links two contiguous Y nucleotides together link three or four contiguous Y nucleotides.

61. The oligonucleotide of claim 58.(a) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each MsPA;(b) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each OiPS;(c) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each PS2; or(d) the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides are each PN.

62. The oligonucleotide polypeptide conjugate of claim 58, wherein the 1 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides comprise 2 to 4 stabilizing intemucleoside linkages that each links two contiguous Y nucleotides, wherein the 2 to 4 stabilizing intemucleoside linkages comprise at least two different stabilizing intemucleoside linkages.

63. The oligonucleotide of any one of claims 47-62, wherein u is an integer from 10 to 12.

64. The oligonucleotide of claim 47, wherein:t is 3;each X nucleoside is an LNA;u is an integer from 10 to 16;v is 3;each Z is an LNAeach stabilizing intemucleoside linkage is a PS2, MsPA, PN, or OiPS; andeach intemucleoside linkage that is not a PS2. MsPA, PN. and OiPS is a phosphorothioate.

65. The oligonucleotide polypeptide conjugate of claim 64, wherein 2 to 3 intemucleoside linkages adjacent to X nucleosides are stabilizing intemucleoside linkages; 2 to 3 intemucleoside linkages adjacent to Z nucleosides are stabilizing intemucleoside linkages;and 1 to 4 intemucleoside linkages adjacent to Y nucleosides are stabilizing intemucleoside linkages.

66. The oligonucleotide of any one of claims 47-65, wherein the locations of one or more of the PS2, MsPA, PN, and OiPS present in the oligonucleotide were first identified in an oligonucleotide catabolism assay.

67. The oligonucleotide of claim 66, wherein the locations of two or more of the PS2, MsPA, PN, and OiPS present in the oligonucleotide were first identified in an oligonucleotide catabolism assay.

68. The oligonucleotide of claim 66, wherein the location of each of the PS2, MsPA, PN, and OiPS present in the oligonucleotide was first identified in an oligonucleotide catabolism assay.

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