Oligonucleotide fragments and methods of preparing RNAi agents using same

Abstract

Disclosed are intermediate compounds (i.e., oligonucleotide fragments), or pharmaceutically acceptable salts thereof, useful in the preparation of RNAi agents. In addition, the present invention discloses methods of making single stranded oligonucleotides by linking two or more intermediate compounds described herein by a hybrid chemical-enzymatic pathway.

Description

[0001] References to related applications

[0002] This application claims all Paris Convention benefits and priority to U.S. Provisional Patent Application No. 63 / 434,661, filed on December 22, 2022.

[0003] Reference to sequence listings submitted electronically

[0004] The present invention is submitted with a sequence listing in ST.26 XML format. The sequence listing is provided as a file named "30019_US_PRI", which was created on October 24, 2023 and is 35 kilobytes (kb). The sequence listing information in ST.26 XML format is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates generally to biology, chemistry, and medicine, and more particularly to methods for synthesizing oligonucleotides via hybrid chemical-enzymatic pathways. Background Art

[0006] Oligonucleotides are used in various biological and biochemical applications. Of concern herein are the uses of oligonucleotides as therapeutic agents, such as RNA activation (RNAa), RNA editing (RNAe), and RNA interference (RNAi). The widespread use of oligonucleotides increases the demand for their rapid, inexpensive, and efficient synthesis methods.

[0007] Oligonucleotides can be synthesized by a variety of methods known in the art, particularly by solid phase synthesis by repeated coupling of nucleoside phosphoramidites. See, for example, Beaucage and Caruthers (1981) Tetrahedron Letters 22: 1859-1862; McBride and Caruthers (1983) Tetrahedron Letters 24: 245-248; Sinha et al. (1984) Nucleic Acids Res. 12: 4539-4557; and Beaucage and Iyer (1992) Tetrahedron 48: 2223-2311.

[0008] However, alternative methods for preparing oligonucleotides and their intermediates are needed to achieve pharmaceutically elegant production with commercially desirable purity. Likewise, efficient methods and stable intermediates are needed to efficiently provide oligonucleotides with fewer purification steps. Summary of the Invention

[0009] To address this need, the present invention describes nucleotide-based intermediate compounds (i.e., oligonucleotide fragments), as well as methods for preparing single-stranded (ss) oligonucleotides by ligating multiple oligonucleotide fragments herein, and ultimately preparing double-stranded (ds) therapeutic oligonucleotides, such as RNAi agents.

[0010] Regarding intermediate compounds, the present invention describes an oligonucleotide fragment having a nucleotide sequence selected from any one of SEQ ID NOs: 5 to 37.

[0011] Regarding the method, the present invention describes a method for preparing a nucleic acid having a nucleotide sequence of SEQ ID NO: 1, the method comprising the steps of ligating a combination of the following oligonucleotide fragments having a nucleotide sequence selected from the group consisting of:

[0012] (a) SEQ ID NOs: 5, 6 and 7;

[0013] (b) SEQ ID NOs: 5, 10, and 11;

[0014] (c) SEQ ID NOs: 5, 12, and 13;

[0015] (d) SEQ ID NOs: 7, 14, and 15;

[0016] (e) SEQ ID NOs: 7, 18 and 19;

[0017] (f) SEQ ID NOs: 7, 22, and 23; and

[0018] (g) SEQ ID NOs: 5, 26 and 27.

[0019] Furthermore, the present invention describes a method for preparing a nucleic acid having the nucleotide sequence of SEQ ID NO: 2, the method comprising the step of ligating a combination of the following oligonucleotide fragments having a nucleotide sequence selected from the group consisting of:

[0020] (a') SEQ ID NOs: 8 and 9;

[0021] (b') SEQ ID NOs: 16 and 17;

[0022] (c') SEQ ID NOs: 20 and 21; and

[0023] (d') SEQ ID NOs: 24 and 25.

[0024] The above method may include the additional step of annealing SEQ ID NO: 1 and SEQ ID NO: 2 to form an RNAi agent that modulates expression of the apolipoprotein (a) gene (LPA).

[0025] Furthermore, the present invention describes a method for preparing a nucleic acid having the nucleotide sequence of SEQ ID NO: 3, the method comprising the step of ligating a combination of the following oligonucleotide fragments having a nucleotide sequence selected from the group consisting of:

[0026] (a) SEQ ID NOs: 7, 32 and 33.

[0027] Furthermore, the present invention describes a method for preparing a nucleic acid having the nucleotide sequence of SEQ ID NO: 4, comprising the step of ligating a combination of the following oligonucleotide fragments having a nucleotide sequence selected from the group consisting of:

[0028] (a') SEQ ID NOs: 34 and 35.

[0029] The above method may include the additional step of annealing SEQ ID NO: 3 and SEQ ID NO: 4 to form an RNAi agent that modulates expression of the angiopoietin-like protein 3 gene (ANGPTL3).

[0030] In any of the above methods, the ligation can be mediated by an enzyme. In some cases, the enzyme is a ligase, such as a naturally occurring ligase or a non-naturally occurring ligase. In other cases, the ligase is a DNA ligase. In some cases, the ligase is an RNA ligase.

[0031] The advantage of the method herein includes process improvement, for example, the shorter fragments initially produced via solid phase oligonucleotide synthesis (SPOS) allow increased purity and higher productive rate. In the case of shorter fragments, more approach flexibility can be obtained to incorporate modified nucleotides, and the fragment structure can be redesigned to solve more difficult link segments.

[0032] Advantages of the methods herein include improved control strategies for impurities during synthesis, which may include improved detection and characterization of impurities at the fragment stage and improved final impurity profiles of crude oligonucleotide duplexes.

[0033] Advantages of the methods herein include improved final duplex purity, as certain classes of fragment impurities are discarded based on their inability to participate in the ligation step or their reduced adherence to complementary principles during fragment self-assembly in the annealing step.

[0034] Advantages of the methods herein include a reduction in the unit operations required to generate oligonucleotide duplexes, as a single step for forming double-stranded material from fragment building blocks allows the reduction of downstream operations to a single chromatography and ultrafiltration (whereas conventional methods require separate steps for each strand).

[0035] Advantages of the methods herein include that the synthesis of shorter fragments via SPOS allows for reduced washing cycles and reagent volumes, resulting in a lower process mass intensity (PMI).

[0036] Advantages of the methods herein include that in the case of shorter fragment intermediates, the impact of synthesis failures during fragment manufacture is reduced, resulting in a reduction in overall cost and manufacturing cycle impact.

[0037] Advantages of our approach include shorter fragment intermediates that are more amenable to new synthetic manufacturing platforms, and enzymatic assembly of oligonucleotide duplexes allows for the introduction of other innovative technologies for downstream unit operations.

[0038] Advantages of the methods herein include flexibility in supply chain and logistics by using several independent segments for the manufacturing process.

[0039] Advantages of the methods herein include the additional flexibility of the supply chain resulting from utilizing the same delivery platform for the sequences to enable the design of conserved fragments having the same nucleotide sequence composition.

[0040] Advantages of the methods herein include that using parallel manufacturing of segments can reduce manufacturing cycle time by parallel processing of segments.

[0041] Advantages of the methods herein include that the current good manufacturing practice (cGMP) enzymatic ligation step can be performed in a solvent-free facility under water-based conditions without the need for specialized equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Advantages, effects, features and objects other than those set forth above will become more apparent upon consideration of the following detailed description, which makes reference to the following drawings, in which:

[0043] Figure 1 Shown are diagrams of the structures of exemplary RNAi agents (SEQ ID NOs: 1 and 2) that have a nicked tetracyclic structure and modulate LPA expression.

[0044] Figure 2 Shown are diagrams of the structures of exemplary RNAi agents (SEQ ID NOs: 3 and 4) that have a nicked tetraloop structure and modulate ANGTPL3 expression. DETAILED DESCRIPTION

[0045] Overview

[0046] International Patent Application Publication No. WO 2022 / 032288 describes an RNAi agent (e.g., LPA-3291-M1) that can be used to alleviate, prevent, and / or treat diseases, disorders, and / or conditions associated with LPA expression (i.e., reducing the levels of LPA mRNA and Apo(a) protein, as well as reducing Lp(a) levels). The RNAi agent includes an N-acetylgalactosamine (GalNAc) ligand to target the RNAi agent to the asialoglycoprotein receptor (ASGPR).

[0047] International Patent Application Publication No. WO 2021 / 188795 describes an RNAi agent (e.g., GalXC-1412) that can be used to alleviate, prevent, and / or treat diseases, disorders, and / or conditions associated with ANGPTL3 expression (i.e., reducing the levels of ANGPTL3 mRNA and ANGPTL3 protein). The RNAi agent includes a GalNAc ligand to target the RNAi agent to the ASGPR.

[0048] Abbreviations and definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of incretin analogs, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0050] Furthermore, unless the context clearly requires that one and only one of the element be present, referring to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present. Thus, the indefinite article "a" or "an" generally means "at least one."

[0051] Certain abbreviations used herein are defined as follows:

[0052] "A" refers to adenosine; "G" refers to guanosine; "U" refers to uridine; "C" refers to cytosine; "fX" refers to 2'-fluoro-nucleotides (e.g., fA, fG, fU, fC); "fX" refers to 2'-fluoro-nucleotides (e.g., fA, fG, fU, fC); s ” refers to 2′-fluoro nucleotides linked via phosphorothioate bonds; “mX” refers to 2′-O-methyl nucleotides (eg, mA, mG, mU, mC); “mX s ” refers to 2'-O-methyl nucleotides (e.g. mA) linked via phosphorothioate bonds. S 、mA S 、mU S 、mC S ); "adem A" refers to 2'-O-GalNAc-modified adenosine (sodium salt equivalent), which has the following structure:

[0053]

[0054] “ACN” refers to acetonitrile (C2H3N); “ANGPTL3” refers to angiopoietin-like protein 3 gene; “ASGPR” refers to asialoglycoprotein receptor; “cGMP” refers to current good manufacturing practice; “CV” refers to column volume; “Da” refers to Dalton; “DCA” refers to dichloroacetic acid (C2H2Cl2O2); “DEA” refers to diethylamine (C4H 11 N); "DIPEA" refers to N,N-diisopropylethylamine (C8H 19 N); “Dmt” refers to 4,4'-dimethoxytrityl; “DNA” refers to deoxyribonucleic acid; “ds” refers to double-stranded; “DTT” refers to 1,4-dithiothreitol; “EDTA” refers to ethylenediaminetetraacetic acid (C 10 H 16 N2O8); "EtOH" refers to ethanol (C2H6O); "ETT" refers to 5-(ethylthio)-1H-tetrazole (C3H6N4S); "GalNAc" refers to N-acetylgalactosamine, which has the following structure:

[0055]

[0056] "HFIP" refers to hexafluoroisopropanol (C3H2F6O); "HPLC" refers to high performance liquid chromatography; "LPA" refers to apolipoprotein (a) gene; and "meMOP mU" refers to 4'-O-monomethylphosphonate-2'-O-methyluridine (sodium salt equivalent), which has the following structure:

[0057]

[0058] “MWCO” refers to molecular weight cutoff; “NAD” refers to nicotinamide adenine dinucleotide; “p” refers to 5′ phosphate cap; “PA” refers to phosphoramidite; “PO” refers to phosphodiester; “PS” refers to phosphorothioate; “PMI” refers to process mass intensity; “RISC” refers to RNA-induced silencing complex; “RNA” refers to ribonucleic acid; “RNAa” refers to RNA activation; “RNAe” refers to RNA editing; “RNAi” refers to RNA interference; “SPOS” refers to solid-phase oligonucleotide synthesis; “ss” refers to single-stranded; “TFF” refers to tangential flow filtration; “T m ” refers to melting temperature; “UPLC” refers to ultraperformance liquid chromatography; “UF / DF” refers to ultrafiltration and diafiltration; and “UV” refers to ultraviolet light.

[0059] Certain definitions used herein are defined as follows:

[0060] As used herein, "about" means within a statistically significant range of one or more values ​​(e.g., a stated concentration, length, molecular weight, pH, pressure, sequence identity, time range, temperature, or volume). This value or range may be within an order of magnitude of the stated value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The permissible variations encompassed by "about" will depend on the particular system under study and will be readily understood by those skilled in the art.

[0061] As used herein, "annealing" or the like means hybridizing to a complementary oligonucleotide in a sequence-specific manner. Annealing conditions will depend on the melting temperature (T m ) and will be apparent to those skilled in the art. For example, the annealing temperature may be lower than the T of the hybridizing oligonucleotide. m Alternatively, the annealing temperature can be close to the T of the hybridized oligonucleotide. m (e.g., + / - about 1°C, 2°C, or 3°C). The annealing temperature is typically no greater than the T of the hybridizing oligonucleotide. m About 10℃ higher.

[0062] As used herein, "antisense strand" or "guide strand" means an ss oligonucleotide that is complementary to a region of a target sequence (e.g., a target sequence in an mRNA). Similarly, and as used herein, "sense strand" or "follower strand" means an ss oligonucleotide that is complementary to a region of the antisense strand.

[0063] As used herein, "asialoglycoprotein receptor" or "ASGPR" means a bipartite C-type lectin formed by a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2).

[0064] As used herein, a "chemically synthesized" oligonucleotide refers to an oligonucleotide produced by using a chemical reaction, e.g., without the use of an enzyme. Methods for chemically synthesizing oligonucleotides such as RNA molecules are known in the art, specifically the chemical synthesis methods described in Verma & Eckstein (1998) or as described herein. In general, dsRNA constructs can be synthesized using SPOS (see, e.g., Usman et al. (1987) J. Am. Chem. Soc. 109:7845-7854, U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,008,400; 6,111,086; 6,117,657; 6,353,098; 6,362,323; 6,437,117; and 6,469,158; and Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441; see also Beaucage and Caruthers (1981) Tetrahedron Letters 22: 1859-1862; McBride and Caruthers (1983) Tetrahedron Letters 24: 245-248; Sinha et al. (1984) Nucleic Acids Res. 12: 4539-4557 and Beaucage and Iyer (1992) Tetrahedron 48: 2223-2311); and International Patent Application Publication Nos. 2005 / 070859 and 2012 / 157723.

[0065] As used herein, "complementary" means a structural relationship between two nucleotides that allows the two nucleotides to form base pairs with each other (e.g., on two opposing nucleic acids or on opposing regions of an ss nucleic acid (e.g., a hairpin). For example, the purine nucleotides of an oligonucleotide that are complementary to the pyrimidine nucleotides of the opposing oligonucleotide can base pair together by forming hydrogen bonds with each other. Complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two oligonucleotides can have regions of multiple nucleotides that complement each other to form a complementary region, as described herein.

[0066] As used herein, "deoxyribonucleotide" means a nucleotide that has a hydrogen instead of a hydroxyl group at the 2' position of its pentose sugar, as compared to a ribonucleotide. A modified deoxyribonucleotide has a modification or substitution of one or more atoms other than the hydroxyl group at the 2' position, including modifications or substitutions in or of a nucleobase, sugar, or phosphate group.

[0067] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" means an oligonucleotide in double-stranded form. The complementary base pairing of the double-stranded region of a ds oligonucleotide can be formed between the antiparallel sequences of nucleotides of a covalently separated nucleic acid. Similarly, the complementary base pairing of the double-stranded region of a ds oligonucleotide can be formed between the antiparallel sequences of nucleotides of a covalently linked nucleic acid. In addition, the complementary base pairing of the double-stranded region of a ds oligonucleotide can be formed by folding (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides of the base-paired ss nucleic acid. A ds oligonucleotide can include two covalently separated nucleic acids that completely form a double strand with each other. However, a ds oligonucleotide can include two covalently separated nucleic acids that partially form a double strand (e.g., have an overhang at one or both ends). A ds oligonucleotide can include antiparallel sequences of partially complementary nucleotides and therefore can have one or more mismatches, which can include internal mismatches or terminal mismatches.

[0068] As used herein, "double-stranded" and "double-stranded region" with respect to nucleic acids (e.g., oligonucleotides) means a structure formed by complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two covalently separated nucleic acids or by a single folded strand (e.g., via a hairpin).

[0069] As used herein, "enzymatic ligation," "enzymatically ligating," and the like mean that the linkage between two adjacent nucleotides is enzymatically formed, wherein the linkage may be a naturally occurring phosphodiester (PO) linkage or a modified linkage including, but not limited to, a phosphorothioate (PS) linkage or a phosphoramidite (PA) linkage.

[0070] As used herein, "enzymatically synthesized" oligonucleotides are oligonucleotides having modifications resulting from the reaction of a nucleic acid with an enzyme, including naturally occurring enzymes or non-naturally occurring enzymes (e.g., kinases, ligases, methyltransferases, nickases, nucleases, phosphatases, sulfurylases, and recombinases). Accordingly, and as used herein, "enzymatic" modifications refer to those modifications resulting from the reaction of a nucleic acid with an enzyme, including naturally occurring and non-naturally occurring enzymes.

[0071] " Ligase " used herein means an enzyme that catalyzes the connection (i.e., covalent connection) of two oligonucleotides, for example, by forming a PO bond between the 3' end of an oligonucleotide (or fragment) and the 5' end of the same or another oligonucleotide (or fragment) to connect. These enzymes are often referred to as DNA ligases or RNA ligases, and are generally members of the enzyme class EC 6.5 defined by the International Union of Biochemistry and Molecular Biology (International Union of Biochemistry and Molecular Biology) (i.e., ligases for forming phosphate bonds). In addition, the ligase herein can connect an unmodified oligonucleotide to another unmodified oligonucleotide, can connect an unmodified oligonucleotide to a modified oligonucleotide (i.e., a modified 5' oligonucleotide is connected to an unmodified 3' oligonucleotide and / or an unmodified 5' oligonucleotide is connected to a modified 3' oligonucleotide), and / or can connect a modified oligonucleotide to another modified oligonucleotide.

[0072] As used herein, a "modified ligase" or "non-naturally occurring ligase" means a ligase that differs from a naturally occurring (ie, wild-type) ligase by one or more amino acid residues.

[0073] As used herein, "modified nucleotides" refer to nucleotides that have one or more chemical modifications when compared to a corresponding reference nucleotide selected from the group consisting of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. The modified nucleotides may be non-naturally occurring nucleotides. The modified nucleotides may have, for example, one or more chemical modifications in their sugars, core bases, and / or phosphate groups. Additionally or alternatively, the modified nucleotides may have one or more chemical moieties that are bound to the corresponding reference nucleotides.

[0074] As used herein, "N-acetylgalactosamine" or "GalNAc" means 2-(acetylamino)-2-deoxy-D-galactose or a derivative thereof, which can be directly or indirectly conjugated to the oligonucleotides herein to target the oligonucleotides to ASGPR.

[0075] As used herein, "nicked tetraloop structure" refers to the structure of a RNAi agent characterized by separated sense and antisense strands, wherein the sense strand has a region complementary to the antisense strand, and wherein at least one strand (typically the sense strand) has a tetraloop configured to stabilize adjacent stem regions formed within the at least one strand.

[0076] As used herein, "non-naturally occurring" means that the oligonucleotide, nucleic acid, peptide, polypeptide, or protein is modified in a manner that is not found or identical to that found in nature but is produced or derived synthetically (i.e., engineered, recombinant, or modified by human manipulation).

[0077] As used herein, "nucleotide" means an organic compound having a nucleoside (a nucleobase such as adenine, cytosine, guanine, thymine, or uracil; and a pentose such as ribose or 2'-deoxyribose) and a phosphate group. "Nucleotide" can serve as a monomeric unit of nucleic acids such as deoxyribonucleic acid (DNA) oligonucleotides and ribonucleic acid (RNA) oligonucleotides.

[0078] As used herein, "oligonucleotide" means a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide may be ss or ds. An oligonucleotide may or may not have a double-stranded region. Examples of oligonucleotides include, but are not limited to, antisense oligonucleotides (ASOs), Dicer substrate interfering RNA (DsiRNA), microRNA (miRNA), short hairpin RNA (shRNA), and small interfering RNA (siRNA).

[0079] As used herein, "overhang" refers to a terminal non-base-paired nucleotide resulting from a strand or region extending beyond the end of a complementary strand (whereby a strand or region forms a duplex with the complementary strand). In some embodiments, the overhang comprises one or more unpaired nucleotides extending from the double-stranded region at the 5' end or 3' end of a ds oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of a ds oligonucleotide.

[0080] As used herein, "pharmaceutically acceptable buffer" means any standard pharmaceutical buffer known to those skilled in the art.

[0081] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar containing 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 a nucleobase, sugar, or phosphate group.

[0082] As used herein, "iRNA," "iRNA agent," "RNAi," "RNAi agent," and "RNA interference agent" mean an oligonucleotide that contains RNA and mediates targeted cleavage of RNA transcripts through RNA interference, such as through the RNA-induced silencing complex (RISC) pathway. The RNAi agent may have a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex. In some cases, the sense and antisense strands of the RNAi agent may be 21-23 nucleotides in length. Alternatively, the sense and antisense strands may be longer, such as 25-36 nucleotides in length, in which case the longer nucleotide sequence is processed first by the Dicer enzyme. RNAi agents direct sequence-specific degradation of mRNA through RNA interference. RNAi agents attenuate, inhibit, modulate, or reduce gene expression (e.g., ANGPTL3 or LPA expression herein) in a cell, tissue, organ, system, or individual.

[0083] As used herein, a "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide linkages (eg, PO linkages or PS linkages). A strand may have two free ends (eg, a 5' end and a 3' end).

[0084] " Targeting ligand " used herein means a chemical part that promotes oligonucleotides (such as RNAi agents herein) to enter tissues or cells. It can be a compound (such as an amino sugar, carbohydrate, cholesterol, lipid or polypeptide) that selectively binds to a paired compound (such as a receptor) of a target tissue or cell and can be conjugated with another substance to target another substance to a target tissue or cell. For example, in order to target an oligonucleotide to a specific target cell or tissue, a targeting ligand can be conjugated with an oligonucleotide herein. The targeting ligand can selectively bind to a cell surface receptor. Therefore, when conjugated with an oligonucleotide, the targeting ligand helps to deliver the oligonucleotide to a specific cell by selective binding to receptors expressed on the cell surface and by endosome internalization of a complex comprising an oligonucleotide, a targeting ligand and a receptor by the cell. In addition, the targeting ligand can be conjugated to the oligonucleotide via a joint, which is cut after or during cellular internalization to release the oligonucleotide from the targeting ligand in the cell.

[0085] Composition

[0086] Oligonucleotide fragments

[0087] The present invention describes oligonucleotide fragments (ie, intermediate compounds) having the following exemplary sequences / structures:

[0088] Intermediate compound 1:

[0089] 5'mU S-mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU 3′ (SEQ ID NO: 5).

[0090] Intermediate compound 2:

[0091] 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 6).

[0092] Intermediate compound 3:

[0093] 5'p-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3′ (SEQ ID NO: 7).

[0094] Intermediate compound 4:

[0095] 5'p-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3′ (SEQ ID NO: 8).

[0096] Intermediate compound 5:

[0097] 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC-mC-fA 3′ (SEQ ID NO:9).

[0098] Intermediate compound 6:

[0099] 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC 3' (SEQ ID NO: 10)

[0100] Intermediate compound 7:

[0101] 5'p-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC3' (SEQ ID NO: 11).

[0102] Intermediate compound 8:

[0103] 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC 3' (SEQ ID NO: 12).

[0104] Intermediate compound 9:

[0105] 5'p-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 13).

[0106] Intermediate compound 10:

[0107] 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU 3′ (SEQ ID NO: 14).

[0108] Intermediate compound 11:

[0109] 5'p-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 15).

[0110] Intermediate compound 12:

[0111] 5'p-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3′ (SEQ ID NO: 16).

[0112] Intermediate compound 13:

[0113] 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG 3′ (SEQ ID NO: 17).

[0114] Intermediate compound 14:

[0115] 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC 3′ (SEQ ID NO: 18).

[0116] Intermediate compound 15:

[0117] 5'p-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 19).

[0118] Intermediate compound 16:

[0119] 5'p-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3′ (SEQ ID NO: 20).

[0120] Intermediate compound 17:

[0121] 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA 3′ (SEQ ID NO: 21).

[0122] Intermediate compound 18:

[0123] 5'mU S -mU-mG-mC-mC-mA-mA-fG 3′ (SEQ ID NO: 22).

[0124] Intermediate compound 19:

[0125] 5'p-fC-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 23).

[0126] Intermediate compound 20:

[0127] 5'p-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3′ (SEQ ID NO: 24).

[0128] Intermediate compound 21:

[0129] 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC 3′ (SEQ ID NO: 25).

[0130] Intermediate compound 22:

[0131] 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG 3' (SEQ ID NO: 26).

[0132] Intermediate compound 23:

[0133] 5'p-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 27).

[0134] Intermediate compound 24:

[0135] 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA

[0136] -GalNAc]-[ademA-GalNAc]-mG-mG 3′ (SEQ ID NO: 28).

[0137] Intermediate compound 25:

[0138] 5'mC-mU-mG-mC 3' (SEQ ID NO: 29).

[0139] Intermediate compound 26:

[0140] 5'p-mG-mC-mA-mA S -mG S -mG 3′ (SEQ ID NO: 30).

[0141] Intermediate compound 27:

[0142] 5'p-mA-mG-mC-fU-mU-mG 3' (SEQ ID NO: 31).

[0143] Intermediate compound 28:

[0144] 5'mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU 3′ (SEQ ID NO: 32).

[0145] Intermediate compound 29:

[0146] 5'p-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG 3' (SEQ ID NO: 33).

[0147] Intermediate compound 30:

[0148] 5'p-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S -mG 3′ (SEQ ID NO: 34).

[0149] Intermediate compound 31:

[0150] 5'[MePhosphonate-4O-mU S ]-fG S -fU S -fA-fU-mA-fA-mC-mC-fU 3′ (SEQ ID NO: 35).

[0151] Intermediate compound 32:

[0152] 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA

[0153] -GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3′ (SEQ ID NO: 36).

[0154] Intermediate compound 33:

[0155] 5' loop-(mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-

[0156] [ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC)3' (SEQ ID NO: 37).

[0157] As described above and in detail below, certain combinations of oligonucleotide fragments herein can be linked together and used to prepare a first RNAi agent having a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2. Other combinations of oligonucleotide fragments herein can be linked together and used to prepare a second RNAi agent having a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4.

[0158] RNAi agents

[0159] As described above, certain combinations of oligonucleotide fragments (ie, intermediate compounds) herein can be linked to form the RNAi agents herein.

[0160] First RNAi agents that can be formed from certain oligonucleotide fragments herein include Figure 1 The first RNAi agent comprises a sense strand having the nucleotide sequence of SEQ ID NO: 1 and an antisense strand having the nucleotide sequence of SEQ ID NO: 2. The first RNAi agent is used to alleviate, prevent and / or treat diseases, disorders and / or conditions associated with LPA expression.

[0161] Second RNAi agents that can be formed from certain oligonucleotide fragments herein include Figure 2 The second RNAi agent is used to alleviate, prevent and / or treat diseases, disorders and / or conditions associated with ANGPTL3 expression.

[0162] method

[0163] The method may include the steps described herein, and these steps may (but not necessarily) be performed in the order described. However, other orders are also contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping in time and / or individually or in multiple repetitions. Furthermore, the method may include additional unspecified steps.

[0164] Standard solid-phase oligonucleotide synthesis of intermediate compounds

[0165] Oligonucleotide fragments herein (i.e., intermediate compounds) can be prepared via standard oligonucleotide synthesis methods as known in the art, such as SPOS. Using standard phosphoramidite chemistry technique (amidite chemistry technique), SPOS construction is achieved by sequential coupling using an automated oligonucleotide synthesizer. See, for example, Paredes et al. (2018) Synthesis of Therapeutic Oligonucleotides, Springer Nature Singapore Pte Ltd., Paredes et al. (2017) Comprehensive Medicinal Chemistry III, pp. 233-279. Automated nucleic acid synthesizers including DNA / RNA synthesizers can be purchased from, for example, Applied Biosystems (Foster City, CA), BioAutomation (Irving, TX) and GE Healthcare Life Sciences (Pittsburgh, PA); See also International Patent Application Publication No. 2005 / 070859 and No. 2012 / 157723.

[0166] Those skilled in the art will appreciate that additional methods and / or techniques for synthesizing oligonucleotides can be used. In addition, various synthesis steps can be carried out in an alternative order or sequence to obtain the desired compound. Other synthetic chemical transformations, protecting groups (such as for the hydroxyl, amino group, etc. present on the base) and protecting group methods (protection and deprotection) for synthesizing oligonucleotides are known in the art, and are described in, for example, Larock, " Comprehensive Organic Transformations ", VCH Publishers (1989); Greene and Wuts, " Protective Groups in Organic Synthesis ", 2nd edition, John Wiley & Sons (1991); Fieser and Fieser, " Fieser & Fieser's Reagents for Organic Synthesis ", John Wiley & Sons (1994); and Paquette, " Encyclopedia of Reagents for Organic Synthesis ", John Wiley & Sons (1995).

[0167] In brief, during SPOS, nucleoside phosphoramidite structural unit can be added to solid support to prepare oligonucleotide with desired length and order in continuous circulation.Each circulation is made up of several chemical reactions: detritylation, coupling, oxidation or thiolation and end-blocking.After the synthesis of given oligonucleotide is completed, it is discharged from solid support and can remove protecting group in the same step.

[0168] For detritylation, the initial resin can be swollen with ACN and subsequently treated with 10% DCA in toluene.

[0169] The crude oligonucleotide fragments produced can then be purified using chromatography to separate the full-length oligonucleotide and its associated impurities. Finally, pure oligonucleotide solid material is obtained by desalting and further freeze-drying.

[0170] Hybrid solid-phase and enzymatic ligation to form RNAi agents

[0171] Certain combinations of oligonucleotide fragments herein (i.e., intermediate compounds) prepared by the SPOS described above can be combined according to methods known to those skilled in the art to obtain RNAi agents of SEQ ID NOs: 1 and 2. Thus, the methods described herein can include synthesizing independent oligonucleotide fragments and then ligating such fragments to form RNAi agents of SEQ ID NOs: 1 and 2.

[0172] For example, an exemplary method for preparing the oligonucleotide of SEQ ID NO: 1 includes at least the step of ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 5, 6, and 7. In some cases, the fragments may be ligated in the following order: SEQ ID NO: 5 to SEQ ID NO: 6 to SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0173] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 5, 10, and 11. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 5 to SEQ ID NO: 10 to SEQ ID NO: 11 (i.e., from the 5' end to the 3' end).

[0174] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 5, 12, and 13. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 5 to SEQ ID NO: 12 to SEQ ID NO: 13 (i.e., from the 5' end to the 3' end).

[0175] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 7, 14, and 15. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 14 to SEQ ID NO: 15 to SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0176] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 7, 18, and 19. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 18 to SEQ ID NO: 19 to SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0177] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 7, 22, and 23. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 22 to SEQ ID NO: 23 to SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0178] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 5, 26, and 27. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 5 to SEQ ID NO: 26 to SEQ ID NO: 27 (i.e., from the 5' end to the 3' end).

[0179] Likewise, an exemplary method of making the oligonucleotide of SEQ ID NO: 2 comprises the step of ligating together at least two (2) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 8 and 9. In some cases, the fragments may be ligated in the following order: SEQ ID NO: 9 to SEQ ID NO: 8 (i.e., from the 5' end to the 3' end).

[0180] Alternatively, the oligonucleotide of SEQ ID NO: 2 can be made by ligating together two (2) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 16 and 17. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 17 to SEQ ID NO: 16 (i.e., from the 5' end to the 3' end).

[0181] Alternatively, the oligonucleotide of SEQ ID NO: 2 can be made by ligating together two (2) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 20 and 21. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 21 to SEQ ID NO: 20 (i.e., from the 5' end to the 3' end).

[0182] Alternatively, the oligonucleotide of SEQ ID NO: 2 can be made by ligating together two (2) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 24 and 25. In some cases, the fragments can be ligated in the following order: SEQ ID NO: 25 to SEQ ID NO: 24 (i.e., from the 5' end to the 3' end).

[0183] In addition, certain combinations of the oligonucleotide fragments herein (i.e., intermediate compounds) prepared by the SPOS described above can be combined according to methods known to those skilled in the art to obtain the RNAi agents of SEQ ID NOs: 3 and 4. Briefly, the method can include synthesizing independent oligonucleotide fragments, followed by ligating such fragments, thereby forming the RNAi agents of SEQ ID NOs: 3 and 4.

[0184] For example, an exemplary method for preparing the oligonucleotide of SEQ ID NO: 3 includes at least the step of ligating together the following three (3) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 7, 32, and 33. In some cases, the fragments may be ligated in the following order: SEQ ID NO: 32 to SEQ ID NO: 33 to SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0185] Likewise, an exemplary method for preparing the oligonucleotide of SEQ ID NO: 4 comprises at least the step of ligating together two (2) oligonucleotide fragments, wherein such fragments have the nucleotide sequences set forth in SEQ ID NOs: 34 and 35. In some cases, the fragments may be ligated in the following order: SEQ ID NO: 35 to SEQ ID NO: 34 (i.e., from the 5' end to the 3' end).

[0186] In any of the above methods, the ligation can be performed in an aqueous solution, such as a reaction buffer. In some cases, the solution can be an acetate buffer, a carbonate buffer, a citrate buffer, or a phosphate buffer, such as a Tris buffer. Additionally, the solution can have a pH of about 5 to about 9, about 6 to about 8, or about 7. In other cases, the pH can be about 5, about 6, about 7, about 8, or about 9.

[0187] Additionally, the aqueous solution may include a cofactor (eg, adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD)) and a divalent metal salt (eg, MgCl2).

[0188] In addition, the oligonucleotide fragment can be present in an aqueous solution at a concentration of about 1 μM to about 100,000 μM (100 mM). In some cases, the oligonucleotide fragment concentration can be about 100 μM to about 90,000 μM, about 1,000 μM to about 80,000 μM, about 2,000 μM to about 70,000 μM, about 3,000 μM to about 60,000 μM, about 4,000 μM to about 50,000 μM, about 5,000 μM to about 40,000 μM, about 6,000 μM to about 30,000 μM, about 7,000 μM to about 20,000 μM, about 8,000 μM to about 10,000 μM or about 9,000 μM. In other cases, the oligonucleotide fragment concentration can be from about 100 μM to about 200 μM, from about 200 μM to about 300 μM, from about 300 μM to about 400 μM, from about 400 μM to about 500 μM, from about 500 μM to about 600 μM, from about 600 μM to about 700 μM, from about 700 μM to about 800 μM, from about 800 μM to about 900 μM, from about 900 μM to about 1,000 μM, from about 1,000 μM to about 2,000 μM, from about 2,000 μM to about 3,000 μM, from about 3,000 μM to about 4,000 μM, from about 4,000 μM to about 5,000 μM, from about 5,000 μM to about 6,000 μM, from about 6,000 μM to about 7 About 7,000 μM, about 7,000 μM to about 8,000 μM, about 8,000 μM to about 9,000 μM, about 9,000 μM to about 10,000 μM, about 10,000 μM to about 20,000 μM, about 20,000 μM to about 30,000 μM, about 30,000 μM to about 40,000 μM, about 40,000 μM to about 50,000 μM, about 50,000 μM to about 60,000 μM, about 60,000 μM to about 70,000 μM, about 70,000 μM to about 80,000 μM, about 80,000 μM to about 90,000 μM, or about 90,000 μM to about 100,000 μM.In yet other cases, the concentration of the oligonucleotide fragments may be about 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, about 1,000 μM, about 1,500 μM, about 2,000 μM. , about 2,500 μM, about 3,000 μM, about 3,500 μM, about 4,000 μM, about 4,500 μM, about 5,000 μM, about 5,500 μM, about 6,000 μM, about 6,500 μM, about 7,000 μM, about 7,500 μM, about 8,000 μM, about 8,500 μM, about 9,000 μM, about 9,500 μM, about 10,000 μM, about 10,500 μM, about 11,000 μM, about 11,500, about 12,000 μM, about 12,500 μM, about 13,000 μM, about 13,500, about 14,000 μM, about 14,500, about 15,000 μM, about 15,500 μM, about 16,000 μM, about 16,500 μM, about 17,000 μM, about 17,500 μM, about 18,000 μM, about 18,500 μM, about 19,000 μM, about 19,500 μM, about 2 In some embodiments, the oligonucleotide fragments can be at the same concentration as the other oligonucleotide fragments. In other embodiments, the oligonucleotide fragments can be at different concentrations than the other oligonucleotide fragments.

[0189] In addition, the ligation can be carried out at a reaction temperature sufficient to activate the enzyme, which can be from about 2°C to about 50°C. In some cases, the reaction temperature is from about 5°C to about 45°C, from about 10°C to about 40°C, from about 15°C to about 35°C, from about 20°C to about 30°C, or from about 25°C. In other cases, the reaction temperature is from about 5°C to about 10°C, from about 10°C to about 15°C, from about 15°C to about 20°C, from about 20°C to about 25°C, from about 25°C to about 30°C, from about 30°C to about 35°C, from about 35°C to about 40°C, from about 40°C to about 45°C, or from about 45°C to about 50°C. In yet other cases, the reaction temperature is from about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, or about 50°C.

[0190] In some cases, the reaction time can be from about 2 hr to about 70 hr, from about 4 hr to about 68 hr, from about 6 hr to about 66 hr, from about 8 hr to about 64 hr, from about 10 hr to about 62 hr, from about 12 hr to about 60 hr, from about 14 hr to about 58 hr, from about 16 hr to about 56 hr, from about 18 hr to about 54 hr, from about 20 hr to about 52 hr, from about 22 hr to about 54 hr, from about 24 hr to about 52 hr, from about 26 hr to about 50 hr, from about 28 hr to about 48 hr, from about 30 hr to about 46 hr, from about 32 hr to about 44 hr, from about 34 hr to about 42 hr, from about 36 hr to about 40 hr or from about 38 hr. In other cases, the reaction time can last from about 2 hr to about 10 hr, from about 10 hr to about 20 hr, from about 20 hr to about 30 hr, from about 30 hr to about 40 hr, from about 40 hr to about 50 hr, from about 50 hr to about 60 hr, or from about 60 hr to about 70 hr. In still other cases, the reaction time may last about 2 hr, about 4 hr, about 6 hr, about 8 hr, about 10 hr, about 12 hr, about 14 hr, about 16 hr, about 18 hr, about 20 hr, about 22 hr, about 24 hr, about 26 hr, about 28 hr, about 30 hr, about 32 hr, about 34 hr, about 36 hr, about 38 hr, about 40 hr, about 42 hr, about 44 hr, about 46 hr, about 48 hr, about 50 hr, about 52 hr, about 54 hr, about 56 hr, about 58 hr, about 60 hr, about 62 hr, about 64 hr, about 66 hr, about 68 hr, about 70 hr, or about 72 hr.

[0191] In the above method, the ligation step can be performed by an enzyme. In some cases, the enzyme is a ligase, such as a DNA ligase or an RNA ligase. In some cases, the ligase is a naturally occurring (i.e., wild type) ligase. In other cases, the ligase is a non-naturally occurring (i.e., modified) ligase. Examples of ligases that can be used in the method include, but are not limited to, T4 RNA ligase 1 or T4 RNA ligase 2. In some cases, the ligase is T4 RNA ligase 1. In other cases, the ligase is T4 RNA ligase 2.

[0192] RNA ligases are commercially available from sources such as Ajinomoto, Almac, Codexis, New England Biolabs, Takara, and ThermoFisher Scientific.

[0193] The enzyme activity may be from about 0.01 U / μL to about 1 U / μL. In some cases, the activity may be from about 0.05 U / μL to about 0.95 U / μL, from about 0.1 U / μL to about 0.9 U / μL, from about 0.15 U / μL to about 0.85 U / μL, from about 0.2 U / μL to about 0.7 U / μL, from about 0.25 U / μL to about 0.65 U / μL, from about 0.3 U / μL to about 0.6 U / μL, from about 0.35 U / μL to about 0.55 U / μL, from about 0.4 U / μL to about 0.5 U / μL, or from about 0.45 U / μL. In other cases, the activity can be about 0.01 U / μL, about 0.02 U / μL, about 0.03 U / μL, about 0.04 U / μL, about 0.05 U / μL, about 0.06 U / μL, about 0.07 U / μL, about 0.08 U / μL, about 0.09 U / μL, about 0.1 U / μL, about 0.15 U / μL, about 0.2 U / μL, about 0.25 U / μL. L, about 0.3 U / μL, about 0.35 U / μL, about 0.4 U / μL, about 0.45 U / μL, about 0.5 U / μL, about 0.55 U / μL, about 0.6 U / μL, about 0.65 U / μL, about 0.7 U / μL, about 0.75 U / μL, about 0.8 U / μL, about 0.85 U / μL, about 0.9 U / μL, about 0.95 or about 1 U / μL.

[0194] Alternatively, the enzyme concentration may be from about 0.01 g / L to about 10 g / L. In some cases, the enzyme concentration may be from about 0.05 g / L to about 9.9 g / L, from about 0.1 g / L to about 9.8 g / L, from about 0.2 g / L to about 9.7 g / L, from about 0.3 g / L to about 9.6 g / L, from about 0.4 g / L to about 9.5 g / L, from about 0.5 g / L to about 9.4 g / L, from about 0.6 g / L to about 9.3 g / L, from about 0.7 g / L to about 9.2 g / L, from about 0.8 g / L to about 9.1 g / L, from about 0.9 g / L to about 9 g / L, from about 1 g / L to about 8.9 g / L, from about 1.1 g / L to about 8.8 g / L, about 1.2 g / L to about 8.7 g / L, about 1.3 g / L to about 8.6 g / L, about 1.4 g / L to about 8.5 g / L, about 1.5 g / L to about 8.4 g / L, about 1.6 g / L to about 8.3 g / L, about 1.7 g / L to about 8.2 g / L, about 1.8 g / L to about 8.1 g / L, about 1.9 g / L to about 8 g / L, about 2 g / L to about 7.9 g / L, about 2.1 g / L to about 7.8 g / L, about 2.2 g / L to about 7.7 g / L, about 2.3 g / L to about 7.6 g / L, about 2.4 g / L / L to about 7.5g / L, about 2.5g / L to about 7.4g / L, about 2.6g / L to about 7.3g / L, about 2.7g / L to about 7.2g / L, about 2.8g / L to about 7.1g / L, about 2.9g / L to about 7g / L, about 3g / L to about 6.9g / L, about 3.1g / L to about 6.8g / L, about 3.2g / L to about 6.7, about 3.3g / L to about 6.6g / L, about 3.4g / L to about 6.5g / L, about 3.5g / L to about 6.4g / L, about 3.6g / L to about 6.3g / L, about From about 3.7 g / L to about 6.2 g / L, from about 3.8 g / L to about 6.1 g / L, from about 3.9 g / L to about 6 g / L, from about 4 g / L to about 5.9 g / L, from about 4.1 g / L to about 5.8 g / L, from about 4.2 g / L to about 5.7 g / L, from about 4.3 g / L to about 5.6 g / L, from about 4.4 g / L to about 5.5 g / L, from about 4.5 g / L to about 5.4 g / L, from about 4.6 g / L to about 5.3, from about 4.7 g / L to about 5.2 g / L, from about 4.8 g / L to about 5.1 g / L, and from about 4.9 g / L to about 5 g / L.In other cases, the concentration can be about 0.01 g / L, about 0.02 g / L, about 0.03 g / L, about 0.04 g / L, about 0.05 g / L, about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1.4 g / L, about 1.5 g / L, about 1.6 g / L, about 1.7 g / L, about 1.8 g / L, about 1.9 g / L, about 2 g / L, about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, about 3 g / L, about 3.1 g / L, about 3.2 g / L, about 3.3 g / L, about 3.4 g / L, about 3.5 g / L, about 3.6 g / L, about 3.7 g / L, about 3.8 g / L, about 3.9 g / L, about 4 g / L, about 4.1 g / L, about 4.2 g / L, about 4.3 g / L, about 4.4 g / L, about 4.5 g / L, about 4.6 g / L, about 4,7, about 4.8g / L, about 4.9g / L, about 5g / L, about 5.1g / L, about 5.2g / L, about 5.3g / L, about 5.4g / L, about 5.5g / L, about 5.6g / L, about 5.7g / L, about 5.8g / L, about 5.9g / L, about 6g / L, about 6.1g / L, about 6.2g / L, about 6.3g / L, about 6.4g / L, about 6.5g / L, about 6.6g / L, about 6.7g / L, about 6.8g / L, about 6.9g / L, about 7g / L, about 7.1g / L, about 7.2g / L, about 7.3g / L, about 7 .4 g / L, about 7.5 g / L, about 7.6 g / L, about 7.7 g / L, about 7.8 g / L, about 7.9 g / L, about 8 g / L, about 8.1 g / L, about 8.2 g / L, about 8.3 g / L, about 8.4 g / L, about 8.5 g / L, about 8.6 g / L, about 8.7 g / L, about 8.8 g / L, about 8.9 g / L, about 9 g / L, about 9.1 g / L, about 9.2 g / L, about 9.3 g / L, about 9.4 g / L, about 9.5 g / L, about 9.6 g / L, about 9.7 g / L, about 9.8 g / L, about 9.9 g / L or about 10 g / L. In certain instances, the concentration is about 0.025 g / L, about 0.1 g / L, about 0.3 g / L, or about 1 g / L.

[0195] The above methods may further include the step of annealing the sense and antisense strands to form an RNAi agent. In some cases, SEQ ID NO: 1 and SEQ ID NO: 2 are annealed to form an RNAi agent that modulates LPA expression, such that the complementary nucleotides in each strand hybridize / base pair with each other according to methods known to those skilled in the art. In other cases, SEQ ID NO: 3 and SEQ ID NO: 4 are annealed to form an RNAi agent that modulates ANGPTL3 expression, such that the complementary nucleotides in each strand hybridize / base pair with each other according to methods known to those skilled in the art.

[0196] Other methods / uses

[0197] The RNAi agents herein can be used in a variety of therapeutic applications. For example, the RNAi agents of SEQ ID NOs: 1 and 2 can be used in methods for alleviating, preventing, and / or treating diseases, disorders, and / or conditions associated with LPA expression, wherein such methods comprise at least the step of administering to a subject in need of such treatment an effective amount of the RNAi agents of SEQ ID NOs: 1 and 2, or a pharmaceutically acceptable salt thereof.

[0198] Likewise, the RNAi agents of SEQ ID NOs: 3 and 4 can be used in methods for alleviating, preventing and / or treating diseases, disorders and / or conditions associated with ANGPTL3 expression, wherein such methods comprise at least the step of administering to a subject in need of such treatment an effective amount of the RNAi agents of SEQ ID NOs: 3 and 4, or a pharmaceutically acceptable salt thereof.

[0199] Example

[0200] The following non-limiting examples are offered for purposes of illustration and not limitation.

[0201] Development and synthesis of oligonucleotide fragments

[0202] Example 1: SPOS of Intermediate Compound 1

[0203] Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU 3′ (SEQ ID NO: 5) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL 2′-OMe U 250 polystyrene resin (246 μmol / g, about 750 mg, about 185 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed using the conditions outlined in Table 1.

[0204] Table 1: Method parameters used for SPOS

[0205]

[0206]

[0207]

[0208] Table 2: Reagents used during SPOS

[0209]

[0210] Resin cleavage and separation: After synthesis, nitrogen was passed through the resin-bound oligonucleotide fragment intermediate until constant mass was achieved. Intermediate compound 1 was cleaved from the resin, and the nucleobases were globally deprotected at 38°C using H2O containing concentrated NH4OH for 18 hours. 20 mL NH4OH / g resin was loaded into a reduced pressure reaction vial containing dried resin (approximately 1.5 g / batch). The used resin was filtered and rinsed with 2 × 20 mL of 1:1 EtOH:H2O. The filtrate was collected in a round-bottom flask, and NH3 was removed by rotary evaporation. Three batches of intermediate compound 1 were combined for downstream processing.

[0211] Tangential Flow Filtration (TFF): PendoTECH TFF system was used to desalt the intermediate compound 1 and exchange the NH3 salt for the Na salt. Two Omega PES membranes (0.1 m 2 , 1kDa MWCO) of Pall T series filter cartridge. Before treatment, the membrane was conditioned with H2O (about 5 L). The intermediate compound 1 solution was concentrated to about 50 mL and then diafiltered 10 times using 0.5M NaCl solution (500 mL) to convert it into sodium phosphate. The intermediate compound 1 sodium salt was subjected to water diafiltration until the permeate conductivity was less than 50 μS / cm. The intermediate compound 1 sodium salt retentate and the membrane were rinsed with water 3 times. Intermediate compound 1 was lyophilized and separated into crude oligonucleotide (oligo) powder.

[0212] Analysis: Water's Acquity UPLC system equipped with a tunable UV (TUV) detector was used to assess the purity of intermediate Compound 1. The mobile phase, column, gradient, and general parameters for chromatography are summarized in Tables 3 and 4.

[0213] Table 3: Chromatography mobile phases and columns

[0214]

[0215] Table 4: Chromatographic gradient parameters

[0216] Time (min) ％A ％B initial 99.0 1.0 25.00 75.7 24.3 25.10 0.0 100.0 27.00 0.0 100.0 27.10 99.0 1.0 30.00 99.0 1.0

[0217] Results: Intermediate compound 1 (1.89 g, 90.21% by UPLC, expected exact mass = 4585.706 Da, observed exact mass = 4585.702 Da) was prepared as a crude sodium salt.

[0218] Example 2: SPOS of Intermediate Compound 2

[0219] Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 6) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL 2′-OMe G(iBu)250 polystyrene resin (249 μmol / g, about 850 mg, about 212 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in

[0220] Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0221] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0222] Analysis: Analysis was performed as described in Example 1.

[0223] TFF: TFF was performed as described in Example 1.

[0224] Results: Intermediate compound 2 (1.89 g, 95.87% by UPLC, expected exact mass = 3362.594 Da, observed exact mass = 3362.588 Da) was prepared as a crude sodium salt.

[0225] Example 3: SPOS of Intermediate Compound 3

[0226] Synthesis: 5'p-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 7) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL 2′-OMe C(Ac)250 polystyrene resin (257 μmol / g, about 800 mg, about 206 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0227] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0228] TFF: TFF was performed as described in Example 1.

[0229] Analysis: Analysis was performed as described in Example 1; however, the mobile phase, column, and gradient are summarized in Table 5.

[0230] Table 5: Chromatography mobile phases and columns

[0231]

[0232] Results: Intermediate compound 3 (2.54 g, 88.22% by UPLC, expected exact mass = 5298.322 Da, observed exact mass = 5298.318 Da) was prepared as a crude sodium salt.

[0233] Example 4: SPOS of Intermediate Compound 4

[0234] Synthesis: 5'p-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3' (SEQ ID NO: 8) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL 2′-OMe G(iBu)250 polystyrene resin (249 μmol / g, about 800 mg, about 200 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0235] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0236] TFF: TFF was performed as described in Example 1.

[0237] Analysis: Analysis was performed as described in Example 3.

[0238] Results: Intermediate compound 4 (2.00 g, 89.03% by UPLC, expected exact mass = 4140.661 Da, observed exact mass = 4140.652 Da) was prepared as a crude sodium salt.

[0239] Example 5: SPOS of Intermediate Compound 5

[0240] Synthesis: 5'[MePhosphonate-4O-mU S ]-fA S -fG S-fA-fU-mG-fA-mC-mC-fA3′ (SEQ ID NO: 9) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL 2′-Fluoro A(bz)250 polystyrene resin (229 μmol / g, about 850 mg, about 195 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0241] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0242] TFF: TFF was performed as described in Example 1.

[0243] Analysis: Analysis was performed as described in Example 3.

[0244] Results: Intermediate compound 5 (1.57 g, 92.84% by UPLC, expected exact mass = 3376.432 Da, observed exact mass = 3376.426 Da) was prepared as a crude sodium salt.

[0245] Example 6: SPOS of Intermediate Compound 6

[0246] Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC 3' (SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8031 g, 277.9 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in

[0247] Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0248] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0249] TFF: The intermediate compound 6 was desalted and the NH3 salt was exchanged for the sodium salt using the Millipore Cogent μScale system. Membrane (0.02m 2, 2kDa MWCO) is equipped on the system. Before treatment, the membrane is conditioned with H2O (about 0.5L). The intermediate compound 6 solution is concentrated to about 20mL and then diafiltered 10 times using 0.5M NaCl solution (200mL) to convert it into sodium phosphate. The intermediate compound 6 sodium salt is subjected to water diafiltration until the permeate conductivity is less than 50μS / cm. The intermediate compound 6 sodium salt retentate and the membrane are rinsed three times with water. Intermediate compound 6 is lyophilized, and the crude oligonucleotide powder is dissolved in milliQ water (13.22mL, OD / mL=1678.7) to produce a stock solution.

[0250] Analysis: Analysis was performed as described in Example 3.

[0251] Results: Intermediate compound 6 (0.783 g by optical density, 94.65% by UPLC, expected accurate mass = 4000.708 Da, observed accurate mass = 4000.719 Da) was prepared as a crude sodium salt.

[0252] Example 7: SPOS of Intermediate Compound 7

[0253] Synthesis: 5'p-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 11) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HLUnylinker 350 polystyrene resin (346 μmol / g, 0.7503 g, 259.6 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0254] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0255] TFF: TFF was performed as described in Example 6.

[0256] Analysis: Analysis was performed as described in Example 3.

[0257] Results: Intermediate compound 7 (0.819 g by optical density, 89.44% by UPLC, expected accurate mass = 4660.208 Da, observed accurate mass = 4660.209 Da) was prepared as a crude sodium salt.

[0258] Example 8: SPOS of Intermediate Compound 8

[0259] Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC 3' (SEQ ID NO: 12) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8007 g, 277.0 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA

[0260] Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0261] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0262] TFF: TFF was performed as described in Example 6.

[0263] Analysis: Analysis was performed as described in Example 3.

[0264] Results: Intermediate compound 8 (0.719 g by optical density, 95.15% by UPLC, expected accurate mass = 3681.651 Da, observed accurate mass = 3681.655 Da) was prepared as a crude sodium salt.

[0265] Example 9: SPOS of Intermediate Compound 9

[0266] Synthesis: 5'p-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 13) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HLUnylinker 350 polystyrene resin (346 μmol / g, 0.7454 g, 257.9 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0267] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0268] TFF: TFF was performed as described in Example 6.

[0269] Analysis: Analysis was performed as described in Example 3.

[0270] Results: Intermediate compound 9 (0.896 g by optical density, 90.24% by UPLC, expected accurate mass = 4979.265 Da, observed accurate mass = 4979.264 Da) was prepared as a crude sodium salt.

[0271] Example 10: SPOS of Intermediate Compound 10

[0272] Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU 3′ (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8066 g, 279.1 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0273] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0274] TFF: PendoTECH and Millipore Cogent μScale TFF systems were used to desalt the intermediate compound 10 and exchange the NH3 salt for the Na salt. Membrane (0.02m 2 , 2kDa MWCO) is equipped on the system. Before treatment, the membrane is conditioned with H2O (about 0.5L). The intermediate compound 10 solution is concentrated to about 30mL and then diafiltered 10 times using 0.5M NaCl solution (300mL) to convert it into sodium phosphate. The intermediate compound 10 sodium salt is subjected to water diafiltration until the permeate conductivity is less than 50μS / cm. The intermediate compound 10 sodium salt retentate and the membrane are rinsed three times with water. Intermediate compound 10 is lyophilized, and the crude oligonucleotide powder is dissolved in milliQ water (14.05mL, OD / mL=1368.3) to produce a stock solution.

[0275] Analysis: Analysis was performed as described in Example 3.

[0276] Results: Intermediate compound 10 (0.655 g by optical density, 94.69% by UPLC, expected accurate mass = 3239.518 Da, observed accurate mass = 3239.522 Da) was prepared as a crude sodium salt.

[0277] Example 11: SPOS of Intermediate Compound 11

[0278] Synthesis: 5'p-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 15) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8010 g, 277.1 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0279] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0280] TFF: TFF was performed as described in Example 10.

[0281] Analysis: Analysis was performed as described in Example 3.

[0282] Results: Intermediate compound 11 (0.930 g by optical density, 93.06% by UPLC, expected accurate mass = 4708.782 Da, observed accurate mass = 4708.791 Da) was prepared as a crude sodium salt.

[0283] Example 12: SPOS of Intermediate Compound 12

[0284] Synthesis: 5'p-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3' (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8027 g, 277.7 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0285] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0286] TFF: TFF was performed as described in Example 10.

[0287] Analysis: Analysis was performed as described in Example 3.

[0288] Results: Intermediate compound 12 (0.899 g by optical density, 82.82% by UPLC, expected accurate mass = 5440.871 Da, observed accurate mass = 5440.883 Da) was prepared as a crude sodium salt.

[0289] Example 13: SPOS of Intermediate Compound 13

[0290] Synthesis: 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG 3′ (SEQ ID NO: 17) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8036 g, 278.0 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0291] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0292] TFF: TFF was performed as described in Example 10.

[0293] Analysis: Analysis was performed as described in Example 3.

[0294] Results: Intermediate compound 13 (0.388 g by optical density, 94.34% by UPLC, expected accurate mass = 2076.222 Da, observed accurate mass = 2076.226 Da) was prepared as a crude sodium salt.

[0295] Example 14: SPOS of Intermediate Compound 14

[0296] Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC 3′ (SEQ ID NO: 18) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8043 g, 278.3 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0297] Resin cleavage and separation: After synthesis, nitrogen was passed through the resin-bound intermediate oligonucleotide fragment (1.7125 g, 3.26 g / mmol mass gain) until constant mass was achieved. Intermediate compound 14 was cleaved from the resin and the nucleobases were globally deprotected using H2O containing concentrated NH4OH at 38°C for 18 hr. 20 mL NH4OH / g resin was charged into a reduced pressure reaction vial containing the dried resin. The spent resin was filtered and rinsed with 2×5 mL of concentrated NH4OH. The filtrate was collected in a 50 mL Falcon tube and Concentrate to dryness in an EZ-2 Elite centrifugal evaporation system. Intermediate compound 14 was reconstituted in nuclease-free water (15 mL, OD / mL=1076.7) to the desired concentration for subsequent enzymatic ligation reaction.

[0298] Analysis: Analysis was performed as described in Example 3.

[0299] Results: Intermediate compound 14 (0.388 g by optical density, 94.34% by UPLC, expected accurate mass = 2076.222 Da, observed accurate mass = 2076.226 Da) was prepared as a crude ammonium salt.

[0300] Example 15: SPOS of Intermediate Compound 15

[0301] Synthesis: 5'p-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 19) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7019 g, 242.9 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0302] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0303] Analysis: Analysis was performed as described in Example 3.

[0304] Results: Intermediate compound 15 (0.824 g by optical density, 90.12% by UPLC, expected accurate mass = 5016.803 Da, observed accurate mass = 5016.811 Da) was prepared as a crude ammonium salt.

[0305] Example 16: SPOS of Intermediate Compound 16

[0306] Synthesis: 5'p-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3' (SEQ ID NO: 20) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7036 g, 243.4 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0307] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0308] Analysis: Analysis was performed as described in Example 3.

[0309] Results: Intermediate compound 16 (0.698 g by optical density, 90.09% by UPLC, expected accurate mass = 5109.823 Da, observed accurate mass = 5109.830 Da) was prepared as a crude ammonium salt.

[0310] Example 17: SPOS of Intermediate Compound 17

[0311] Synthesis: 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA 3′ (SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8041 g, 278.2 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0312] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0313] Analysis: Analysis was performed as described in Example 3.

[0314] Results: Intermediate compound 17 (0.414 g by optical density, 93.23% by UPLC, expected accurate mass = 2407.270 Da, observed accurate mass = 2407.274 Da) was prepared as a crude ammonium salt.

[0315] Example 18: SPOS of Intermediate Compound 18

[0316] Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG 3′ (SEQ ID NO: 22) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8010 g, 277.1 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0317] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0318] Analysis: Analysis was performed as described in Example 3.

[0319] Results: Intermediate compound 18 (0.536 g by optical density, 96.44% by UPLC, expected accurate mass = 2624.460 Da, observed accurate mass = 2624.463 Da) was prepared as a crude ammonium salt.

[0320] Example 19: SPOS of Intermediate Compound 19

[0321] Synthesis: 5'p-fC-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG 3' (SEQ ID NO: 23) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.6999 g, 242.2 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0322] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0323] Analysis: Analysis was performed as described in Example 3.

[0324] Results: Intermediate compound 19 (0.850 g by optical density, 88.94% by UPLC, expected accurate mass = 5323.840 Da, observed accurate mass = 5323.849 Da) was prepared as a crude ammonium salt.

[0325] Example 20: SPOS of Intermediate Compound 20

[0326] Synthesis: 5′p-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG 3' (SEQ ID NO: 24) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7008 g, 242.5 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0327] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0328] Analysis: Analysis was performed as described in Example 3.

[0329] Results: Intermediate compound 20 (0.685 g by optical density, 89.06% by UPLC, expected accurate mass = 4790.766 Da, observed accurate mass = 4790.771 Da) was prepared as a crude ammonium salt.

[0330] Example 21: SPOS of Intermediate Compound 21

[0331] Synthesis: 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC 3′ (SEQ ID NO: 25) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8031 g, 277.9 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA

[0332] Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0333] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 14.

[0334] Analysis: Analysis was performed as described in Example 3.

[0335] Results: Intermediate compound 21 (0.608 g by optical density, 92.67% by UPLC, expected accurate mass = 2726.327 Da, observed accurate mass = 2726.328 Da) was prepared as a crude ammonium salt.

[0336] Example 22: SPOS of Intermediate Compound 22

[0337] Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG 3' (SEQ ID NO: 26) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8081 g, 279.6 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0338] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0339] TFF: The intermediate compound 22 was desalted and the NH4 salt was exchanged for the Na salt using the Millipore Cogent μScale TFF system. Membrane (0.02m 2 , 2kDa MWCO) is equipped on the system. Before treatment, the membrane is conditioned with H2O (about 0.5L). The intermediate compound 22 solution is concentrated to about 40mL and then diafiltered 10 times using 0.5M NaCl solution (400mL) to convert it into sodium phosphate. The intermediate compound 22 sodium salt is subjected to water diafiltration until the permeate conductivity is less than 60μS / cm. The intermediate compound 22 sodium salt retentate and the membrane are rinsed twice with water. Intermediate compound 22 is lyophilized, and the crude oligonucleotide powder is dissolved in milliQ water (60.24mL, OD / mL=415.07) to produce a stock solution.

[0340] Analysis: Analysis was performed as described in Example 3.

[0341] Results: Intermediate compound 22 (0.881 g by optical density, 94.11% by UPLC, expected accurate mass = 4359.771 Da, observed accurate mass = 4359.778 Da) was prepared as a crude sodium salt.

[0342] Example 23: SPOS of Intermediate Compound 23

[0343] Synthesis: 5'p-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 27) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HLUnylinker 350 polystyrene resin (346 μmol / g, 0.8043 g, 278.3 μmol) was loaded onto a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0344] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0345] TFF: TFF was performed as described in Example 22.

[0346] Analysis: Analysis was performed as described in Example 3.

[0347] Results: Intermediate compound 23 (0.853 g by optical density, 86.25% by UPLC, expected accurate mass = 4301.145 Da, observed accurate mass = 4301.149 Da) was prepared as a crude sodium salt.

[0348] Example 24: SPOS of Intermediate Compound 24

[0349] Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG 3' (SEQ ID NO: 28) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL 2′-OMe G(Ac)250 polystyrene resin (247 μmol / g, about 650 mg, about 161 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0350] Resin cleavage and separation: Resin cleavage and separation were performed as described in Example 1. In contrast to Example 1, only two batches were prepared in this example and the two batches were combined before TFF.

[0351] TFF: TFF was performed as described in Example 22.

[0352] Analysis: Analysis was performed as described in Example 1.

[0353] Results: Intermediate compound 24 (1.08 g, 94.38% by UPLC, expected exact mass = 7325.687 Da, observed exact mass = 7325.680 Da) was isolated as a crude sodium salt.

[0354] Example 25: SPOS of Intermediate Compound 25

[0355] Synthesis: 5'mC-mU-mG-mC 3' (SEQ ID NO: 29) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. Briefly, SPOS was performed using preloaded resin (loading factor 0.250 mmol / g) under the conditions described in Tables 6 and 7 below.

[0356] Table 6: SPOS method parameters

[0357]

[0358]

[0359] Table 7: Reagents used during SPOS

[0360]

[0361] TFF: TFF was performed as described in Example 22.

[0362] Analysis: Analysis was performed as described in Example 1.

[0363] Results: Intermediate compound 25 (0.70 g, 96.7% by UPLC, expected accurate mass = 1335.228 Da) was isolated as a crude sodium salt.

[0364] Example 26: SPOS of Intermediate Compound 26

[0365] Synthesis: 5'p-mG-mC-mA-mA S -mG S -mG 3′ (SEQ ID NO: 30) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS as described in Example 25.

[0366] TFF: TFF was performed as described in Example 22.

[0367] Analysis: Analysis was performed as described in Example 1.

[0368] Results: Intermediate compound 26 (0.45 g, 98.6% by UPLC, expected exact mass = 2133.35 Da, observed exact mass = 2133.31 Da) was isolated as a crude sodium salt.

[0369] Example 27: SPOS of Intermediate Compound 27

[0370] Synthesis: 5′p-mA-mG-mC-fU-mU-mG 3′ (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS as described in Example 25.

[0371] TFF: TFF was performed as described in Example 22.

[0372] Analysis: Analysis was performed as described in Example 1.

[0373] Results: Intermediate compound 27 (0.85 g, 95.3% by UPLC, expected exact mass = 2026.32 Da, observed exact mass = 2026.29 Da) was isolated as a crude sodium salt.

[0374] Example 28: SPOS of Intermediate Compound 28

[0375] Synthesis: 5'mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU 3′ (SEQ ID NO: 32) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, about 750 mg, about 260 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1. Compared to Example 1, 2 batches were produced and each batch of resin was divided for 2 separate separation methods.

[0376] Resin Cleavage and Isolation: Following synthesis, nitrogen was passed through the resin-bound intermediate oligonucleotide fragment until a constant mass was achieved. Intermediate compound 32 was cleaved from the resin, and the nucleobases were globally deprotected using concentrated NH4OH in HO at 38°C for 18 hr. 20 mL of NH4OH / g of resin was charged to a reduced pressure reaction vial containing the dried resin.

[0377] • Isolation Method A: The spent resin was filtered and rinsed with 2 x 20 mL of 1:1 EtOH:H2O. The filtrate was collected in a round bottom flask and NH3 was removed by rotary evaporation. The two batches of intermediate compound 28 were combined for subsequent processing. The TFF system desalted the intermediate compound 28 and exchanged the ammonium salt for the sodium salt. Two OmegaPES membranes (0.1 m 2 , 1kDa MWCO) Pall T series filter cartridge. Before treatment, use H2O conditioning membrane. Intermediate compound 28 solution is concentrated to about 80mL, and then 0.5M NaCl solution is used to perform 7.5 times diafiltration (600mL) to convert it into sodium phosphate. Intermediate compound 28 sodium salt is subjected to water diafiltration until the permeate conductivity is less than 50μS / cm. Collect intermediate compound 28 sodium salt retentate and membrane water rinse several times (a total of about 300mL). Intermediate compound 28 is lyophilized and separated into crude oligonucleotide powder.

[0378] Isolation Method B: The spent resin was filtered and rinsed with 2 x 5 mL of concentrated NH4OH. The filtrate was collected in a 50 mL Falcon tube. The sample was concentrated to dryness using a Genevac EZ-2 Elite centrifugal evaporation system. Intermediate compound 28 was reconstituted in nuclease-free water to the desired concentration for subsequent enzymatic ligation reactions.

[0379] TFF: TFF was performed as described in Example 28, Isolation Method A.

[0380] Analysis: Analysis was performed as described in Example 1.

[0381] Results: Intermediate compound 28 (1.08 g, 90.47% by UPLC, expected exact mass = 4679.783 Da, observed exact mass = 4679.776 Da) was isolated as a crude sodium salt.

[0382] Example 29: SPOS of Intermediate Compound 29

[0383] Synthesis: 5'p-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG 3' (SEQ ID NO: 33) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, about 850 mg, about 295 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA

[0384] Plus 100 synthesizer. SPOS was performed as described in Example 1. Compared to Example 1, two batches were produced and each batch of resin was divided for two separate separation methods described in Example 28.

[0385] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 28.

[0386] TFF: TFF was performed as described in Example 22.

[0387] Analysis: Analysis was performed as described in Example 3.

[0388] Results: Intermediate compound 29 (1.20 g, 95.74% by UPLC, expected exact mass = 3386.605 Da, observed exact mass = 3386.601 Da) was isolated as a crude sodium salt.

[0389] Example 30: SPOS of Intermediate Compound 30

[0390] Synthesis: 5′p-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S -mG 3' (SEQ ID NO: 34) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, about 800 mg, about 277 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA

[0391] Plus 100 synthesizer. SPOS was performed as described in Example 1. Compared to Example 1, two batches were produced and each batch of resin was divided for two separate separation methods described in Example 28.

[0392] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 28.

[0393] TFF: TFF was performed as described in Example 22.

[0394] Analysis: Analysis was performed as described in Example 3.

[0395] Results: Intermediate compound 30 (1.10 g, 93.47% by UPLC, expected exact mass = 4039.589 Da, observed exact mass = 4039.584 Da) was isolated as a crude sodium salt.

[0396] Example 31: SPOS of Intermediate Compound 31

[0397] Synthesis: 5'[MePhosphonate-4O-mU S ]-fG S -fU S-fA-fU-mA-fA-mC-mC-fU 3′ (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, about 800 mg, about 277 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA Plus 100 synthesizer. SPOS was performed as described in Example 1. Compared to Example 1, two batches were produced and each batch of resin was divided for two separate separation methods described in Example 28.

[0398] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 28.

[0399] TFF: TFF was performed as described in Example 22.

[0400] Analysis: Analysis was performed as described in Example 3.

[0401] Results: Intermediate compound 31 (1.20 g, 89.24% by UPLC, expected exact mass = 3314.383 Da, observed exact mass = 3314.377 Da) was isolated as a crude sodium salt.

[0402] Example 32: SPOS of Intermediate Compound 32

[0403] Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 36) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. HL Unylinker 350 polystyrene resin (346 μmol / g, about 600 mg, about 208 μmol) was loaded into a stainless steel column (6.3 CV, 20 mm diameter), which was then installed in an AKTA

[0404] Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0405] Resin cleavage and isolation: Resin cleavage and isolation were performed as described in Example 1.

[0406] TFF: TFF was performed as described in Example 22.

[0407] Analysis: Analysis was performed as described in Example 1.

[0408] Results: Intermediate compound 32 (1.05 g by optical density, 71.55% by UPLC, expected accurate mass = 8642.905 Da, observed accurate mass = 8642.919 Da) was isolated as a crude sodium salt.

[0409] Enzymatic ligation of oligonucleotide fragments

[0410] Example 33: Comparison of pure oligonucleotide fragments versus crude oligonucleotide fragments in enzymatic ligation catalyzed by RNA ligase to form RNAi agents

[0411] Method 1 (pure oligonucleotide fragments): The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 1 g / L of the first RNA ligase (Almac) to ligate purified intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) in the presence of 2 mM ATP and 10 mM MgCl2. A 10 mg / mL enzyme stock solution was prepared by dissolving RNA ligase 1 in nuclease-free water. In a 2 mL HPLC vial, reaction buffer (800 μl) was prepared by adding nuclease-free water (295.3 μl) containing Tris-HCl, pH 7.5 (1000 mM, 40 μl), MgCl2 (100 mM, 80 μl), KCl (2000 mM, 40 μl), DTT (100 mM, 8 μl), ATP (10 mM, 160 μl), intermediate compound 1 (3.7 mM, 21.6 μl), intermediate compound 2 (4.7 mM, 17.0 μl), intermediate compound 3 (2.9 mM, 27.6 μl), intermediate compound 4 (4.5 mM, 17.8 μl), intermediate compound 5 (6.3 mM, 12.7 μl) and RNA ligase (10 mg / mL, 80 μl). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. Place a 2 mL HPLC vial at 35°C. (500 rpm) for 3 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0412] Method 2 (crude oligonucleotide fragments): Alternatively, the first RNAi agent was synthesized using 1 g / L of the first RNA ligase to ligate crude intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) in the presence of ATP (2 mM) and MgCl2 (10 mM). A 10 mg / mL enzyme stock solution was prepared by dissolving the RNA ligase in nuclease-free water. In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (312.3 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 80 μL), KCl (2000 mM, 40 μL), DTT (100 mM, 8 μL), ATP (10 mM, 160 μL), intermediate compound 1 (4.5 mM, 17.8 μL), intermediate compound 2 (6.8 mM, 11.8 μL), intermediate compound 3 (3.6 mM, 22.2 μL), intermediate compound 4 (5.2 mM, 15.4 μL), intermediate compound 5 (6.4 mM, 12.5 μL) and RNA ligase (10 mg / mL, 80 μL). The reaction mixture was thoroughly mixed by pipetting the solution gently above the HPLC column. Place a 2 mL HPLC vial in an Eppendorf tube at 35 °C. (500 rpm) for 3 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0413] Results: Ligation of method 1 yielded 1.66 mg of the first RNAi agent (81.87% by UPLC), and ligation of method 2 yielded 1.66 mg of the first RNAi agent (83.44% by UPLC).

[0414] Example 34: Comparison of pure oligonucleotide fragments versus crude oligonucleotide fragments in enzymatic ligation catalyzed by RNA ligase to form RNAi agents

[0415] Method 1 (pure oligonucleotide fragments): The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to ligate purified intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) in the presence of ATP (0.4 mM) and MgCl2 (2.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (622.9 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 20 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 40 μL), intermediate compound 1 (5.4 mM, 14.8 μL), intermediate compound 2 (7.4 mM, 10.8 μL), intermediate compound 3 (4.7 mM, 17.0 μL), intermediate compound 4 (6.0 mM, 13.3 μL) and intermediate compound 5 (7.5 mM, 10.8 μL). Enzyme working solution (0.125 g / L, 224 μ L) is prepared by diluting RNA ligase (3.5 g / L, 8 μ L) in enzyme storage buffer (216 μ L). Enzyme working solution (200 μ L) is added to reaction buffer (800 μ L). The reaction mixture is fully mixed by slowly pipetting the solution up and down. At 37 ° C, 2 mL HPLC bottles are placed in Eppendorf (500 rpm) for 2 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0416] Method 2 (crude oligonucleotide fragments): Alternatively, the first RNAi agent was synthesized using 0.025 g / L of a second RNA ligase to catalyze the ligation of crude intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) in the presence of ATP (0.4 mM) and MgCl2 (2.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (622.9 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 20 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 40 μL), intermediate compound 1 (5.4 mM, 14.8 μL), intermediate compound 2 (7.4 mM, 10.8 μL), intermediate compound 3 (4.7 mM, 17.0 μL), intermediate compound 4 (6.0 mM, 13.3 μL) and intermediate compound 5 (7.5 mM, 10.8 μL). Enzyme working solution (0.125 g / L, 224 μ L) is prepared by diluting RNA ligase (3.5 g / L, 8 μ L) in enzyme storage buffer (216 μ L). Enzyme working solution (200 μ L) is added to reaction buffer (800 μ L). The reaction mixture is fully mixed by slowly pipetting the solution up and down. At 37 ° C, 2 mL HPLC bottles are placed in Eppendorf (500 rpm) for 2 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0417] Results: Ligation of method 1 yielded 1.66 mg of the first RNAi agent (89.43% by UPLC), and ligation of method 2 yielded 1.66 mg of the first RNAi agent (92.58% by UPLC).

[0418] Example 35: Standard conditions for enzymatic ligation using RNA ligase to form RNAi agents

[0419] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.1 g / L of a first RNA ligase (Almac) to ligate intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) in the presence of ATP (2 mM) and MgCl2 (10 mM). A 10 mg / mL enzyme stock solution was prepared by dissolving the RNA ligase in nuclease-free water. In a 2 mL HPLC vial, reaction buffer (1000 μL) was prepared by adding nuclease-free water (181.8 μL) containing Tris-HCl, pH 7.5 (1000 mM, 50 μL), MgCl2 (100 mM, 100 μL), KCl (2000 mM, 50 μL), DTT (100 mM, 10 μL), ATP (10 mM, 200 μL), intermediate compound 1 (4.5 mM, 88.9 μL), intermediate compound 2 (6.8 mM, 58.8 μL), intermediate compound 3 (3.6 mM, 111.1 μL), intermediate compound 4 (5.2 mM, 76.9 μL), intermediate compound 5 (6.4 mM, 62.5 μL) and RNA ligase (10 mg / mL, 10 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. Place a 2 mL HPLC vial at 35°C. (500 rpm) for 24 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0420] Results: The ligation yielded 8.29 mg of the first RNAi agent (91.63% by UPLC).

[0421] Example 36: Standard conditions for enzymatic ligation using RNA ligase to form RNAi agents

[0422] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L second RNA ligase (Codexis) to ligate intermediate compounds 1, 2, 3, 16, and 17 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (171.0 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 1 (4.5 mM, 88.9 μL), intermediate compound 2 (6.8 mM, 58.8 μL), intermediate compound 3 (3.6 mM, 111.1 μL), intermediate compound 4 (5.2 mM, 76.9 μL) and intermediate compound 5 (6.4 mM, 62.5 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 5% flask at 37°C. (500 rpm) for 24 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0423] Results: This ligation yielded 8.29 mg of the first RNAi agent (90.24% by UPLC).

[0424] Example 37: Enzymatic ligation using RNA ligase to form RNAi agents

[0425] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to ligate intermediate compounds 1, 6, 7, 5, and 5 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (298.7 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 1 (4.5 mM, 88.9 μL), intermediate compound 6 (16.1 mM, 24.8 μL), intermediate compound 7 (15.1 mM, 26.5 μL), intermediate compound 4 (5.9 mM, 67.8 μL) and intermediate compound 5 (6.4 mM, 62.5 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 5% flask at 37°C. (500 rpm) for 23 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0426] Results: The ligation yielded 8.29 mg of the first RNAi agent (87.34% by UPLC).

[0427] Example 38: Enzymatic ligation using RNA ligase to form RNAi agents

[0428] Synthesis: A first RNAi agent having a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L second RNA ligase (Codexis) to ligate intermediate compounds 1, 8, 9, 4, and 5 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (307.9 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 1 (4.5 mM, 88.9 μL), intermediate compound 8 (21.3 mM, 18.8 μL), intermediate compound 9 (17.2 mM, 23.3 μL), intermediate compound 4 (5.9 mM, 67.8 μL) and intermediate compound 5 (6.4 mM, 62.5 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 5% flask at 37°C. (500 rpm) for 23 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0429] Results: The ligation yielded 8.29 mg of the first RNAi agent (88.80% by UPLC).

[0430] Example 39: Enzymatic ligation using RNA ligase to form RNAi agents

[0431] Synthesis: A first RNAi agent having a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to catalyze the ligation of intermediate compounds 10, 11, 3, 12, and 13 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (298.7 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 10 (15.1 mM, 26.5 μL), intermediate compound 11 (12.7 mM, 31.5 μL), intermediate compound 3 (3.1 mM, 129.0 μL), intermediate compound 12 (9.2 mM, 43.5 μL) and intermediate compound 13 (10.0 mM, 40.0 μL). Enzyme working solution (0.125 g / L, 224 μ L) is prepared by diluting RNA ligase (3.5 g / L, 8 μ L) in enzyme storage buffer (216 μ L). Enzyme working solution (200 μ L) is added to reaction buffer (800 μ L). The reaction mixture is fully mixed by slowly pipetting the solution up and down. At 37 ° C, 2 mL HPLC bottles are placed in Eppendorf (500 rpm) for 24 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0432] Results: The ligation yielded 8.29 mg of the first RNAi agent (78.71% by UPLC).

[0433] Example 40: Enzymatic ligation using RNA ligase to form RNAi agents

[0434] The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to ligate intermediate compounds 14, 15, 3, 16, and 17 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (295.2 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 14 (13.3 mM, 30.1 μL), intermediate compound 15 (10.7 mM, 37.4 μL), intermediate compound 3 (3.1 mM, 129.0 μL), intermediate compound 16 (9.3 mM, 43.0 μL) and intermediate compound 17 (11.6 mM, 34.5 μL). Enzyme working solution (0.125 g / L, 224 μ L) is prepared by diluting RNA ligase (3.5 g / L, 8 μ L) in enzyme storage buffer (216 μ L). Enzyme working solution (200 μ L) is added to reaction buffer (800 μ L). The reaction mixture is fully mixed by slowly pipetting the solution up and down. At 37 ° C, 2 mL HPLC bottles are placed in Eppendorf (500 rpm) for 24 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0435] Results: The ligation yielded 8.29 mg of the first RNAi agent (90.44% by UPLC).

[0436] Example 41: Enzymatic ligation using RNA ligase to form RNAi agents

[0437] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to ligate intermediate compounds 18, 19, 3, 20, and 21 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (307.2 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 18 (14.4 mM, 27.8 μL), intermediate compound 19 (10.5 mM, 38.1 μL), intermediate compound 3 (3.1 mM, 129.0 μL), intermediate compound 20 (9.8 mM, 40.8 μL) and intermediate compound 21 (15.2 mM, 26.3 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 5% flask at 37°C. (500 rpm) for 24 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0438] Results: The ligation yielded 8.29 mg of the first RNAi agent (89.18% by UPLC).

[0439] Example 42: Enzymatic ligation using RNA ligase to form RNAi agents

[0440] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to ligate intermediate compounds 1, 22, 23, 4, and 5 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (13.2 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 1 (4.1 mM, 97.6 μL), intermediate compound 22 (3.3 mM, 121.2 μL), intermediate compound 23 (2.0 mM, 200.0 μL), intermediate compound 4 (5.5 mM, 72.7 μL) and intermediate compound 5 (6.2 mM, 64.5 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase 2 (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 50 mL PBS container at 37°C. (500 rpm) for 23 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0441] Result: The ligation reaction failed because the ligation between intermediate compound 22 and intermediate compound 23 did not occur; therefore, the ligation reaction failed to produce the desired sense strand of SEQ ID NO: 1.

[0442] Example 43: Enzymatic ligation using RNA ligase to form RNAi agents

[0443] Synthesis: A second RNAi agent having a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4 was synthesized using 0.4 g / L of a first RNA ligase (Almac) to ligate crude intermediate compounds 28, 29, 3, 30, and 31 (0.4 mM) in the presence of ATP (2 mM) and MgCl2 (10 mM). A 10 mg / mL enzyme stock solution was prepared by dissolving the RNA ligase in nuclease-free water. In a 2 mL HPLC vial, reaction buffer (1000 μL) was prepared by adding nuclease-free water (145.5 μL) containing Tris-HCl, pH 7.5 (1000 mM, 50 μL), MgCl2 (100 mM, 100 μL), KCl (2000 mM, 50 μL), DTT (100 mM, 10 μL), ATP (10 mM, 200 μL), intermediate compound 28 (4.4 mM, 90.9 μL), intermediate compound 29 (6.3 mM, 63.5 μL), intermediate compound 3 (3.5 mM, 114.3 μL), intermediate compound 30 (4.8 mM, 83.3 μL), intermediate compound 31 (6.4 mM, 62.5 μL) and RNA ligase (10 mg / mL, 30 μL). The reaction mixture was mixed thoroughly by pipetting the solution up and down gently. A 2 mL HPLC vial was placed at 35°C. (500 rpm) for 19 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0444] Results: This ligation yielded 8.29 mg of the second RNAi agent (84.93% by UPLC).

[0445] Example 44: Enzymatic ligation using RNA ligase to form RNAi agents

[0446] Synthesis: A second RNAi agent having a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4 was synthesized using 0.025 g / L of a second RNA ligase (Codexis) to ligate intermediate compounds 28, 29, 3, 30, and 31 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (154.7 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 28 (4.4 mM, 90.9 μL), intermediate compound 29 (6.3 mM, 63.5 μL), intermediate compound 3 (3.5 mM, 114.3 μL), intermediate compound 30 (4.8 mM, 83.3 μL) and intermediate compound 31 (6.4 mM, 62.5 μL). Enzyme working solution (0.125 g / L, 224 mL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 50 mL PBS container at 37°C. (500 rpm) for 20 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0447] Results: This ligation yielded 8.29 mg of the second RNAi agent (82.91% by UPLC).

[0448] Example 45: Enzymatic ligation using RNA ligase to form intermediate compound 33

[0449] Synthesis: Intermediate compound 33 (SEQ ID NO: 37) was synthesized using 0.025 g / L second RNA ligase (Codexis) to connect intermediate compounds 2 and 3 (0.08 mM) in the presence of ATP (0.4 mM) and MgCl2 (2.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding nuclease-free water (655.6 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 20 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 40 μL), intermediate compound 2 (6.8 mM, 11.8 μL) and intermediate compound 3 (3.6 mM, 22.2 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase 2 (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in a 50 mL PBS container at 37°C. (500 rpm) for 2 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0450] Result: The ligation yielded 0.69 mg of intermediate compound 33 (94.58% by UPLC).

[0451] Example 46: Inhibition of the formation of intermediate compound 33 during enzymatic ligation using RNA ligase when forming an RNAi agent

[0452] Synthesis: A second RNAi agent having the sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO: 4 was synthesized using 0.025 g / L second RNA ligase (Codexis) to ligate intermediate compounds 28, 29, 3, 30, and 31 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 2 mL HPLC vial, reaction buffer (685.7 μL) was prepared by adding nuclease-free water (154.7 μL) containing Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl2 (100 mM, 30 μL), DTT (100 mM, 10.4 μL), ATP (10 mM, 150.4 μL), intermediate compound 28 (4.4 mM, 90.9 μL), intermediate compound 29 (6.3 mM, 63.5 μL), intermediate compound 30 (4.8 mM, 83.3 μL), and intermediate compound 31 (6.4 mM, 62.5 μL). Enzyme working solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) in enzyme storage buffer (216 μL). Enzyme working solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by slowly pipetting the solution up and down. A 2 mL HPLC vial was placed in an Eppendorf The reaction was cooled to room temperature and intermediate compound 3 (3.5 mM, 114.3 μL) was placed in a 2 mL HPLC vial, which was returned to the Eppendorf vial at 37°C. The reaction was cooled to room temperature and quenched with EDTA (26.7 mM, 3 mL).

[0453] Results: This ligation yielded 8.29 mg of the second RNAi agent (93.97% by UPLC).

[0454] Example 47: Enzymatic Ligation Amplification Using RNA Ligase to Form RNAi Agents

[0455] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of a third RNA ligase (Codexis) to ligate intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) in the presence of ATP (1.5 mM) and MgCl2 (3.0 mM). In a 200 mL pressure vial, reaction buffer (96.52 mL) was prepared by adding nuclease-free water (21.52 mL) containing Tris-HCl, pH 7.5 (1000 mM, 4.83 mL), MgCl2 (100 mM, 3.62 mL), DTT (100 mM, 1.25 mL), ATP (10 mM, 18.15 mL), intermediate compound 1 (4.5 mM, 10.72 mL), intermediate compound 2 (6.7 mM, 7.20 mL), intermediate compound 3 (3.6 mM, 13.41 mL), intermediate compound 4 (6.0 mM, 8.04 mL) and intermediate compound 5 (6.2 mM, 7.78 mL). The enzyme working solution (0.125 g / L, 24.38 mL) was prepared by diluting RNA ligase (10.16 g / L, 300 μL) in enzyme storage buffer (24.08 mL). The enzyme working solution (24.13 mL) was added to the reaction buffer (96.52 mL). The reaction mixture was thoroughly mixed by gentle inversion. The 200 mL pressure vessel was equipped with a magnetic stirring bar (250 rpm) and an adapter containing a pressure relief valve, a pressure gauge, and a thermocouple to monitor the internal reaction temperature. The reactants were heated to 37° C. in a water bath for 21 hr, with the temperature of the immersed copper coil controlled by a ThermoFisherHaake TM Phoenix II cooler / circulator control. The reaction was quenched with EDTA (26.7 mM, 360 mL). After 21 hr, most of the intermediate compounds 1, 2, 3, 16 and 17 were consumed to obtain the crude first RNAi agent (0.727 g, 92.72% (IM1) by optical density).

[0456] Results: The ligation yielded 0.587 g of purified second RNAi agent (89.46% by UPLC (IM2)).

[0457] Example 48: Enzymatic Ligation Amplification Using RNA Ligase to Form RNAi Agents

[0458] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 1 g / L of a fourth RNA ligase (Codexis) to ligate intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) in the presence of ATP (2.0 mM) and MgCl2 (10.0 mM). A 10 mg / mL enzyme stock solution was prepared by dissolving RNA ligase (125.6 mg) in 50 mM Tris HCl, pH 7.5 (12.56 mL). In a 200 mL pressure vial, reaction buffer (120.7 mL) was prepared by adding nuclease-free water (53.8 mL) containing Tris-HCl, pH 7.5 (1000 mM, 6.0 mL), MgCl2 (100 mM, 12.1 mL), KCl (2000 mM, 6.0 mL), DTT (100 mM, 1.2 mL), ATP (100 mM, 2.4 mL), intermediate compound 1 (8.1 mM, 6.0 mL), intermediate compound 2 (12.1 mM, 4.0 mL), intermediate compound 3 (6.2 mM, 7.8 mL), intermediate compound 4 (10.0 mM, 4.8 mL), intermediate compound 5 (10.8 mM, 4.5 mL) and RNA ligase (10 mg / mL, 12.1 mL). The reaction mixture was mixed thoroughly by gentle inversion. The 200 mL pressure vessel was equipped with a magnetic stir bar (250 rpm) and an adapter containing a pressure relief valve, a pressure gauge, and a thermocouple to monitor the internal reaction temperature. The reaction was heated to 37°C in a water bath for 23.5 hr, where the temperature of the immersed copper coil was controlled by a ThermoFisher Haake TM Phoenix II cooler / circulator control. The reaction was quenched with EDTA (26.7 mM, 54.38 mL). After 21 hr, most of the intermediate compounds 1, 2, 3, 4, and 5 were consumed to obtain the crude first RNAi agent (1.01 g by optical density, 86.44% by UPLC (IM1)).

[0459] Results: The ligation yielded 0.682 g of purified second RNAi agent (90.39% by UPLC (IM2)).

[0460] Example 49: SPOS of Intermediate Compound 1-5 on a 15 or 22 mmol scale

[0461] Synthesis: Intermediate compounds 1, 2, 4 and 5, or pharmaceutically acceptable salts thereof, were synthesized by SPOS at a 22 mmol scale according to the process parameters outlined in Table 8. Similarly, intermediate compound 3, or a pharmaceutically acceptable salt thereof, was synthesized by SPOS at a 15 mmol scale according to the process parameters outlined in Table 8. All syntheses were performed on an AKTA OligoPilot 400 synthesizer.

[0462] Table 8: Optimization method parameters of SPOS

[0463]

[0464]

[0465]

[0466]

[0467]

[0468] After synthesis, the crude oligonucleotide fragments bound by nitrogen drying resin are used until constant mass is reached. Ammonolysis reaction (ammonolysis) is carried out by adding concentrated NH4OH (100ml / mmol) to the crude oligonucleotide fragments on the resin. Material is carried out to the cracking and deprotection conditions shown in Table 9. After 16hr, the resin used is filtered from the ammonolysis solution to obtain the crude deprotected oligonucleotide fragments in solution form.

[0469] Table 9: C&D conditions for intermediate compounds 1-5

[0470]

[0471] Results: The synthesis results after cleavage and deprotection (C&D) are shown in Table 10.

[0472] Table 10: SPOS results of crude intermediate compounds 1-5 after C&D

[0473]

[0474] The ammonia solution was further processed by ultrafiltration and diafiltration (UF / DF) using a Sartorius TFF system equipped with a Sartorius 2kDa MCWO membrane. Salt exchange was performed using a 0.5M NaCl solution. The solution was desalted until the final conductivity value was obtained. The detailed parameters of the UF / DF process are listed in Table 11. After UF / DF, the product was isolated using lyophilization to obtain the intermediate compound 1-5 in the form of a sodium salt. The final yield and purity of the intermediate compound 1-5 are listed in Table 12.

[0475] Table 11: UF / DF parameters for intermediate compounds 1-5.

[0476]

[0477]

[0478] Table 12: Results of Intermediate Compounds 1-5

[0479]

[0480] Example 50: Enzymatic Ligation Amplification Using RNA Ligase to Form RNAi Agents

[0481] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.1 g / L fifth RNA ligase (Codexis, cell-free extract) to ligate intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) in the presence of ATP (2.0 mM) and MgCl2 (10.0 mM). In a 2 L pressure vial, reaction buffer (845.0 mL) was prepared by adding nuclease-free water (602.4 mL) containing Tris-HCl, pH 7.5 (1 M, 42.30 mL), MgCl2 (1 M, 8.50 mL), KCl (6.299 g), DTT (130.0 mg), ATP disodium salt hydrate (931.0 mg), intermediate compound 1 (7.92 mM, 42.67 mL), intermediate compound 2 (11.92 mM, 28.35 mL), intermediate compound 3 (6.02 mM, 56.15 mL), intermediate compound 4 (9.60 mM, 35.21 mL) and intermediate compound 5 (11.49 mM, 29.41 mL). RNA ligase (84.5 mg) was added to the reaction buffer. The 2 L pressure vessel was equipped with a magnetic stir bar (150 rpm) and an adapter containing a pressure relief valve, a pressure gauge, and a thermocouple to monitor the internal reaction temperature. The reaction was heated to 37° C. for 25 hr in a water bath where the temperature of the immersed copper coil was controlled by a ThermoFisher Haake TM Phoenix II cooler / circulator control. After 25 hr, the reaction was cooled to room temperature and quenched with EDTA (0.5 M, 34 mL). Intermediate compounds 1, 2, 3, 4, and 5 were consumed primarily to give the crude first RNAi agent (83.04% (IM2) by UPLC).

[0482] Results: After purification, the ligation yielded 6.62 g (93.82% yield) of potency-corrected RNAi agent (87.66% by UPLC (IM2), 92.91% by native UPLC method).

[0483] Example 51: 100g Enzymatic Ligation Scale-Up Using RNA Ligase to Form RNAi Agents

[0484] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.1 g / L fifth RNA ligase (Codexis, cell-free extract) to ligate intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) in the presence of ATP (2.0 mM) and MgCl2 (10.0 mM). In a 22 L three-necked jacketed reactor, reaction buffer (12.066 L) was prepared by adding nuclease-free water (10.016 L) containing Tris-HCl, pH 7.5 (1 M, 603.275 mL), MgCl2 (1 M, 120.655 mL), KCl (89.948 g), DTT (1.861 g), ATP disodium salt hydrate (13.300 g), intermediate compound 1 (15.37 mM, 314.001 mL), intermediate compound 2 (22.97 mM, 210.109 mL), intermediate compound 3 (15.26 mM, 316.265 mL), intermediate compound 4 (17.37 mM, 277.847 mL), and intermediate compound 5 (23.23 mM, 207.757 mL). RNA ligase (1.207 g) was added to the reaction buffer. A 22 L three-neck jacketed reactor was equipped with an overhead stirrer (80 rpm), a septum, a nitrogen line, and a thermocouple to monitor the internal reaction temperature. A ThermoFisher Haake TM The Phoenix II cooler / circulator was vertical to the fittings on the jacket of the reactor. The reactants were heated to 37°C for 24.5 hours. After 24.5 hours, the reaction was quenched with EDTA (0.5 M, 482.62 mL) and allowed to cool to room temperature. Using a peristaltic pump, the crude reaction mixture was filtered through a Millipak 40 Gamma Gold Capsule with a sterile Durapore membrane (0.22 μm, PVDF). Intermediate compounds 1, 2, 3, 4, and 5 were primarily consumed to obtain a crude first RNAi agent (84.1% (IM2) based on UPLC).

[0485] Results: After purification, the ligation yielded 92.20 g (76.16% yield, extrapolated, corrected for purity and water content) of the RNAi agent as the sodium salt (88.87% by UPLC (IM2), 93.40% by native UPLC method).

[0486] Example 52: Standard Conditions for Enzymatic Ligation Using RNA Ligase to Form RNAi Agents, Four-Fragment Method

[0487] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.1 g / L of fifth RNA ligase (Codexis, cell-free extract) to ligate intermediate compounds 1, 32, 4, and 5 (0.4 mM) in the presence of ATP (2 mM) and MgCl2 (10 mM). A 10 mg / mL enzyme stock solution was prepared by dissolving RNA ligase in nuclease-free water. In a 2 mL HPLC vial, reaction buffer (1000 μL) was prepared by adding nuclease-free water (197.1 μL) containing Tris-HCl, pH 7.5 (1000 mM, 50 μL), MgCl2 (100 mM, 100 μL), KCl (2000 mM, 50 μL), DTT (100 mM, 10 μL), ATP (100 mM, 20 μL), intermediate compound 1 (7.92 mM, 50.5 μL), intermediate compound 32 (0.92 mM, 435.9 μL), intermediate compound 4 (9.60 mM, 41.7 μL), intermediate compound 5 (11.49 mM, 34.8 μL) and RNA ligase (10 mg / mL, 10 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. Place a 2 mL HPLC vial in an Eppendorf tube at 37°C. (500 rpm) for 23 hr. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0488] Results: The ligation yielded 8.29 mg (theoretical) of RNAi agent (88.04% (IM1) by UPLC).

[0489] Example 53: Enzymatic ligation using commercial RNA ligase to form RNAi agents

[0490] Synthesis: The first RNAi agent having the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2 was synthesized using 0.025 g / L of sixth RNA ligase (New England Biolabs, M0239) to ligate intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) in the presence of ATP (2 mM) and MgCl2 (10 mM). In a 2 mL HPLC vial, reaction buffer (500 μL) was prepared by adding nuclease-free water (280.1 μL) containing Tris-HCl, pH 7.5 (1000 mM, 25 μL), MgCl2 (100 mM, 50 μL), KCl (2000 mM, 25 μL), DTT (100 mM, 5 μL), ATP (100 mM, 10 μL), intermediate compound 1 (15.37 mM, 13.0 μL), intermediate compound 2 (22.97 mM, 8.7 μL), intermediate compound 3 (15.26 mM, 13.1 μL), intermediate compound 4 (17.37 mM, 11.5 μL), intermediate compound 5 (23.23 mM, 8.6 μL) and RNA ligase (0.25 mg / mL, 50 μL). The reaction mixture was mixed thoroughly by pipetting the solution up and down gently. A 2 mL HPLC vial was placed in an Eppendorf (500 rpm) for 20 hr. The reaction was quenched with EDTA (26.7 mM, 1.5 mL).

[0491] Results: This ligation yielded 4.15 mg (theoretical) of RNAi agent (84.71% (IM2) by UPLC).

[0492] sequence

[0493] The following nucleotide and / or amino acid sequences are referred to in the present invention and are provided below for reference.

[0494] SEQ ID NO: 1—Artificial nucleic acid #1 (36 nt)

[0495] mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG- mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0496] SEQ ID NO:2—Artificial nucleic acid #2 (22 nt)

[0497] [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0498] SEQ ID NO:3–Union fragment#3(36nt)

[0499] mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG- mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0500] SEQ ID NO:4–Union fragment#4(22nt)

[0501] [MePhosphonate-4O-mU S ]-fG S -fU S -fA-fU-mA-fA-mC-mC-fU-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S -mG

[0502] SEQ ID NO:5–United fragment 1(14nt)

[0503] mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU

[0504] SEQ ID NO:6–Free fragment 2(10nt)

[0505] p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0506] SEQ ID NO:7–United fragment 3(12nt)

[0507] p-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0508] SEQ ID NO:8—Intermediate compound 4 (12 nt)

[0509] p-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0510] SEQ ID NO:9—Intermediate compound 5 (10 nt)

[0511] [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC-mC-fA

[0512] SEQ ID NO:10—Intermediate compound 6 (12 nt)

[0513] p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC

[0514] SEQ ID NO:11—Intermediate compound 7 (10 nt)

[0515] p-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0516] SEQ ID NO:12—Intermediate compound 8 (11 nt)

[0517] p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC

[0518] SEQ ID NO:13—Intermediate compound 9 (11 nt)

[0519] p-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0520] SEQ ID NO:14—Intermediate Compound 10 (10 nt)

[0521] mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU

[0522] SEQ ID NO:15—Intermediate Compound 11 (14 nt)

[0523] p-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0524] SEQ ID NO:16—Intermediate compound 12 (16 nt)

[0525] p-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0526] SEQ ID NO:17—Intermediate Compound 13 (6-nt)

[0527] [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG

[0528] SEQ ID NO:18—Intermediate compound 14 (9 nt)

[0529] mU S -mU-mG-mC-mC-mA-mA-fG-fC

[0530] SEQ ID NO:19—Intermediate Compound 15 (15nt)

[0531] p-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0532] SEQ ID NO:20—Intermediate compound 16 (15 nt)

[0533] p-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0534] SEQ ID NO:21—Intermediate compound 17 (7 nt)

[0535] [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA

[0536] SEQ ID NO:22—Intermediate compound 18 (8 nt)

[0537] mU S -mU-mG-mC-mC-mA-mA-fG

[0538] SEQ ID NO:23—Intermediate compound 19 (16 nt)

[0539] p-fC-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0540] SEQ ID NO:24—Intermediate compound 20 (14 nt)

[0541] p-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0542] SEQ ID NO:25—Intermediate compound 21 (8 nt)

[0543] [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC

[0544] SEQ ID NO:26—Intermediate compound 22 (13 nt)

[0545] p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG

[0546] SEQ ID NO:27—Intermediate compound 23 (9 nt)

[0547] p-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0548] SEQ ID NO:28—Intermediate compound 24 (18 nt)

[0549] p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA

[0550] -GalNAc]-[ademA-GalNAc]-mG-mG

[0551] SEQ ID NO:29—Intermediate compound 25 (4 nt)

[0552] mC-mU-mG-mC

[0553] SEQ ID NO:30—Intermediate compound 26 (6 nt)

[0554] p-mG-mC-mA-mA S -mG S -mG

[0555] SEQ ID NO:31—Intermediate compound 27 (6 nt)

[0556] p-mA-mG-mC-fU-mU-mG

[0557] SEQ ID NO:32—Intermediate compound 28 (14 nt)

[0558] mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU

[0559] SEQ ID NO:33—Intermediate compound 29 (10 nt)

[0560] p-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG

[0561] SEQ ID NO:34—Intermediate compound 30 (12 nt)

[0562] p-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S -mG

[0563] SEQ ID NO:35—Intermediate compound 31 (10 nt)

[0564] [MePhosphonate-4O-mU S ]-fG S -fU S -fA-fU-mA-fA-mC-mC-fU

[0565] SEQ ID NO:36—Intermediate compound 32 (22 nt)

[0566] p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0567] SEQ ID NO:37—Intermediate compound 33 (22 nt)

[0568] Ring-(mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC)

Claims

1. An oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 5 to 37, or a pharmaceutically acceptable salt thereof.

2. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

5.

3. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

6. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

7. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

8. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

9. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

10. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

11.

9. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

12.

10. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

13. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

14.

12. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

15.

13. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

16. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

17.

15. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

18. The oligonucleotide according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

19.

17. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

20.

18. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

21.

19. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

22.

20. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

23.

21. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

24.

22. The oligonucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO:

25.

23. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

26.

24. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

27.

25. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

28.

26. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

29.

27. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

30.

28. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

31.

29. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

32.

30. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

33.

31. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

34.

32. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

35.

33. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

36.

34. The oligonucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

37.

35. A method for preparing the nucleic acid of SEQ ID NO: 1, the method comprising the following steps: Ligate three oligonucleotide fragments selected from the group consisting of: (a) SEQ ID NOs: 5, 6 and 7, (b) SEQ ID NO: 5, 10 and 11, (c) SEQ ID NOs: 5, 12 and 13, (d) SEQ ID NOs: 7, 14 and 15, (e) SEQ ID NOs: 7, 18 and 19, (f) SEQ ID NOs: 7, 22 and 23, and (g) SEQ ID NOs: 5, 26 and 27.

36. The method of claim 35, wherein the three oligonucleotide fragments are (a) SEQ ID NOs: 5, 6, and 7, and wherein the fragments are SEQ ID NOs: 5 to 6 to 7 linked from the 5' end to the 3' end.

37. The method of claim 35, wherein the three oligonucleotide fragments are (b) SEQ ID NOs: 5, 10, and 11, and wherein the fragments are SEQ ID NO: 5 to SEQ ID NO: 10 to SEQ ID NO: 11 linked from the 5' end to the 3' end.

38. The method of claim 35, wherein the three oligonucleotide fragments are (c) SEQ ID NOs: 5, 12, and 13, and wherein the fragments are SEQ ID NO: 5 to SEQ ID NO: 12 to SEQ ID NO: 13 linked from the 5' end to the 3' end.

39. The method of claim 35, wherein the three oligonucleotide fragments are (d) SEQ ID NOs: 7, 14, and 15, and wherein the fragments are SEQ ID NO: 14 to SEQ ID NO: 15 to SEQ ID NO: 7 linked from the 5' end to the 3' end.

40. The method of claim 35, wherein the three oligonucleotide fragments are (e) SEQ ID NOs: 7, 18, and 19, and wherein the fragments are SEQ ID NO: 18 to SEQ ID NO: 19 to SEQ ID NO: 7 linked from the 5' end to the 3' end.

41. The method of claim 35, wherein the three oligonucleotide fragments are (f) SEQ ID NOs: 7, 22, and 23, and wherein the fragments are SEQ ID NO: 22 to SEQ ID NO: 23 to SEQ ID NO: 7 linked from the 5' end to the 3' end.

42. The method of claim 35, wherein the three oligonucleotide fragments are (g) SEQ ID NOs: 5, 26, and 27, and wherein the fragments link SEQ ID NO: 5 to SEQ ID NO: 26 to SEQ ID NO: 27 from the 5' end to the 3' end.

43. A method for preparing the nucleic acid of SEQ ID NO: 2, the method comprising the following steps: Ligate two oligonucleotide fragments selected from: (a') SEQ ID NOs: 8 and 9; (b') SEQ ID NOs: 16 and 17; (c') SEQ ID NOs: 20 and 21; and (d') SEQ ID NOs: 24 and 25.

44. The method of claim 43, wherein the two oligonucleotide fragments are (a') SEQ ID NOs: 8 and 9, and wherein the fragments link SEQ ID NO: 9 to SEQ ID NO: 8 from the 5' end to the 3' end.

45. The method of claim 43, wherein the two oligonucleotide fragments are (b') SEQ ID NOs: 16 and 17, and wherein the fragments link SEQ ID NO: 17 to SEQ ID NO: 16 from the 5' end to the 3' end.

46. ​​The method of claim 43, wherein the two oligonucleotide fragments are (c') SEQ ID NOs: 20 and 21, and wherein the fragments link SEQ ID NO: 21 to SEQ ID NO: 20 from the 5' end to the 3' end.

47. The method of claim 43, wherein the two oligonucleotide fragments are (d') SEQ ID NOs: 24 and 25, and wherein the fragments link SEQ ID NO: 25 to SEQ ID NO: 24 from the 5' end to the 3' end.

48. A method for preparing the nucleic acid of SEQ ID NO: 3, the method comprising the following steps: (a) connecting three oligonucleotide fragments of SEQ ID NOs: 7, 32, and 33, wherein the fragments connect SEQ ID NO: 32 to SEQ ID NO: 33 to SEQ ID NO: 7 from the 5' end to the 3' end.

49. A method for preparing the nucleic acid of SEQ ID NO: 4, the method comprising the following steps: Connecting (a') two oligonucleotide fragments of SEQ ID NOs: 34 and 35, wherein the fragment connects SEQ ID NO: 35 to SEQ ID NO: 34 from the 5' end to the 3' end.

50. A method for preparing an RNAi agent having a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2, the method comprising the steps of: The sense strand of SEQ ID NO: 1 was formed by ligating three oligonucleotide fragments selected from the group consisting of: (a) SEQ ID NOs: 5, 6, and 7, wherein the fragment is formed by linking SEQ ID NO: 5 to SEQ ID NO: 6 to SEQ ID NO: 7 from the 5' end to the 3' end, (b) SEQ ID NOs: 5, 10, and 11, wherein the fragment is formed by linking SEQ ID NO: 5 to SEQ ID NO: 10 to SEQ ID NO: 11 from the 5' end to the 3' end, (c) SEQ ID NOs: 5, 12, and 13, wherein the fragment is formed by linking SEQ ID NO: 5 to SEQ ID NO: 12 to SEQ ID NO: 13 from the 5' end to the 3' end, (d) SEQ ID NOs: 7, 14, and 15, wherein the fragment is formed by linking SEQ ID NO: 14 to SEQ ID NO: 15 to SEQ ID NO: 7 from the 5' end to the 3' end, (e) SEQ ID NOs: 7, 18, and 19, wherein the fragment is formed by linking SEQ ID NO: 18 to SEQ ID NO: 19 to SEQ ID NO: 7 from the 5' end to the 3' end, (f) SEQ ID NOs: 7, 22, and 23, wherein the fragment is SEQ ID NO: 22 to SEQ ID NO: 23 to SEQ ID NO: 7 connected from the 5' end to the 3' end, and (g) SEQ ID NOs: 5, 26, and 27, wherein the fragment is formed by linking SEQ ID NO: 5 to SEQ ID NO: 26 to SEQ ID NO: 27 from the 5' end to the 3' end; The antisense strand of SEQ ID NO: 2 was formed by ligating two oligonucleotide fragments selected from: (a') SEQ ID NOs: 8 and 9, wherein the fragment is formed by linking SEQ ID NO: 9 to SEQ ID NO: 8 from the 5' end to the 3' end, (b') SEQ ID NOs: 16 and 17, wherein the fragment is formed by linking SEQ ID NO: 17 to SEQ ID NO: 16 from the 5' end to the 3' end, (c') SEQ ID NOs: 20 and 21, wherein the fragment is formed by linking SEQ ID NO: 21 to SEQ ID NO: 20 from the 5' end to the 3' end, and (d') SEQ ID NOs: 24 and 25, wherein the fragment is formed by linking SEQ ID NO: 25 to SEQ ID NO: 24 from the 5' end to the 3' end; and The RNAi agent is formed by annealing complementary nucleotides of the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO:

2.

51. A method for preparing an RNAi agent having a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4, the method comprising the steps of: Forming the sense strand of SEQ ID NO: 3 by ligating (a) three oligonucleotide fragments of SEQ ID NO: 7, 32, and 33, wherein the fragments are 5' to 3' linked from SEQ ID NO: 32 to SEQ ID NO: 33 to SEQ ID NO: 7; forming an antisense strand of SEQ ID NO: 4 by ligating (a') two oligonucleotides of SEQ ID NO: 34 and 35, wherein the fragment ligates SEQ ID NO: 35 to SEQ ID NO: 34 from the 5' end to the 3' end; and The RNAi agent is formed by annealing complementary nucleotides of the sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO:

4.

52. The method of any one of claims 35 to 51, wherein the ligation step is mediated by an enzyme.

53. The method of claim 52, wherein the enzyme is deoxyribonucleic acid (DNA) ligase or ribonucleic acid (RNA) ligase.

54. The method of claim 52, wherein the enzyme is an RNA ligase and is selected from RNA ligase 1 and RNA ligase 2.

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