Novel oligonucleotides bearing a "2-propyl" internucleoside linkage

Abstract

Disclosed herein are nucleic acids containing a modified internucleotide linkage, which is the 2-propyl (PrON) linkage, to increase durability and exposure of therapeutic oligonucleotides, as well as nucleotides, phosphoramidites, and other intermediate compounds useful for making the same.

Description

NOVEL SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30776_WO” created 30-May-2025 and is 426 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The present invention is directed to nucleic acids, including RNA molecules such as therapeutic RNA molecules, comprising a novel internucleotide linkage, termed a “2-propyl” linkage, and nucleotides and intermediates for making the same. These “2-propyl” nucleotides are termed “PrON” herein. The present invention is also directed to novel nucleosides comprising a PrON moiety. The present invention is also directed to oligonucleotides, such as RNAi agents, comprising at least one PrON, including RNAi agents capable of degrading angiotensinogen (AGT) gene mRNA. BACKGROUND OF THE INVENTION

[0003] Molecules comprising oligonucleotides that modulate mRNA levels of genes have made significant contributions in the study of molecular biology and increasingly show promise as therapies. RNA interference (RNAi), a highly conserved regulatory mechanism in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA molecules, is the mechanism that underlies a number of approved therapeutics and numerous others in the clinic.

[0004] As RNA is relatively unstable, much effort has been expended in increasing stability and potency of potential therapeutic RNA molecules with cellular and organ safety. Organisms including humans harbor numerous threats to RNA durability, including exonucleases and endonucleases such as RNase H family endonucleases. These enzymes degrade both natively- produced and exogenous RNA molecules. Low durability of RNA can result in low exposure of the therapeutic oligonucleotide to its intended target.

[0005] Stability-enhancing modifications available in the art include phosphorothioate (PS) internucleotide linkages and extended nucleic acid (exNA) as described in International PatentPublication No. WO2021 / 195533. As the advances, however, more options for extending the half-life of therapeutic oligonucleotides, such as RNAi agents is needed. In particular, modifications which reduce degradation products,(for example, metabolic byproducts of one or more molecular weights resulting from the delivery and processing of therapeutic oligonucleotides.)

[0006] The angiotensinogen (AGT) gene encodes the protein angiotensinogen. This protein is part of the renin-angiotensin system, which regulates blood pressure and the balance of fluids and salts in the body. Dysregulation of AGT expression can give rise to a number deleterious conditions, including hypertension. Current treatment for hypertension includes antihypertensive drugs, with first-line therapies typically including daily beta-blockers. There remains a need for therapeutic agents that can inhibit or adjust the expression of AGT for treating hypertension and hypertension-related disorders, e.g. by utilizing RNAi. In particular there remains a need for pharmaceutical options for patients that does not require daily dosing. SUMMARY

[0007] In one aspect, the present disclosure provides an oligonucleotide of Formula I: wherein:R1is selected from the group consisting of H, OR2, NH2, SR2, F, and Cl, wherein R2is selected from the group consisting of H and C1-C20 alkyl, or B is a nucleobase; and m and n are each independently selected from any whole number from 0 to 40; wherein when a is 2, both B can be the same, or can be different; and wherein the oligonucleotide optionally comprises at least one of an abasic moiety and an inverted abasic moiety.

[0008] In one embodiment, the present disclosure provides a double-stranded RNA molecule which includes a first oligonucleotide as described above, and a second oligonucleotide of 15 to 30 nucleotides in total length, wherein the first oligonucleotide and the second oligonucleotide form a duplex region. In one aspect, the first oligonucleotide is an antisense strand of an RNAi agent, and the second oligonucleotide is a sense strand of an RNAi agent.

[0009] In another embodiment, the present provides a compound of Formula II:

[0010] wherein B is a nucleobase and protecting group.

[0011] In another embodiment, theprovides a compound of Formula III:wherein B is a nucleobase and Y is an organic protecting group.

[0012] In another embodiment, the present disclosure provides a compound of Formula IV: wherein B is a nucleobase, Y1is first organic protecting group, and Y2is asecond organic protecting group.

[0013] In another embodiment, the present disclosure provides a compound of Formula V: wherein B is a nucleobase and Y isgroup.

[0014] In another embodiment, the present provides an AGT RNAi agent or a pharmaceutical composition comprising the same which includes the compound of Formula I. In some embodiments, the AGT RNAi agent is of the formula R-L-D, wherein R is a double stranded RNA (dsRNA) comprising a sense strand an antisense strand, wherein the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, wherein optionally one or more nucleotides of the sense strand and the antisense strands are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified nucleotide linkages; L is a linker; and D is a delivery moiety, such as a GalNAc delivery moiety. DETAILED DESCRIPTION

[0015] The present disclosure provides nucleic acids including the PrON moiety as an internucleotide linkage, and nucleotides, phosphoramidites, and other molecules for making the same.

[0016] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0017] As used herein, the term “alkyl” means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms. For example, “C1-C20 alkyl” means a radical having 1-20 carbon atoms in a linear or branched arrangement.

[0018] As used herein, “RNAi” refers to RNA interference, a process that results in sequence- specific degradation of a messenger RNA (mRNA), such as via the RNA-induced silencing complex (RISC) pathway. RNAi is achieved through the use of an “RNAi agent,” also referred to interchangeably herein as “iRNA,” “iRNA agent,” and “RNA interference agent,” each of which means an agent that mediates sequence-specific degradation of a target mRNA by RNA interference. In some embodiments, the RNAi agent has a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex (e.g., a double stranded RNA). In some embodiments, the RNAi agent includes a delivery moiety for targeting various cells or tissues. In some instances, the sense strand has a delivery moiety conjugated to the 3’ end of the sense strand or a nucleotide of the sense strand.

[0019] The RNAi agents herein comprise a sense strand and an antisense strand, wherein each is an oligonucleotide. As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase such as, for example, adenine, cytosine, guanine, thymine, or uracil;and a pentose sugar such as, for example, or 2'-deoxyribose) and a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0020] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can be a non-naturally occurring nucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[0021] As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. Typically, an oligonucleotide is a short nucleic acid compound, less than about 100 nucleotides in length. An oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (DsiRNA), RNA aptamer, or antisense oligonucleotide (ASO).

[0022] As used herein, “ribonucleotide” means a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0023] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5’ end and a 3’ end).

[0024] As used herein, “ANG8” refers to an ANGPTL8 mRNA transcript. The nucleic acid sequence of human ANG8 mRNA can be found at NM_018687.7 (SEQ ID NO: 2). The amino acid sequence of human ANG8 protein can be found at NP_061157.3. “ANG8” and “ANGPTL8” are used interchangeably herein.

[0025] As used herein, “HMGCR” refers to an HMGCR mRNA transcript. The nucleic acid sequence of human HMGCR mRNA can be found at NM_000859.3 (SEQ ID NO: 1). The amino acid sequence of human HMGCR protein can be found at NP_000850.1.

[0026] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotidelinkage having a phosphodiester bond. A internucleotide linkage can be a non-naturally occurring linkage.

[0027] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, means a structure formed through complementary base pairing of two antiparallel sequences of nucleotides (i.e., in opposite directions, whether formed by two separate nucleic acid strands or by a single, folded strand (such as via a hairpin). In some instances, a duplex will arise by pairing of a sense strand to an antisense strand. A duplex may form despite not having full complementarity between the two strands, or when an abasic nucleotide is present. A Duplex No:, as shown herein, e.g., in Table 1, Table 2, Table 3, or Table 4, corresponds to a specific sense and antisense strand that comprise a given RNAi agent.

[0028] As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein. A natural or canonical nucleotide can be complementary to a modified nucleotide; for example, a natural nucleotide bearing adenine as its nucleobase is complementary to a PrON with a uracil nucleobase.

[0029] As used herein, “region of complementarity” means a nucleotide sequence of a nucleic acid (e.g., a double stranded oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, an oligonucleotide herein includes a targeting sequence having a region of complementary to a mRNA target sequence.

[0030] As used herein, “antisense strand” means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sense strand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.

[0031] As used herein, “overhang” means the unpaired nucleotide or nucleotides that protrude from the duplex structure of a double stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from a duplex region at the 5’ terminus or 3’ terminus of adouble stranded oligonucleotide. The can be a 3’ or 5’ overhang on the antisense strand or sense strand of a double stranded oligonucleotide. For example, the sense strand and the antisense strand may have 5’ or 3’ overhangs of 1 to 5 nucleotides.

[0032] In one aspect, the present disclosure provides an oligonucleotide, or salt thereof, of Formula I: wherein:R1is selected from the group SR2, F, and Cl, wherein R2is selected from the group consisting of H and C1-C20 alkyl; and B is a nucleobase.

[0033] In some aspects, the oligonucleotide or salt thereof comprises Formula VI: wherein:each X1is an independently selected nucleotide; each X2 is an independently selected nucleotide; each Q is PO2X5wherein X5is O or S, or, when n is 0, Q may be H; and m and n are each independently selected from any integer from 0 to 40.

[0034] In certain aspects, the oligonucleotide or salt thereof comprises Formula VII:VII.

[0035] In some aspects, the oligonucleotide or salt thereof comprises Formula VIIa: In other aspects, the oligonucleotide or Formula VIIb:

[0036] In some aspects, the oligonucleotide or salt thereof comprises Formula VIII:wherein X5is selected from O and S.

[0037] In certain aspects, the oligonucleotide or salt thereof is of Formula VIIIa:B In another embodiment, the oligonucleotide or salt thereof is of Formula VIIIb: In another embodiment, theFormula VIIIc: In another embodiment, theFormula VIIId:

[0038] In one embodiment of the X5is O. In another embodiment of the oligonucleotide or salt thereof, X5is S.

[0039] In one aspect, the oligonucleotide or salt thereof is of Formula Ia: O B

[0040] In an aspect, the oligonucleotide or salt thereof is of Formula VIa: X1m O B

[0041] In an aspect, theFormula VIIc:

[0042] In one embodiment, the oligonucleotide or salt thereof is of Formula VIId:

[0043] In one embodiment, the is of Formula VIIe:

[0044] In one embodiment, theis of Formula VIIIe: .

[0045] In one embodiment, theis of Formula VIIIf:

[0046] In another embodiment, the or salt thereof is of Formula VIIIg:

[0047] In another embodiment, is of Formula VIIIh:

[0048] In another embodiment,is of the formula VIIIi:

[0049] In one embodiment, m andfrom any whole number from 0 to 20.

[0050] In an embodiment with X1 and / or X2, nucleotide X1 or X2 is independently selected. In one embodiment, a nucleotide X1may include the 5’ end of the oligonucleotide. In an embodiment, a nucleotide X2 may include the 3’ end of the oligonucleotide.

[0051] In an embodiment, each B is independently selected from the group consisting of hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6- dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil. In a particular embodiment, B is independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil. In one embodiment, each B is uracil. For example, when “rPrON” or “sPrON” is used with regard to a nucleic acid of the sequence listing, the nucleobase B is uracil, though other PrON molecules (e.g., those with nucleobase cytosine) have been made and are disclosed herein.

[0052] In one aspect, X5is O. In another embodiment, X5is S.

[0053] In one embodiment, the oligonucleotide or salt thereof is 15 to 30 nucleotides in total length. In one embodiment, the oligonucleotide is 18 to 30 nucleotides in total length. In an embodiment, the oligonucleotide is 18 to 23 nucleotides in total length.

[0054] In one aspect, the oligonucleotide may include at least one modification selected from the group consisting of 2’-O-methoxy, 2’-O-methyl, 2’-fluoro, phosphorothioate, 2’-deoxy, abasic moiety, inverted abasic moiety, glycol nucleic acid (GNA), R-PrON (that is PrON in which the branched methyl of the internucleotide linkage is in the R configuration), and S-PrON (that is, PrON in which the branched methyl of the internucleotide linkage is in the S configuration); that is, R-PrON includes the following structure: , and S-PrON includes the following.

[0055] In one embodiment, themodified nucleotide at the following positions:2, 3, 7, 14, and 16 from the 5’ end; 2, 5, 7, 14, and 16 from the 5’ end; 2, 3, 8, 14, and 16 from the 5’ end; 2, 5, 8, 14, and 16 from the 5’ end; 2, 6, 14, and 16 from the 5’ end; or 2, 14, and 16 from the 5’ end of the oligonucleotide.

[0056] In one embodiment, the oligonucleotide includes 2’O-methyl modified nucleotides at all the other positions of the oligonucleotide.

[0057] In one embodiment, the present disclosure provides a double-stranded RNA molecule or salt thereof which includes a first oligonucleotide as described above, and a second oligonucleotide of 15 to 30 nucleotides in total length, wherein the first oligonucleotide and the second oligonucleotide form a duplex region. In one aspect, the first oligonucleotide is an antisense strand of an RNAi agent, and the second oligonucleotide is a sense strand of an RNAi agent.

[0058] In one aspect, the duplex region is 15 to 23 base pairs in length.

[0059] In one embodiment, the antisense strand has a 3’ overhang of 1 or 2 nucleotides. In one aspect, the overhang includes 1 or 2 PrON.

[0060] In one aspect, the sense strand of the double-stranded RNA molecule includes a delivery moiety at its 3’ end. In one aspect, the delivery moiety is of Formula IX:wherein E is a point at which the delivery moiety is conjugated to the sense strand.

[0061] In another embodiment, the present disclosure provides a compound of Formula II:wherein B is a nucleobase and Y is an organic protecting group.

[0062] In one aspect, the compound is of Formula IIa:

[0063] In one aspect, the organic ether (MOM),methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p-Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS). In a particular embodiment, the organic protecting group is TBDMS.

[0064] In one embodiment, B is selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5- hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil. In a specific embodiment, B is uracil.

[0065] In another embodiment, the present disclosure provides a compound of Formula III:wherein B is a nucleobase and Y is an organic protecting group.

[0066] In one embodiment, the compound is of Formula IIIa:

[0067] In a specific embodiment, the compound is of Formula IIIb:

[0068] In another embodiment, the IIIc:

[0069] In one embodiment, the organic is methoxylmethyl ether (MOM),methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p-Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS). In certain embodiments, the organic protecting group can be a benzoyl group or a silyl group. In a specific embodiment, the organic protecting group is TBDMS.

[0070] In one embodiment, B is selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5- hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil. In one embodiment, B is uracil.

[0071] In another embodiment, the present disclosure provides a compound of Formula IV:wherein B is a nucleobase, Y1 is selected from H and a first organic protecting group, and Y2 is a second organic protecting group.

[0072] In one embodiment, the compound is of Formula IVa:

[0073] In one embodiment, the IVb:

[0074] In another embodiment, the IVc:

[0075] In one aspect, Y1 is H.

[0076] In one aspect, Y1 is a first organic protecting group, and Y2 is a second organic protecting group.

[0077] In one embodiment, the first organic protecting group and the second organic protecting group are each independently selected from the group consisting of methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p- Methoxybenzyl ether (PMB), p-Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS).

[0078] In one embodiment, Y2 is DMT.

[0079] In an embodiment, Y1is TBDMS.

[0080] In one aspect, B is selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5- hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil. In a particular embodiment, B is uracil.

[0081] In another embodiment, the present provides a compound of Formula V: wherein B is a nucleobase and Y is group.

[0082] In one embodiment, the Va:

[0083] In one embodiment, theVb:

[0084] In one embodiment, theVc:

[0085] wherein B is a nucleobase group.

[0086] In an embodiment, the

[0087] In an embodiment, the

[0088] In one aspect, the organicether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p-Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP),tetrahydrofuran (THF), trityl (Tr), or (TMS). In a specific embodiment, the organic protecting group is DMT.

[0089] In one embodiment, B is selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5- hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil. In a specific embodiment, B is uracil.

[0090] In one embodiment, the present disclosure provides an AGT RNAi agent capable of reducing expression of AGT mRNA, and which contains at least one PrON. In one embodiment, the

[0091] Disclosed herein are oligonucleotides including at least one PrON, including single- stranded RNA molecules, double-stranded RNA molecules, and RNAi agents. The 2-propyl internucleotide linker represents a departure from natural internucleotide linkages and as such, provides an RNA molecule in which it is included with potentially increased durability because of, for example, lesser susceptibility to degradation by nucleases. The PrON provides an increased distance between a nucleotide and the phosphate in the 3’ direction, and also provides a branched alkyl by virtue of the methyl group, reducing the chances that the moiety will be recognized by a nuclease or even fit within its binding pocket. When used in the context of a therapeutic molecule, a PrON-containing oligonucleotide can possess higher durability than an oligonucleotide which does not include a PrON, and better exposure to target mRNA. In some instances, the resistance to degradation conferred by PrON can potentially reduce the number and type of metabolic byproducts (fragmented RNA molecules).

[0092] In certain embodiments, an oligonucleotide of the present disclosure has one PrON. In another embodiment, the oligonucleotide has two PrONs. In another embodiment, the oligonucleotide includes three PrONs, or four PrONs, or five PrONs. A PrON may be incorporated at the 5’ end of an oligonucleotide in one embodiment. In another embodiment, the PrON is incorporated or at the 3’ end of the oligonucleotide. In another embodiment, the PrON is incorporated at an internal portion of the oligonucleotide. A PrON may be included in a duplex region of a double-stranded RNA in one embodiment. In another embodiment, the PrON or PrONs may be present in an overhang portion of the dsRNA.

[0093] In an oligo nucleotide with multiple PrONs, the PrONs can be located in consecutive positions, or may instead be spaced apart (that is, separated by at least one non-PrON nucleotide.)

[0094] In some embodiments, the PrON as listed herein has a uracil nucleobase. In some instances such a moiety may be referred to as “2-propyl-O-uridine.”

[0095] The a PrON includes a stereocenter. the PrON is located at a position other than the 5’ end of an oligonucleotide, this stereocenter is present in the interculeotide linker. In some instances, the PrON introduced into an oligonucleotide has the R stereochemical configuration. In another embodiment, the PrON included in an oligonucleotide has the S stereochemical configuration. In an embodiment wherein the oligonucleotide has multiple PrONs, the stereochemical configuration of each PrON is selected independently. For example, in an oligonucleotide containing two PrONs, the configuration of the two PrONs in a 5’ to 3’ direction may be R / R, R / S, S / R, or S / S. In one instance, such an oligonucleotide has these two PrONs in consecutive positions. In some embodiments, the two PrONs of such a nucleotide are in adjacent or consecutive positions, such as in a 3’ overhang in the antisense strand of a dsRNA duplex.

[0096] The PrON as envisaged herein can be used in an oligonucleotide with other modified internucleotide linkers, including but not limited to phosphorothioate linkers. The PrON can be used with other modified nucleotides as disclosed herein.

[0097] In one aspect, the oligonucleotide as provided herein includes PrONs in nonconsecutive positions. In an example of an oligonucleotide having two PrONs in such a fashion is provided as Formula X:In this formula, X6is one or more nucleotides.

[0098] The skilled artisan will be capable of envisioning formulae for oligonucleotides including three, four, or five PrONs, in view of Formula X above.

[0099] Further provided herein is a pharmaceutical composition for use as a therapy, or for treatment of a disease or a condition, which includes an oligonucleotide as described herein. In one embodiment, the pharmaceutical composition may include a salt of the oligonucleotide. RNA is a polyanion owing to the negative charges present on the phosphate groups of thebackbone, and these charges may be balanced provision of positive charges, yielding a salt of the RNA. In one instance, the salt is a sodium salt. In another instance, the salt is a potassium salt. In another embodiment, the salt may include multiple different cationic species.

[0100] In one example, RNAi agents targeting HMGCR and containing PrON have been made and characterized. Nucleic acid sequences related to HMGCR and the experiments described herein are provided in Tables 1 and 2 below. TABLE 1: Unmodified sense strand and antisense strand nucleic acid sequences of HMGCR RNAi agents dsRNA SEQ SEQ Transcript NO: Sense Strand 5' to 3' ID Antisense Strand 5' to 3' ID Position 000859.3 2434 2423 2424TABLE 2: Modified sense strand and antisense strand nucleic acid sequences of HMGCR RNAi agents dsRNA Strand Oligo Sequence 5' to 3' SEQ ID NO: 9 10 9 11 9 12 9 13 9 14 9 15 16 17 16 18dsRNA Strand Oligo Sequence 5' t SEQ ID NO: o 3' NO: 16 19 16 20 21 22 21 23 21 24 21 25 9 26 9 27 9 28 9 29 930strand; “AS” means the antisense strand; rPrON indicates PrON with R stereocenter in the internucleotide linkage; sPrON indicates PrON with S stereocenter in the internucleotide linkage.

[0101] In one example, RNAi agents targeting ANGPTL8 and containing PrON have been made and characterized. Nucleic acid sequences related to ANGPTL8 and the experiments described herein are provided in Tables 3 and 4 below. TABLE 3: Unmodified sense strand and antisense strand nucleic acid sequences of ANGPTL8 RNAi agents SE pt 87SE Q S Transcript dsRNA EQ ' ' ' ' Position 87TABLE 4: Modified sense strand and antisense strand nucleic acid sequences of ANGPTL8 RNAi agents Q ID NO: 34 35 34 36 34 37 34 38 34 39 34 40linkage; “S” means the sense strand; “AS” means the antisense strand; rPrON indicates PrON with R stereocenter in the internucleotide linkage; sPrON indicates PrON with S stereocenter in the internucleotide linkage; 5ex indicates exNA. PREPARATIONS

[0102] The compounds of the present disclosure may be prepared by following the schemes and preparations detailed below. These schemes and preparations are not limiting in their scope.

[0103] Certain abbreviations are defined as follows: “AS” refers to antisense strand; “CT” refers to cycle threshold; “DCM” refers to dichloromethane; “DMF” refers to dimethylformamide; “DIEA” refers to N,N-diisopropylethylamine; “PE” refers to petroleum ether; “TFA” refers to trifluoroacetic acid; “DMT” refers to dimethoxytrityl; “DMTCl” refers to dimethoxytrityl chloride; “DMSO” refers to dimethyl sulfoxide; “dsRNA” refers to double stranded ribonucleicacid; “EtOH” refers to ethanol; “EtOAc” ethyl acetate; “hiPSC” refers to human induced pluripotent stem cell; “MeCN” refers to acetonitrile; “MeOH” refers to methanol and methyl alcohol; “PBS” phosphate-buffered saline; “PCR” refers to polymerase chain reaction; “RT- PCR” refers to reverse transcription polymerase chain reaction; “siRNA” refers to small interfering RNA; “SS” refers to sense strand; “TEA” refers to triethylamine; “IBX” refers to 2- iodoxybenzoic acid “9-BBN” refers to 9-borabicyclo[3.3.1]nonane; “2, 4, 6 TMP” refers to 2, 4, 6 trimethylpyridine; “TBAF” refers to tetra-butylammonium fluoride; “TBDMSCl” refers to tert-butyldimethylsilyl chloride and “TBDMS” refers to tert-butyldimethylsilyl. Scheme 1(1), the conditions of which will be known by one skilled in the art. Step B shows the conversion of compound (2) to compound (3) by protection of the 3’ alcohol using TBDMSCl in an appropriate solvent such as DMF. For step C, the DMT group was removed from compound (3) to give compound (4), the conditions of which will be known by one skilled in the art. Step D depicts the oxidation of compound (4), using IBX in an appropriate solvent such as EtOAc, to provide compound (5). Conversion of compound (5) to compound (6) via a Wittig reaction, in step E, was accomplished using methyltriphenylphosphonium bromide in an appropriate solvent such as THF. Hydroboration of compound (6) in step F, using 9-BBN in an appropriate solventsuch as THF, gave compound (7) which was oxidized in step G, using IBX and an appropriate solvent such as ACN, to provide compound (8). A Grignard reaction, depicted in step H, was used to convert compound (8) to compound (9) using methylmagnesium bromide in an appropriate solvent such as THF. DMT protection of compound (9), depicted in Step I, provided compound (10) using DMTCl, 2, 4, 6-TMP, and AgNO3 in an appropriate solvent such as DCM. The TBDMS group was removed from compound (10) to provide compound (11), the conditions of which will be known by one skilled in the art. Scheme 2(12) and (13) as diastereomers. In step B, the 3’ alcohol of compound (12) was phosphitylated with 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile in an appropriate solvent such as dichloromethane to provide compound (14). Step C, using the same conditions as step B, provided compound (15) from compound (13).1 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0106] DIEA (50.1 g, 67.5 mL,-phenyl-methyl]-4- methoxy-benzene (72.2 g, 213 mmol) were added to a solution of 1-((2R,3R,4R,5R)-4-hydroxy- 5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (50.0 g, 194 mmol) dissolved in DCM (500 mL). The mixture was stirred at ambient temperature for 15 hours. The reaction mixture was quenched with water (500 ml) and the organic layer was removed. The aqueous layer was extracted three times with DCM (200 ml). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / PE, to give the title compound (100 g, 82%) as a yellow solid. ES / MS (m / z): 583.3 (M+23). Preparation 2 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0107] Imidazole (216 g, 3.18 mol) and tert- (47.96 g, 52.9 mL, 318 mmol) were added to a solution of 1-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2- yl)pyrimidine- 2,4(1H,3H)-dione (100.0 g, 159 mmol) in DMF (500 mL). The mixture was stirred under nitrogen at 50 °C for 15 hours. After cooling to ambient temperature, the mixture was quenched with water (1 L). The aqueous layer was extracted three times with EtOAc (800 ml). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / PE, to give the title compound (99 g, 86%) as a yellow solid. ES / MS (m / z): 697.4 (M+23). Preparation 3 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione OH O O NH O

[0108] TFA (59.0 g, 40 ml, 520of 1-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (99.0 g, 136 mmol) dissolved in DCM (1000 mL). The mixture was stirred under nitrogen at ambient temperature for 2 hours, cooled to 0 °C in an ice bath, and then quenched by the addition of aqueous sodium bicarbonate (300 mL). The aqueous was then extracted 3 times with DCM (200 mL) and the organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 80% EtOAc / PE, to give the title compound (30 g, 57%) as a yellow solid. ES / MS (m / z): 373.1 (M+1).4 (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4- methoxytetrahydrofuran-2-carbaldehyde

[0109] IBX (43.8 g, 156 mmol) 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- - 2-yl)pyrimidine- 2,4(1H,3H)- dione (30.0 g, 78.1 mmol) in EtOAc (400 mL). The mixture was stirred under nitrogen at 80 °C for 5 hours. The reaction mixture was then cooled to ambient temperature, filtered, and concentrated under reduced pressure to give to give the title compound (33 g, 86%) as a pink solid. ES / MS (m / z): 371.1 (M+1). Preparation 5 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-methoxy-5-vinyltetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione

[0110] N-Butyllithium (14.6 g,hexane) was added to a solution of methyltriphenylphosphonium bromide (81.4 g, 228 mmol) in THF (600 mL) under nitrogen at - 70 °C. The solution was warmed to 0 °C, stirred for 30 minutes, and (2S,3S,4R,5R)-3-((tert- butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4- methoxytetrahydrofuran-2-carbaldehyde (37.5 g, 75.9 mmol) dissolved in THF (300 mL) was then added. The mixture was then warmed to ambient temperature and stirred under nitrogen for 16 hours. After quenching with saturated ammonium chloride (1200 ml) the mixture was further diluted with water (500 ml). The aqueous was extracted 3 times with EtOAc (1000 ml) and the organic layers were combined, washed with brine (800 ml), dried over sodium sulfate, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 0%to 50% EtOAc / PE, to give the title compound g, 56%) as a white solid. ES / MS (m / z): 369.6 (M+1). Preparation 6 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0111] 1-((2R,3R,4R,5R)-4-((tert- methoxy-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (11.1 g, 28.6 mmol) was dissolved in THF (200 mL) under nitrogen and the solution was cooled to 0 °C. 9-BBN in THF (45.2 g, 143 mmol, 0.5M in THF) was added to the solution and it was stirred at 0 °C for 30 minutes. The mixture was then warmed to ambient temperature. After stirring for 16 hours, the solution was cooled to 0 °C and MeOH (12 mL) was added dropwise. When gas evolution had ceased, water (6.0 mL) was added followed by a mixture of sodium hydroxide (2.29 g, 28.6 mL, 57.2 mmol) and hydrogen peroxide (22.7 g, 20.5 ml, 200 mmol). The ice bath was then removed, and the mixture was stirred vigorously at ambient temperature for 2 hours. Aqueous sodium sulfite (400 mL) was then added, and the mixture was extracted 3 times with EtOAc (500 mL). The organic layers were then combined, washed 2 times with brine (200 ml), dried over sodium sulfate, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 50% EtOAc / PE, to give the title compound (8.6 g, 78%) as a colorless oil. ES / MS (m / z): 387.2 (M+1). Preparation 7 2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 4-methoxytetrahydrofuran-2-yl)acetaldehyde O O

[0112] 1-((2R,3R,4R,5R)-4-((tert- oxy)-5-(2-hydroxyethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)- dione (12.0 g, 22.4 mmol) was dissolved in MeCN (150 mL) and IBX (12.5 g, 44.7 mmol) was added. After stirring at 80 °C for 1 hour, the mixture was filtered through a Celite pad and concentrated to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 60% EtOAc / PE, to give the title compound (8.4 g, 86%) as a white solid. ES / MS (m / z): 385.1 (M+1). Preparation 8 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxypropyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0113] Methylmagnesium3M in THF) was added to a solution of 2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2- yl)acetaldehyde (3.20 g, 7.32 mmol) in THF (100 mL) at 0 °C under nitrogen. The solution was stirred at 0 °C for 1 hour. The reaction mixture was quenched by the addition of aqueous saturated ammonium chloride (150 mL) and then diluted with water (100 mL). The aqueous was extracted with EtOAc (200 mL). The organic layer was washed with brine (100 mL), dried over sodium sulfate, filtered, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 20% to 60% EtOAc / PE to give the title compound (1.8 g, 61%) as a colorless oil. ES / MS (m / z): 401.1 (M+1). Preparation 9 1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dioneO

[0114] Silver nitrate (15.2 g, (14.6 g, 15.9 mL, 119 mmol), and 4,4'-(chloro(phenyl) g, 59.7 mmol) were added to a solution of 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-((RS)-2-hydroxypropyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (5.90 g, 11.9 mmol) in DCM (120 mL) at 0 °C. The ice bath was removed, and the mixture was stirred at ambient temperature for 16 hours under nitrogen. The reaction mixture was quenched by the addition of water (200 mL) and the aqueous was then extracted three times with EtOAc (200 mL). The organic layers were then combined, washed 2 times with brine (200 ml), dried over sodium sulfate, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 70% EtOAc / PE, to give the title compound (11 g, 74%) as an orange solid. ES / MS (m / z): 725.3 (M+23). Preparation 10 1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0115] 1M in THF) was added to a solution of 1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (10.0 g,9.70 mmol) in THF (100 mL) and the stirred at ambient temperature under nitrogen. After 30 minutes of stirring, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / PE, to give the title compound (5.0 g, 80%) as a white solid. ES / MS (m / z): 587.2 (M-1). Preparation 11 1-((2R,3R,4R,5R)-5-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione and 1-((2R,3R,4R,5R)-5-((R)-2- (bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3-methoxytetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione

[0116] 1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (5.00 g, 8.15 mmol) was purified by SFC (Condition: CO2-EtOH; Column: Daicel Chiralpak IBN 250 mm X 50 mm X 10 um; Begin B: 50%; End B: 50%; Gradient Time(min): 150 min; Flowrate: 200mL / min.) to give 1- ((2R,3R,4R,5R)-5-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (first eluting isomer, 1.85 g, 38%) and 1-((2R,3R,4R,5R)-5-((R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (second eluting isomer, 3.01 g, 61%) as white solids. ES / MS (m / z): 587.2 (M-1). Preparation 12 (2R,3R,4R,5R)-2-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-5-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0117] 1-((2R,3R,4R,5R)-5- methoxy)propyl)-4-hydroxy- 3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.85 g, 3.14 mmol) was dissolved in a mixture of DIEA (1.22 g, 1.64 mL, 9.43 mmol) in DCM (25 mL) and the solution was chilled to 0 °C in an ice bath. 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (1.64 g, 1.54 mL, 6.91 mmol) was then added dropwise and the ice bath was removed. The mixture was stirred at ambient temperature for 1.5 hours and the solvent was then removed under vacuum at 25 °C to leave a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / hexanes (eluents contain 1% TEA) to give the title compound (1.54 g, 62%) as an off-white foam.31P NMR (DMSO) δ 148.90, 148.80; ES / MS (m / z): 789.4 (M+1). Preparation 13(2R,3R,4R,5R)-2-((R)-2-(bis(4- (phenyl)methoxy)propyl)-5-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl (2-cyanoethyl)

[0118] 1-((2R,3R,4R,5R)-5-((R)-2-(bis(4--4-hydroxy- 3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.01 g, 5.11 mmol) was dissolved in a mixture of DIEA (1.98 g, 2.67 mL, 15.3 mmol) in DCM (40 mL) and the solution was chilled to 0 °C in an ice bath. 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (2.66 g, 2.51 mL, 11.2 mmol) was then added dropwise and the ice bath was removed. The mixture was stirred at ambient temperature for 1.5 hours and the solvent was then removed under vacuum at 25 °C to leave a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / hexanes (eluents contain 1% TEA) to give the title compound (3.8 g, 95%) as an off-white foam.31P NMR (DMSO) δ 149.06, 148.99; ES / MS (m / z): 789.4 (M+1). Preparation 14 N-(1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)acetamide

[0119] 2, 4, 6- was added to a solution of 1-((2R,3R,4R,5R)-5-(2- propyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (115 g, 163 mmol), triethylamine (33.1 g, 327 mmol), and DMAP (40.0 g, 327 mmol) dissolved in ACN (800 ml) at 0 °C. The ice bath was removed, and the reaction mixture was stirred at ambient temperature for 2 hours. Ammonium hydroxide (101 g, 834 mmol, 29%) was then added, and the mixture was allowed to stir at ambient temperature for an additional two hours. After stirring was complete, the solution was diluted with water (500 ml), extracted twice with ethyl acetate (500 ml), and the combined organic was washed with brine (500 ml), dried over sodium sulfate and concentrated under reduced pressure to give a brown oil. The oil was dissolved in pyridine (1.22 L), acetic anhydride (76.4 g, 249 mmol) was added, and the solution was stirred at ambient temperature for 2 hours. The solution was then diluted with water (500 ml), extracted twice with ethyl acetate (500 ml), and the combined organic was washed with brine (500 ml), dried over sodium sulfate, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography using 20% to 50% EtOAc / PE to give the title compound (100 g, 27%). ES / MS (m / z): 744.4 (M+1).

[0120] Conversion of uracil nucleobase to cytosine may also be conducted generally as described in WO2019 / 217459. Preparation 15 N-(1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide

[0121] TBAF (700 g, 269 ((2R,3R,4R,5R)-5-(2- (bis(4-methoxyphenyl) - oxy)-3- methoxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (100 g, 134 mmol) dissolved in THF (700 ml) and the mixture was stirred at ambient temperature for 2 hours. The mixture was then diluted with water (500 ml), extracted twice ethyl acetate (500 ml), and the combined organic was washed with brine (500 ml), dried over sodium sulfate, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography using 20% to 100% EtOAc / PE to give the title compound (67 g, 79%) as an off- white solid. ES / MS (m / z): 630.2 (M+1). Preparation 16 N-(1-((2R,3R,4R,5R)-5-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide and N-(1- ((2R,3R,4R,5R)-5-((R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide

[0122] N-(1-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (67 g, 106 mmol) waspurified by SFC (Condition: CO2-IPA:ACN; Daicel Chiralpak IM 250 mm X 25 mm X 10 um; Begin B: 50%; End B: 50%) to give N-(1-((2R,3R,4R,5R)-5-((S)-2-(bis(4- methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)acetamide (17.3 g, 26%) and N-(1-((2R,3R,4R,5R)-5-((R)-2-(bis(4- methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)acetamide (32.0 g, 48%) as off-white solids. ES / MS (m / z): 630.3 (M+1). EXAMPLE 1 Synthesis of oligonucleotides

[0123] The sense strand and antisense strand of an RNAi agent including at least one PrON can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.

[0124] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP- HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a duplex.

[0125] In other embodiments disclosed herein are RNAi agents having a formula of R-L-D (Formula XI), wherein R is a double stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the antisense strand is complementary to target mRNA, wherein L is a bond or a linker, and wherein D is a delivery moiety of Formula IX:

[0126] In some an RNAi agent. In some embodiments, an AGT RNAi agent may include one PrON, or two PrON. In some embodiments, an AGT RNAi agent may include at least one PrON and may comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex RNA. In one embodiment, the sense strand includes at least one PrON and comprises a first nucleic acid sequence of SEQ ID NO: 42, and a second nucleic acid sequences of SEQ ID NO: 43. In one embodiment, the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 42, and a second nucleic acid sequences of SEQ ID NO: 44. In one embodiment, the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 45, and a second nucleic acid sequences of SEQ ID NO: 47. In one embodiment, the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 45, and a second nucleic acid sequences of SEQ ID NO: 48. In one embodiment, the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 45, and a second nucleic acid sequences of SEQ ID NO: 49. In one embodiment, the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 45, and a second nucleic acid sequences of SEQ ID NO: 50.

[0127] In some embodiments, the RNAi agent disclosed herein comprises a linker. In further embodiments, R is conjugated to Formula IX via a linker. In other further embodiments R is conjugated to Formula IX via a direct bond. In further embodiments, the linker comprises a linker of Formula XII having connection points A and B or the linker comprises Formula XIII having connection points X and Z, and wherein: BO a. the RNAi agent to Formula XII at connection point A and Formula XII is conjugated to a phosphate group at connection point B, and the phosphate group is conjugated to R; or b. the RNAi agent comprises Formula IX conjugated to Formula XIII at connection point X and Formula XIII is conjugated to a phosphate group at connection point Z, and the phosphate group is further conjugated to R.

[0128] In an embodiment in which the RNAi agent of Formula XI includes a linker, and the linker is of, or comprises, Formula XIII, -L-D has the structure of Formula XIV:wherein dsRNA R is connected to Formula XIV at connection point Z via a phosphate or phosphate analog.

[0129] The delivery moiety of Formula IX may be made by synthetic steps and schemes as described in United States Patent Application No.18 / 573,794, filed December 22, 2023, or International Patent Publication No. WO2022 / 271806, each of which is incorporated herein by reference in its entirety.

[0130] Briefly, for the synthesis of GalNAc-conjugated sense strands, a sense strand may first ne synthesized using standard phosphoramidite chemistry. A stock solution of the desired GalNAc ligand-NHS ester (10 mmol / L in acetonitrile; 1 eq) is prepared. Borate buffer (10% v / v; 20x) isadded to oligonucleotide sense strand in an tube, then GalNAc ligand (5 eq) is added. The mixture is shaken at ambient temperature for 16 hours. After this time, the mixture is transferred to a 15 mL falcon tube, ammonium hydroxide (28 mass%) is added, and the mixture is shaken at ambient temperature for 2 hours. The ammonia is then removed in vacuo. The residue is purified by ion-exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15%MeCN / 20mM NaH2PO4, 1M NaBr; 35-55%B over 5 CV at 8 mL / min, column temperature 60 °C. The desired fractions are pooled and desalted by spin-filtration using an Eppendorf centrifuge or desalting column. After desalting, the material is recovered and OD and volume are measured to obtain concentration.

[0131] Alternatively, and particularly in the case of a delivery moiety including a linker of Formula XIII, oligo synthesis may be conducted on a MerMade™ 12 instrument using phosphoramidite chemistry. Sense strands are synthesized from the prefunctionalized GalNAc solid support and antisense strands are synthesized using standard support preloaded with the first nucleotide of the oligo sequence. Antisense strands are prepared using CPG with a universal support or linker. Oligos are cleaved and deprotected using concentrated ammonium hydroxide solution (28% by mass) and purified by ion exchange chromatography using conditions described above. Such deprotection likewise converts the OAc precursor groups of the galactose moieties of the delivery moiety to hydroxyl groups. Cleavage from the CPG also yields a 3’ hydroxyl at the 3’ end of the newly-made RNA strand. Desalting, annealing, and endotoxin testing are conducted.

[0132] To generate the siRNA duplexes of a sense and antisense strand, the following procedures may be performed. To a tube (such as a Falcon tube) containing oligonucleotide sense strand- GalNAc conjugate, the corresponding antisense oligonucleotide (1 eq) is added and vortexed for 10 seconds before spin-filtering through 100K MWCO Amicon filter unit to remove particulates. The filtrate is recovered and concentrated in vacuo on a Genevac evaporator. The residue is reconstituted in 1x PBS, filtered through 0.2 µ filter, and OD and volume are measured to obtain concentration.

[0133] In further embodiments of the RNAi agents disclosed herein, the sense strand and antisense strand each independently comprise one or more modified internucleotide linkages, and each modified internucleotide linkage is a phosphorothioate linkage. In further embodiments, the sense strand and antisense strand each independently comprise four phosphorothioate linkages. In still further embodiments, the two terminal nucleotides at each of the 5’ and 3’ ends of each of the sense and antisense strand are phosphorothioate linkages.

[0134] In certain embodiments, the 5’ of the antisense strand comprises a phosphate group or a phosphate analog.

[0135] In some embodiments, at least one of the sense strand and the antisense strand can include at least one abasic moiety of the formula: , wherein “5’” and “3’” indicate the

[0136] In some embodiments, at least one of the sense strand and the antisense strand can include at least one inverted abasic (iAb) moiety of the formula: , wherein “5’” and “3’” indicate the

[0137] In some embodiments, an extended nucleic acid (or exNA) moiety can be included in at least one of the sense strand and the antisense strand. The exNA-containing nucleic acid can be prepared as described in WO2021 / 195533. The exNA has the following formula: . As used in experiments herein, theis uracil.

[0138] An abasic moiety or an iAb moiety may be present at the 5’ end of an oligonucleotide, or at the 3’ end of an oligonucleotide, or may substitute for a nucleotide at an internal position of the oligonucleotide. Abasic and iAb moieties may be considered modified nucleotides, wherein one of the modifications is a lack of a nucleobase.

[0139] An endotoxin test may be performed using a Limulus amebocyte lysate on an Endosafe®- nexgen PTS instrument.2 In vitro knockdown of mouse HMGCR in wild type mouse primary hepatocytes (MPH) with PrON modified GalNAc-conjugated HMGCR siRNA

[0140] Mouse primary hepatocytes (MPH) were freshly isolated from wildtype mouse and plated on collagen-I 96-well plates (Corning, Part #: 354649) at 15,000 cells per well and various concentration of GalNAc-conjugated HMGCR siRNA (that is, an RNAi agent conjugated with Formula XIV as shown above on the 5’ end of the sense strand) with chemical modification were added in 10 µL of 10X siRNA duplexes in Opti-MEM per well. Dose response experiments were done at 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM final siRNA duplex concentration. Cells were incubated for 24-48 hours prior to RNA isolation.

[0141] Treated cells were lysed directly into the 96 well cell plate and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research, Part #: R1052). The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen, Part #: A39110) and using the following steps in a thermocycler: 37oC for 30 minutes, 95oC for 5 minutes, and 4oC hold. Polymerase Chain Reaction (PCR) was performed via TaqMan® RT PCR (Life Technologies, Part #: 4326708) using the following cycles temperatures and times: 50oC for 2 minutes, 95oC for 10 minutes, 40 cycles of 95oC for 15 seconds and 60oC for 1 minute.

[0142] The mouse HMGCR levels were normalized to mouse Rplp0 (Life Technologies) and represented the relative knockdown of mouse HMGCR mRNA expression as compared to vehicle-treated control cells. IC50 values were calculated using a 4-parameter fit model using XLFit®. Results are presented in Table 5 below. Table 5: IC50 (nM) and Percent knockdown of mouse HMGCR expression in wildtype mouse primary hepatocytes (MPH) dsRNA % maximumdsRNA O: IC ( % maximum N 50 nM) KDLC-MS quantification of PrON modified siRNA in liver tissue

[0143] To study the durability and the target exposure of RNAi agents containing PrON, approximately 100 mg frozen liver were homogenized in lysis buffer (Thermo Scientific) at 100 mg / mL tissue concentration in a Geno / Grinder®. Pooled blank liver homogenates were spiked with standards to generate calibration standards. The standard curve range in liver was 4.48– 50,000 ng / g. All the lysed liver samples were loaded onto equilibrated WAX SPE 96-well plate cartridges (WatersTM). The SPE cartridges were washed two-times with 0.2 mL of 50 mM ammonium acetate in 50:50 (v / v) water / Acetonitrile (pH 5.5). Samples were eluted with 0.075 mL of 50:50 (v / v) 0.1 M ammonium bicarbonate / Acetonitrile (pH = 9.5). The eluents were dried under nitrogen for 1–2 h at 40°C. The dried samples were resuspended in 200 μL of 10:90 (v:v) dimethylsulfoxide:water. A volume of 20 μL was subjected to LC-HRMS (Liquid chromatography-High resolution mass spectrometry) for qualification analysis. The LC–MS mobile phases used were as follows: Mobile Phase A: 15mM TEA (Triethanolamine), 100mM HFIP (1,1,1,3,3,3-Hexafluoroisopropanol) in water; Mobile Phase B: 15mM TEA, 100mM HFIP in methanol. The typical gradient started with 5% mobile phase B, progressed to 40% B over 4 min and increased to 98% B in 0.25 min. The column was washed with mobile phase B for 0.5 min. The column was re-equilibrated with 5% B for 1.0 min. The flow rate was 0.25 mL / min; column temperature was 80°C. The Exploris™ mass spectrometer was set at full scan monitoring mode and negative ionization mode. dsRNA NO: 22, which does not include PrON, was testedalongside dsRNA NO: 4. dsRNA NO: 4 has same sequence as dsRNA NO: 22, except in dsRNA NO: 4, the nucleotides in the 3’ overhang are replaced by PrONs.

[0144] The results in Table 6 below show that no degradation products of the PrON-containing dsRNA (dsRNA NO: 4) are detected even at 8 weeks. In contrast, measurable degradation products of the dsRNA which does not include PrON (dsRNA NO: 22) are seen.

[0145] In Table 7, exposure in liver to control dsRNA NO: 22 (which does not contain PrON) was evaluated compared to four PrON-containing dsRNA (dsRNA Nos: 4, 5, 6, and 7). At two weeks, exposure values for the PrON-containing dsRNAs are at least five-fold higher than control, and at least ten-fold higher at eight weeks. Table 6 Percentage of AS degradation products detected dsRNA 2w 8w dTable 7 Exposure ng / g dsRNA 2 8EXAMPLE 4 HMGCR siRNA in vivo methods Example 4A: Mouse two week experiment

[0146] GalNAc-siRNAs were tested in male C57bl / 6 mice (Taconic farms). Body weight of mice were measured within one week prior to dosing test article or vehicle. Mice were assignedto groups with similar body weights. Either or GalNac-siRNA test article, at a dose such as 5mg / kg, was administered subcutaneously to mice. Seven days post subcutaneous injection, blood was collected from all mice. Fourteen days post subcutaneous injection mice were euthanized under isoflurane anesthesia, and blood was collected. Liver was collected from the mice and frozen in liquid nitrogen. Livers were homogenized in TriZolTM(Invitrogen) using Lysing Matrix D bead tubes on a FastPrep-24TM(MP Bio). Chloroform was added and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated on column using PureLinkTMPro96 Total RNA purification kit (Invitrogen) according to manufacturer’s protocol and quantified on a NanoDropTM(ThermoFisher). Equal amounts (1ug) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) on Mastercycler® Nexus (Eppendorf). Thermocycler settings are 25°C for 10 min, 37°C for 2 hrs, then 85°C for 5 min. Template cDNA was combined with TaqmanTMUniversal Master Mix and Assays on Demand primer / probesets and RT-PCR was performed on the QuantStudioTMPro7 (ThermoFisher) with the following parameters: 50°C for 2min, 95°C for 10min then 40 cycles of 95°C for 15sec and 60°C for 1min. Fold changes (FC) were calculated as follows: the CT value of mouse Rplp0 was subtracted from CT value of mouse HMGCR to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (average of PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base 2 of the negative delta CT value. Percent mRNA remaining was calculated multiplying the fold change by 100. Data is shown in Table 8. Example 4B: Mouse eight week experiment

[0147] GalNAc-siRNAs were tested in male C57bl / 6 mice (Taconic farms). Mice were weighed within approximately one week prior to dosing test article or vehicle. Body weights of mice were measured and mice were assigned to groups with similar body weight. At the beginning of study (Day 0) either PBS or test article GalNac-siRNA, at doses such as 1.75 or 10mg / kg were administered subcutaneously to mice. At 2 weeks post siRNA administration, three or four mice from each group were euthanized under isoflurane anesthesia, blood was collected, and serum was stored at -80°C for future analysis. Liver was collected from the mice and frozen in liquid nitrogen. Blood was collected from the remaining mice (n=6 / group) at 2, 4, and 6 weeks post siRNA administration under isoflurane anesthesia. Serum was prepared from blood and stored at -80°C for potential future analysis. At 8 weeks post siRNA administration, the remaining mice were euthanized under isoflurane anesthesia. Blood and liver were collected from mice. Liverswere homogenized in TriZolTM(Invitrogen) Lysing Matrix D bead tubes on a FastPrep- 24TM(MP Bio). Chloroform was added and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated on column using PureLinkTMPro96 Total RNA purification kit (Invitrogen) according to manufacturer’s protocol and quantified on a NanoDropTM(ThermoFisher). Equal amounts (1ug) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) on Mastercycler® Nexus (Eppendorf). Thermocycler settings are 25°C for 10 min, 37°C for 2 hrs, then 85°C for 5 min. Template cDNA was combined with TaqmanTMUniversal Master Mix and Assays on Demand primer / probesets and RT-PCR was performed on the QuantStudioTMPro7 (ThermoFisher) with the following parameters: 50°C for 2min, 95°C for 10min then 40 cycles of 95°C for 15sec and 60°C for 1min. Fold changes (FC) were calculated as follows: the CT value of mouse Rplp0 was subtracted from CT value of mouse HMGCR to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (average of PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base 2 of the negative delta delta CT value. Percent mRNA remaining was calculated multiplying the fold change by 100. Data is shown in Table 8. Example 4C: Sterol Panel

[0148] In a 2.0mL TrueTaper® SiliGuard plate was added 1mL of 50:50 dichloromethane:methanol.30 µL of serum or plasma was added to the wells.10µL of internal standard mix was added at a concentration of 1 µg / mL. Then 20 µL of 10 mg / mL Butylated hydroxytoluene solution was added to minimize oxidation. The plate was vortexed for several seconds and a heat sealing aluminum foil was added to the plate. The plate was placed in a sonication bath set to 35°C for 10 minutes. After sonication, the plate was centrifuged at 3300 RPM for 10 minutes at room temperature. The samples were transferred to a new 2.0mL TrueTaper® SiliGuard plate without disrupting the white protein layer at the bottom of the plate. 100uL of 10N KOH solution was added to the new plate and sealed with heat sealing aluminum foil. The plate was placed in the sonication bath, set to 35°C for 90 minutes.500uL of DPBS was added to each well and vortexed for several seconds. The plate was centrifuged and 300uL of the bottom dichloromethane layer was removed to 96-well V-bottom plate with SiliGuard. The plate was dried under a stream of nitrogen.75uL of 90% methanol was added to all wells. The plate was vortexed on a mixer for 5 minutes at 900 RPM after heat sealing. Plates were ran on aAgilent Infinity II autosampler coupled to a 6500 Triple Quad system and all analytes were monitored using multiple reaction monitoring (MRM). Table 8: 2 week 8 week 2 week 8 week Day 0 2 week 4 week 6 week 8 week 10mg / kg 10mg / kg 10mg / kg 10mg / kg 10mg / kg 10mg / kg 10mg / kg 10mg / kg 10mg / kg Mean rol 6In vitro knockdown of human Ang8 in Hep3B cells with GalNAc-conjugated Ang8 siRNA by transfection

[0149] For Hep3B (ATCC, Part #: HB-8064) cells, transfection was carried out by adding 24.7 µL of Opti-MEMTM(Gibco, Part #: 31985062) plus 0.3 µL of LipofectamineTMRNAiMAX (Life Technologies, Part #: 13778-150) per well to 25 µL of each siRNA duplex to an individual well in a 96-well plate (Costar, Part #3596). RNAi duplexes were conjugated with Formula XIV as shown above on the 5’ end of the sense strand. The mixture was then incubated at room temperature for 20 minutes. Fifty microliters of complete growth media without antibiotic containing Hep3B at 400,000 cells / mL were then added to the siRNA mixture. 10 points CRC was done at siRNA duplex final concentration starts at 20nM, then 1:5 serial dilution. Cells were incubated for 24 hours prior to qPCR.

[0150] Treated cells were washed with cold 1xPBS and lysed directly into the 96 well cell plate using TaqManTMFast Advanced Cell-to-CTTMkit (Life Technologies, Part #: AM1729). cDNA was synthesized using the following steps in a thermocycler: 37oC for 30 minutes, 95oC for 5 minutes, and 4oC hold. Polymerase Chain Reaction (PCR) using the following cycles temperatures and times: 50oC for 2 minutes, 95oC for 20 seconds, 40 cycles of 95oC for 1 seconds and 60oC for 20 seconds.

[0151] The human Ang8 levels were to human RPLP0 (Life Technologies) and represented the relative knockdown of human Ang8 mRNA expression as compared to vehicle- treated control cells. Table 9: Hep3B / RNAiMAX dsRNA NO: %KD at 20 nM IC50 (nM)6 ANGPTL8 Liver AAV in CETPA1

[0152] Eight GalNAc-siRNAs were tested in male mice transgenic for human cholesterol ester transfer protein (CETP) and human apolipoprotein A1 (Taconic farms). Mice intended for test article administration were dosed by retro-orbital injection with two adeno-associated virus (AAV) vectors. One vector contained a plasmid with an albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7) (Vector BioLabs). The second vector contained a plasmid with an albumin promoter and the mouse codon optimized sequence of human ANGPTL3 (NP_055310.1) (Vector BioLabs). Four control mice were dosed by retro-orbital injection with an AAV containing a plasmid with an albumin promoter and the firefly luciferase to establish control levels of serum triglyceride. Blood was collected from mice 4 weeks post AAV administration. This was considered the baseline blood collection. Serum was prepared from blood, and triglycerides were measured utilizing a COBAS clinical chemistry analyzer (Roche). ANGPTL3 / 8 serum protein was measured by an in-house ELISA (Meso Scale Diagnostics). Mice were assigned to nine groups with similar serum triglyceride and ANGPTL3 / 8 levels and body weight (n=9). Each group was then randomized again into two groups of n=6 (used for durability) and n=3 (used for maximum knock down at 2 weeks post dose) using the same parameters. Either PBS (Control) or test article GalNac-siRNA at a dose of 1.75mg / kg was administered subcutaneously to mice. The four control mice with the firefly luciferase AAV were untreated with PBS or test articles. At 2 weeks post siRNA administration, n=3 mice were euthanized under isoflurane anesthesia and liver (frozen in liquid nitrogen) and blood were collected to determine maximum knockdown. Blood was collected from the n=6 mice at 3, 6, 9, and 12 weeks post siRNA administration under isoflurane anesthesia. At 15 weeks post siRNA administration, remaining mice were euthanized under isoflurane anesthesia and liver (frozen in liquid nitrogen) and blood was collected. Serum was prepared from blood and triglycerides at all timepoints were measured as before. Livers were homogenized in TriZol (Invitrogen) using Lysing Matrix D bead tubes on a FastPrep-24 (MP Bio). Chloroform was added and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated on columns using RNeasy 96 Universal Tissue kit (Qiagen) according to manufacturer’s protocol and quantitated on the NanoDrop (ThermoFisher). Equal amounts (1ug) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) on Mastercycler Nexus (Eppendorf). Thermocycler settings were 25°C for 10 min, 37°C for 2 hrs, then 85°C for 5 min. Template cDNA was combined with Taqman Universal Master Mixand Assays on Demand primer / probesets Angptl8 Hs00218820_m1 and mouse RPLP0 Mm01974474_gH) and RT-PCR was performed on the ABI Quant Studio 7 Pro (Applied Biosystems) with the following parameters: 50°C for 2 min, 95°C for 10 min then 40 cycles of 95°C for 15 sec and 60°C for 1 min. Fold changes (FC) were calculated as follows: the CT value of mouse Rplp0 was subtracted from CT value of human ANGPTL8 to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base 2 of the negative delta delta CT value. Percent knock down (%KD) was calculated by subtracting FC from one and multiplying by 100 and shown in Table 11. Triglyceride and mRNA fold change data was analyzed for a statistically significant difference from the PBS group at corresponding timepoint using ANOVA and Dunnett’s method where p < 0.05 was considered statistically significant (SAS Institute) Data is shown in Table 10. Table 10: Triglyceride reduction at indicated week following a 1.75 mg / kg subcutaneous dose expressed as percent change vs PBS (numbers in bold indicate p<0.05 by ANOVA with Dunnett’s) Weeks post dose ek 6Table 11: hAngplt8 AAV gene knockdown data measured at 2- and 15-weeks post dose (RPLP0 as housekeeping gene) dsRNA NO mRNA KD mRNA KD30 54 27 93 43Single crystal X-ray diffraction (SCXRD) of N-(1-((2R,3R,4R,5R)-5-((S)-2-(bis(4- methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)benzamide

[0153] Single Crystal X-Ray Diffraction (SCXRD) was carried out on a Bruker D8 VENTURE dual-source diffractometer (serial K209362) equipped with a three-circle goniometer and a Bruker PHOTON II CPAD detector, providing an average area resolution of 7.41 pixels / mm2. CuKα radiation (λ = 1.54178 Å) was exclusively used for these experiments. X-rays were generated by an Incoatec IµS 3.0 microfocus sealed X-ray tube equipped with mirror optics, operating at 50 kV and 1 mA. Data acquisition employed φ and ω scans, with samples mounted on a MiTeGen Nylon Loop using Grade A Immersion Oil, achieving a resolution of up to 0.83 Å; this resolution cutoff is highly dependent on the quality of the measured crystal (volume, disorder, twinning). The experiment was performed at low temperature using an open flow N2cold stream produced by an Oxford Cryostream 800, with all low temperature measurements fixed at 100 K.

[0154] Data collection was managed with APEX5 v2023.9-2, while cell refinement and data reduction were conducted using SAINT V8.40B (Bruker AXS LLC, 2019). An empirical multi- scan absorption correction was applied using TWINABS-2012 / 1 (twinned data). Structure solution was achieved using SHELXT 2018 / 2 (Sheldrick, 2018), followed by refinement with SHELXL-2019 / 1 (Sheldrick, 2019) through the ShelXle visualization software (C.B. Huebschle, 2011). Final publication materials were prepared using Mercury (CCDC, 2024).

[0155] An initial lot of crystals of the title compound crystals were completely redissolved in an arbitrary quantity of MeOH, ensuring full macroscopic dissolution. The test tube was sealed with parafilm, with three small pinholes punctured into the film to allow slow evaporation over the course of two weeks. The newly grown crystals were still microscopically twinned but provided diffraction of sufficient quality for structure solution and refinement. The measured crystal wasclear light colorless in color, plate-like in with an approximate size of 10 x 30 x 130 micron (mounted on a 200 micron nylon loop using Grade A immersion oil).

[0156] X-ray intensity data were measured using Cu Kα radiation (λ = 1.54178 Å). A total of 6067 frames were collected. The total exposure time was 17.99 hours. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a triclinic unit cell yielded a total of 39196 reflections to a maximum θ angle of 68.26° (0.83 Å resolution), of which 3529 were independent (average redundancy 11.107, completeness = 98.7%, R = 11.57%, R = 6.45%) and 2627 (74.44%) were greater than 2σ(F). The final cell constants of a = 4.7875(3) Å, b = 11.8986(9) Å, c = 17.5308(12) Å, α = 102.794(5) °, β = 90.084(5) °, γ = 91.950(5) °, volume = 973.24(12) Å, are based upon the refinement of the XYZ-centroids of 6014 reflections above 20 σ(I) with 5.165° < 2θ < 135.9°. Data were corrected for absorption effects using the Multi-Scan method (TWINABS). The ratio of minimum to maximum apparent transmission was 0.686. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.8950 and 0.9910. The final anisotropic full-matrix least-squares refinement on F with 574 variables converged at R1 = 10.54%, for the observed data and wR2 = 28.98% for all data. The goodness-of-fit was 1.071. The largest peak in the final difference electron density synthesis was 0.411 e- / Å and the largest hole was -0.344 e- / Å with an RMS deviation of 0.091 e- / Å. On the basis of the final model, the calculated density was 1.383 g / cm and F(000), 430 e-. Crystal data and structure refinement parameters are listed in Table 12. Table 12: Crystal data and structure refinement parameters Chemical formula C19.5H25N3O6.5α 102.794(5) β 90.084(5)

[0157] Analysis of the structure showed that the DMT protecting group was cleaved, leaving the ribose 5’-hydroxyl group unprotected. A MeOH molecule was disordered over three positions, with refined occupancies of 0.54167, 0.26416, and 0.19417.

[0158] The refined Flack parameter of -0.6(6) indicated that despite high resolution data (0.83 Å) an absolute structure could not be determined. However, the molecule contains five chiral centers, four of which belong to a ribose sugar moiety with a well-established R configuration.Based on this the relative stereoconfiguration the studied center was assigned as an S through correlation and chemical reasoning. Example 8 AGT RNAi agents including PrON

[0159] In one embodiment, provided herein are AGT RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, and wherein the antisense strand is complementary to a region of AGT mRNA (SEQ ID NO: 41). AGT Transcript: SEQ ID NO:41 (NM_000029.4) 1 gaagaagctg ccgttgttct gggtactaca gcagaagggt atgcggaagc gagcacccca 61 gtctgagatg gctcctgccg gtgtgagcct gagggccacc atcctctgcc tcctggcctg 121 ggctggcctg gctgcaggtg accgggtgta catacacccc ttccacctcg tcatccacaa 181 tgagagtacc tgtgagcagc tggcaaaggc caatgccggg aagcccaaag accccacctt 241 catacctgct ccaattcagg ccaagacatc ccctgtggat gaaaaggccc tacaggacca 301 gctggtgcta gtcgctgcaa aacttgacac cgaagacaag ttgagggccg caatggtcgg 361 gatgctggcc aacttcttgg gcttccgtat atatggcatg cacagtgagc tatggggcgt 421 ggtccatggg gccaccgtcc tctccccaac ggctgtcttt ggcaccctgg cctctctcta 481 tctgggagcc ttggaccaca cagctgacag gctacaggca atcctgggtg ttccttggaa 541 ggacaagaac tgcacctccc ggctggatgc gcacaaggtc ctgtctgccc tgcaggctgt 601 acagggcctg ctagtggccc agggcagggc tgatagccag gcccagctgc tgctgtccac 661 ggtggtgggc gtgttcacag ccccaggcct gcacctgaag cagccgtttg tgcagggcct 721 ggctctctat acccctgtgg tcctcccacg ctctctggac ttcacagaac tggatgttgc 781 tgctgagaag attgacaggt tcatgcaggc tgtgacagga tggaagactg gctgctccct 841 gatgggagcc agtgtggaca gcaccctggc tttcaacacc tacgtccact tccaagggaa 901 gatgaagggc ttctccctgc tggccgagcc ccaggagttc tgggtggaca acagcacctc 961 agtgtctgtt cccatgctct ctggcatggg caccttccag cactggagtg acatccagga 1021 caacttctcg gtgactcaag tgcccttcac tgagagcgcc tgcctgctgc tgatccagcc 1081 tcactatgcc tctgacctgg acaaggtgga gggtctcact ttccagcaaa actccctcaa 1141 ctggatgaag aaactatctc cccggaccat ccacctgacc atgccccaac tggtgctgca 1201 aggatcttat gacctgcagg acctgctcgc ccaggctgag ctgcccgcca ttctgcacac 1261 cgagctgaac ctgcaaaaat tgagcaatga ccgcatcagg gtgggggagg tgctgaacag 1321 catttttttt gagcttgaag cggatgagag agagcccaca gagtctaccc aacagcttaa 1381 caagcctgag gtcttggagg tgaccctgaa ccgcccattc ctgtttgctg tgtatgatca1441 aagcgccact gccctgcact tcctgggccg ccgctgagca cagcatgagg 1501 ccagggcccc agaacacagt gcctggcaag gcctctgccc ctggcctttg aggcaaaggc 1561 cagcagcaga taacaacccc ggacaaatca gcgatgtgtc acccccagtc tcccaccttt 1621 tcttctaatg agtcgacttt gagctggaaa gcagccgttt ctccttggtc taagtgtgct 1681 gcatggagtg agcagtagaa gcctgcagcg gcacaaatgc acctcccagt ttgctgggtt 1741 tattttagag aatgggggtg gggaggcaag aaccagtgtt tagcgcggga ctactgttcc 1801 aaaaagaatt ccaaccgacc agcttgtttg tgaaacaaaa aagtgttccc ttttcaagtt 1861 gagaacaaaa attgggtttt aaaattaaag tatacatttt tgcattgcct tcggtttgta 1921 tttagtgtct tgaatgtaag aacatgacct ccgtgtagtg tctgtaatac cttagttttt 1981 tccacagatg cttgtgattt ttgaacaata cgtgaaagat gcaagcacct gaatttctgt 2041 ttgaatgcgg aaccatagct ggttatttct cccttgtgtt agtaataaac gtcttgccac 2101 aataagcctc caaaaa

[0160] Exemplary unmodified oligonucleotide sequences of AGT RNAi agents are provided in Table 13, and modified sequences including those incorporating at least one PrON are provided in Table 14. Table 13: Exemplary sense strand and antisense strand sequences of AGT RNAi agents Starting target 9.4 52 52Table 14: Modified sense strand and antisense strand sequences of AGT siRNA agents dsRNA strand Modified Sequence 5' to 3' SEQ ID NO: NO:Abbreviations – “m” indicates 2’-OMe; “f” 2’-fluoro; “*” indicates phosphorothioate linkage; “P” indicates 5’-phosphate; “VP” 5’-vinylphosponate; “S” means the sense strand; “AS” means the antisense strand; rPrON indicates PrON with R stereocenter in the internucleotide linkage; sPrON indicates PrON with S stereocenter in the internucleotide linkage. Example 9: In vitro knockdown of human AGT in AAV-AGT mouse primary hepatocytes (MPH) with GalNAc-conjugated AGT siRNA

[0161] Mouse primary hepatocytes (MPH) were freshly isolated from AAV-AGT humanized mouse and plated on collagen-I 96-well plates (Corning, Part #: 354649) at 15,000 cells per well and various concentration of AGT siRNA with chemical modification conjugated to GalNAc (that is, the compound of Formula IX as disclosed herein) were added in 10 µL of 10X siRNA duplexes in Opti-MEMTMper well. Dose response experiments were done at 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM final siRNA duplex concentration. Cells were incubated for 24-48 hours prior to RNA isolation.

[0162] Treated cells were lysed directly into the 96 well cell plate and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research, Part #: R1052). The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen, Part #: A39110) and using the following steps in a thermocycler: 37oC for 30 minutes, 95oC for 5 minutes, and 4oC hold. Polymerase Chain Reaction (PCR) was performed via TaqManTMRT PCR (Life Technologies, Part #: 4326708) using the following cycles temperatures and times: 50oC for 2 minutes, 95oC for 10 minutes, 40 cycles of 95oC for 15 seconds and 60oC for 1 minute.

[0163] The human AGT levels were normalized to mouse Rplp0 (Life Technologies) and represented the relative knockdown of human AGT mRNA expression as compared to vehicle- treated control cells. IC50 values were calculated using a 4-parameter fit model using XLFit®. Table 15: Percent knockdown of human AGT expression in AAV-AGT mouse primary hepatocytes (MPH) Duplex IC50 (nM) % maximum

[0164] All PrON-containing AGT RNAi demonstrated strong knockdown of AGT mRNA in vitro.LISTING SEQ Sequence (5' to 3') ID NOSEQ Sequence (5' to 3') ID NOSEQ Sequence (5' to 3') ID NOSEQ Sequence (5' to 3') ID NO

Claims

CLAIMS We claim:

1. An oligonucleotide, or a salt thereof, comprising the formula: , wherein:R1is selected from the group consisting of H, OR2, NH2, SR2, F, and Cl, wherein R2is selected from the group consisting of H, C1-C20 alkyl; and B is a nucleobase.

2. The oligonucleotide, or salt thereof, of claim 1, comprising the formula: ,wherein: each X1is an independently selected nucleotide; each X2 is an independently selected nucleotide; each Q is PO2X5wherein X5is O or S, or, when n is 0, Q may be H; and m and n are each independently selected from any integer from 0 to 40.

3. The oligonucleotide or salt thereof of claim 1 or claim 2, wherein the oligonucleotide comprises the formula:.

4. The oligonucleotide or of claims 1-3, wherein the oligonucleotide comprises the .

5. The oligonucleotide orof claims 1-3, wherein the oligonucleotide comprises the formula: .

6. The oligonucleotide or salt thereof of any one of claims 1 to 3, wherein the oligonucleotide comprises the formula: ,wherein each B is independently selected.

7. The oligonucleotide or salt thereof of claim 6, wherein the oligonucleotide comprises the formula:B .

8. The oligonucleotide or the oligonucleotide comprises the formula: .

9. The oligonucleotide orthe oligonucleotide comprises the formula: .

10. The oligonucleotide orthe oligonucleotide comprises the formula:.

11. The oligonucleotide or claims 1-10, wherein X5is O.

12. The oligonucleotide or salt thereof of any one of claims 1-10, wherein X5is S.

13. The oligonucleotide or salt thereof of claim 1, wherein the oligonucleotide comprises the formula: O B .

14. The oligonucleotide or salt thereof of claim 1, claim 2, or claim 13, wherein the oligonucleotide comprises the formula: X1m O B .

15. The oligonucleotide or salt thereof of claim 13 or claim 14, wherein the oligonucleotide comprises the formula:.

16. The oligonucleotide or any one of claims 1, 2, or 13-15, wherein the oligonucleotide comprises the formula: .

17. The oligonucleotide or salt thereof of any one of claims 1, 2, or 13-15, wherein the oligonucleotide comprises the formula: .

18. The oligonucleotide or salt thereof of any one of claims 1-3, 6, or 13, wherein the oligonucleotide comprises the formula: ,wherein each B is independently selected.

19. The oligonucleotide or salt of claim 18, wherein the oligonucleotide comprises the formula: , wherein each B is20. The oligonucleotide or salt thereof of claim 18, wherein the oligonucleotide comprises the formula: ,wherein each B is 21. The oligonucleotide or salt thereof of claim 18, wherein the oligonucleotide comprises the formula:, wherein each B is 22. The oligonucleotide or salt thereof of claim 18, wherein the oligonucleotide comprises the formula: .

23. The oligonucleotide or salt thereof of claim 2 or claim 14, wherein m and n are independently selected from any whole number from 0 to 20.

24. The oligonucleotide or salt thereof of any one of claims 1-23, wherein the oligonucleotide has one of a phosphate, a hydroxyl, a phosphate analog, an abasic moiety, and an inverted abasic moiety at its 5’ end.

25. The oligonucleotide or salt thereof of any one of claims 1-24, wherein each B is independently selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5- dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

26. The oligonucleotide or salt thereof of any one of claims 1-25, wherein each B is independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

27. The oligonucleotide or salt thereof of any one of claims 1-26, wherein each B is uracil.

28. The oligonucleotide or salt thereof of any one of claims 1-27, wherein X5is O.

29. The oligonucleotide or salt thereof of any one of claims 1-27, wherein X5is S.

30. The oligonucleotide or salt thereof of any one of claims 1-29, wherein the oligonucleotide is 15 to 30 nucleotides in total length.

31. The oligonucleotide or salt thereof of any one of claims 1-30, wherein the oligonucleotide is 18 to 30 nucleotides in total length.

32. The oligonucleotide or salt thereof of any one of claims 1-31, wherein the oligonucleotide is 18 to 23 nucleotides in total length.

33. The oligonucleotide or salt thereof of any one of claims 1-32, comprising at least one modification selected from the group consisting of 2’-O-methoxy, 2’-O-methyl, 2’- fluoro, phosphorothioate, 2’-deoxy, abasic moiety, inverted abasic moiety, R-PrON, and S-PrON.

34. The oligonucleotide or salt thereof of claim 33, wherein the oligonucleotide comprises 2’-fluoro modified nucleotide at the following positions: a. 2, 3, 7, 14, and 16 from the 5’ end; b. 2, 5, 7, 14, and 16 from the 5’ end; c. 2, 3, 8, 14, and 16 from the 5’ end; d. 2, 5, 8, 14, and 16 from the 5’ end; ore. 2, 6, 14, and 16 from the 5’ the oligonucleotide.

35. The oligonucleotide or salt thereof of claim 34, wherein the oligonucleotide comprises 2’ O-methyl modified nucleotides at all other positions.

36. A double-stranded RNA molecule or salt thereof comprising: a first oligonucleotide comprising an oligonucleotide of any one of claims 1-35; and a second oligonucleotide of 15 to 30 nucleotides in total length; wherein the first oligonucleotide and the second oligonucleotide form a duplex region.

37. The double-stranded RNA molecule or salt thereof of claim 36, wherein the first oligonucleotide is an antisense strand, and the second oligonucleotide is a sense strand.

38. The double-stranded RNA molecule or salt thereof of claim 36, wherein the first oligonucleotide is a sense strand, and the second oligonucleotide is an antisense strand.

39. The double-stranded RNA molecule or salt thereof of any one of claims 36-38, wherein duplex regions is 15 to 23 base pairs in length.

40. The double-stranded RNA molecule or salt thereof of any one of claims 36-39, wherein antisense strand has a 3’ overhang of 1 or 2 nucleotides.

41. The double-stranded RNA molecule or salt thereof of any one of claims 36-40, wherein the sense strand comprises a delivery moiety at its 3’ end.

42. The double stranded RNA molecule or salt thereof of claim 41, wherein the delivery moiety is of the formula:, wherein E is a sense strand.

43. The double standed RNA molecule or salt thereof of claim 41 or claim 42, wherein the linker comprises formula: B wherein the delivery moietypoint A, and the duplex RNA is conjugated to the linker at connection point B.

44. The double standed RNA molecule or salt thereof of claim 41 or claim 42, wherein the linker comprises formula: Zwherein the delivery moiety at connection point X, and the duplex RNA is conjugated to the linker at connection point Z.

45. A compound of the formula:, wherein B is a nucleobase and Y is group.

46. The compound of claim 45, which is of formula: .

47. The compound of claim 45 or claim 46, wherein the organic protecting group is methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert- butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS).

48. The compound of any one of claims 45-47, wherein the organic protecting group is TBDMS.

49. The compound of any one of claims 45-48, wherein B is selected from the group consisting of hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

50. The compound of any one of claims 45-49, wherein B is uracil.

51. A compound of the formula:, wherein B is a nucleobase and Y is group.

52. The compound of claim 51, of the formula: .

53. The compound of claim 51 or claim 52, of formula: .

54. The compound of claim 51 or.

55. The compound of any onewherein the organic protecting group is methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert- butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS).

56. The compound of any one of claims 51 to 55, wherein the organic protecting group is TBDMS.

57. The compound of any one of 51 to 56, wherein B is selected from the group consisting of hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

58. The compound of any one of claims 51 to 57, wherein B is uracil.

59. A compound of formula: , wherein B is a nucleobase, Y1isa first organic protecting group, and Y2 is a second organic protecting group.

60. The compound of claim 59, of formula: .

61. The compound of claim 59 or claim 60, of formula: .

62. The compound of claim 59 or claim 60, of formula:.

63. The compound of any one wherein Y1 is H.

64. The compound of any one of claims 59-63, wherein the first organic protecting group and the second organic protecting group are each independently selected from the group consisting of methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS).

65. The compound of any one of claims 59-64, wherein Y2is DMT.

66. The compound of any one of claims 59-62 and 64-65, wherein Y1is TBDMS.

67. The compound of any one of claims 59-66, wherein B is selected from the group consisting of hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

68. The compound of any one of claims 59-67, wherein B is uracil.

69. A compound of formula:wherein B is a nucleobase and Y group.

70. The compound of claim 69, of formula: .

71. The compound of claim.

72. The compound of claim 69, of formula:wherein B is a nucleobase and Y group.

73. The compound of any one of claims 69, 70, and 72, of formula: .

74. The compound of any72, of formula: .

75. The compound of any one of claims 69-73, wherein the organic protecting group is methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert- butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS).

76. The compound of any one of 69-75, wherein the organic protecting group is DMT.

77. The compound of any one of claims 69-76, wherein B is selected from the group consisting of hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

78. The compound of any one of claims 69-77, wherein B is uracil.

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