Oligonucleotide fragments and methods for producing RNAi agents using the same

Abstract

Intermediate compounds (i.e., oligonucleotide fragments) for making RNAi agents or pharmaceutically acceptable salts thereof are disclosed. Additionally, methods for making single-stranded oligonucleotides by ligating two or more intermediate compounds herein via a hybrid chemical-enzymatic route are disclosed.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the full Paris Convention benefit of and priority to U.S. Provisional Patent Application No. 63 / 434,661, filed December 22, 2022.

[0002] (Reference to electronically submitted sequence listing)

[0001] The present disclosure is filed with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file named "30019_US_PRI" created on October 24, 2023, and is 35 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates generally to biology, chemistry, and medicine, and more particularly to methods for synthesizing oligonucleotides via a hybrid chemical-enzymatic route. [Background technology]

[0004] Oligonucleotides are used in various biological and biochemical applications. Of interest herein is the use of oligonucleotides as therapeutic agents, such as RNA activation (RNAa), RNA editing (RNAe) and RNA interference (RNAi). Such widespread use of oligonucleotides leads to an increasing demand for their rapid, inexpensive and efficient synthesis method.

[0005] Oligonucleotides can be synthesized by many methods known in the art, particularly by solid-phase synthesis through the repeated coupling of nucleoside phosphoramidites.See, for example, Beaucage & Caruthers (1981) Tetrahedron Letters 22:1859-1862; McBride & Caruthers (1983) Tetrahedron Letters 24:245-248; Sinha et al. (1984) Nucleic Acids Res.12:4539-4557 and Beaucage & Iyer (1992) Tetrahedron 48:2223-2311.

[0006] However, alternative methods for producing oligonucleotides and their intermediates are needed to enable industrially pharmaceutically acceptable production at desired purity levels, as well as efficient methods and stable intermediates to efficiently provide oligonucleotides with fewer purification steps. Summary of the Invention

[0007] To address this need, the present disclosure describes nucleotide-based intermediate compounds (i.e., oligonucleotide fragments) as well as methods for generating single-stranded ss oligonucleotides by ligating multiple oligonucleotide fragments herein, ultimately generating double-stranded ds therapeutic oligonucleotides such as RNAi agents.

[0008] With respect to intermediate compounds, the present disclosure describes oligonucleotide fragments having a nucleotide sequence selected from any one of SEQ ID NOs: 5-37.

[0009] With respect to methods, the present disclosure provides a method for making a nucleic acid having the nucleotide sequence of SEQ ID NO:1, comprising: (a) SEQ ID NOs: 5, 6, and 7; (b) SEQ ID NOs: 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) ligating a combination of oligonucleotide fragments having nucleotide sequences selected from SEQ ID NOs: 5, 26, and 27.

[0010] Further disclosed is a method of making a nucleic acid having the nucleotide sequence of SEQ ID NO:2, comprising: (a') SEQ ID NOs: 8 and 9; (b') SEQ ID NOs: 16 and 17; (c') SEQ ID NOs: 20 and 21, and (d') ligating a combination of oligonucleotide fragments having nucleotide sequences selected from SEQ ID NOs: 24 and 25.

[0011] 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 apolipoprotein(a) gene (LPA) expression.

[0012] Additionally, the present disclosure describes a method for producing a nucleic acid having the nucleotide sequence of SEQ ID NO: 3, comprising: (a) ligating a combination of oligonucleotide fragments having nucleotide sequences selected from SEQ ID NOs: 7, 32, and 33.

[0013] Furthermore, the present disclosure describes a method for producing a nucleic acid having the nucleotide sequence of SEQ ID NO: 4, comprising the step of (a') ligating a combination of oligonucleotide fragments having nucleotide sequences selected from SEQ ID NOs: 34 and 35.

[0014] 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 angiopoietin-like 3 gene (ANGPTL3) expression.

[0015] In any of the above methods, the ligation can be mediated by an enzyme. In some instances, the enzyme is a ligase, such as a naturally occurring or non-naturally occurring ligase. In other instances, the ligase is a DNA ligase. In particular instances, the ligase is an RNA ligase.

[0016] Advantages of the methods herein include process improvements such as shorter fragments initially generated via solid phase oligonucleotide synthesis (SPOS), allowing for improved purity and higher yields. With shorter fragments, more pathway flexibility is available for incorporating modified nucleotides and the ability to redesign fragment structures to address more challenging segments of the chain.

[0017] Advantages of the methods herein include improved control strategies for impurities during synthesis, which may include improved detection and characterization of impurities at the fragmentation stage, as well as improved final impurity profiles of crude oligonucleotide duplexes.

[0018] Advantages of the methods herein include improved final duplex purity due to the rejection of certain classes of fragment impurities based on their inability to participate in the ligation step, or reduced adherence to the complementarity principle during fragment self-assembly in the annealing step.

[0019] Advantages of the methods herein include a reduction in the unit operations required to generate oligonucleotide duplexes as a single step used to form duplex material from fragment building blocks, allowing downstream operations to be reduced to a single chromatography and ultrafiltration (versus the need for separate steps for each strand as in conventional approaches).

[0020] Advantages of the methods herein include that the synthesis of short fragments via SPOS allows for shorter wash cycles and reduced reagent amounts, leading to lower process mass intensity (PMI).

[0021] Advantages of the methods herein include the use of shorter fragment intermediates, which reduces the impact of synthetic failures during fragment production, reducing the overall cost and impact of the production cycle.

[0022] Advantages of the methods herein include that shorter fragment intermediates are more amenable to new synthetic manufacturing platforms and that enzymatic assembly of oligonucleotide duplexes allows for the introduction of other innovative technologies for downstream unit operations.

[0023] Advantages of the method of the present invention include flexibility in the supply chain and logistics of the manufacturing process by using several independent pieces.

[0024] Advantages of the methods herein include additional flexibility in the supply chain resulting from sequences utilizing the same delivery platform, allowing for the design of conservative fragments with identical nucleotide sequence composition.

[0025] An advantage of the method of the present invention is that the use of parallel fragment production allows for a shorter production cycle due to parallel processing of the fragments.

[0026] Advantages of the methods of the present invention include that the current Good Manufacturing Practice (cGMP) enzymatic ligation step can be carried out under aqueous-based conditions in a solvent-free facility without the need for specialized equipment. [Brief explanation of the drawings]

[0027] Further advantages, benefits, features and objects will become more readily apparent from a consideration of the following detailed description, which refers to the following drawings. [Figure 1] 1 shows a structural diagram of an exemplary RNAi agent (SEQ ID NOs: 1 and 2) that has a nicked tetraloop structure and regulates LPA expression. [Figure 2]1 shows a structural diagram of an exemplary RNAi agent (SEQ ID NOs: 3 and 4) that has a nicked tetraloop structure and regulates ANGTPL3 expression. DETAILED DESCRIPTION OF THE INVENTION

[0028] overview WO 2022 / 032288 describes RNAi agents (e.g., LPA-3291-M1) that can be used to attenuate, prevent, and / or treat diseases, disorders, and / or conditions associated with LPA expression (i.e., to reduce LPA mRNA and Apo(a) protein levels, and reduce Lp(a) levels). The RNAi agents contain an N-acetylgalactosamine (GalNAc) ligand to target them to the asialoglycoprotein receptor (ASGPR).

[0029] WO 2021 / 188795 describes RNAi agents (e.g., GalXC-1412) that can be used to attenuate, prevent, and / or treat diseases, disorders, and / or conditions associated with ANGPTL3 expression (i.e., to reduce ANGPTL3 mRNA and ANGPTL3 protein levels). The RNAi agent includes a GalNAc ligand to target it to ASGPR.

[0030] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the incretin analogs, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0031] Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0032] Certain abbreviations used herein are defined as follows: "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), and "fX s " refers to a 2'-fluoro nucleotide linked via a phosphorothioate bond, "mX" refers to a 2'-O-methyl nucleotide (e.g., mA, mG, mU, mC), and "mX s " refers to a 2'-O-methyl nucleotide linked via a phosphorothioate bond (e.g., mA S , mA S , mU S , mC S ), and "adem A" refers to 2'-O-GalNAc modified adenosine (sodium salt equivalent), which has the following structure:

[0033] [ka]

[0034] "ACN" refers to acetonitrile (C2H3N), "ANGPTL3" refers to angiopoietin-like 3 gene, "ASGPR" refers to asialoglycoprotein receptor, "cGMP" refers to current good manufacturing practice, "CV" refers to column volume(s), "Da" refers to dalton(s), "DCA" refers to dichloroacetic acid (C2H2Cl2O2), and "DEA" refers to diethylamine (C4H 11 N), and "DIPEA" refers to N,N-diisopropylethylamine (CH 19N), "Dmt" refers to 4,4'-dimethoxytrityl, "DNA" refers to deoxyribonucleic acid, "ds" refers to double-stranded, "DTT" refers to 1,4-dithiothreitol, and "EDTA" refers to ethylenediaminetetraacetic acid (C 10 H 16 "ETT" refers to 5-(ethylthio)-1H-tetrazole (C3H6N4S), and "GalNAc" refers to N-acetylgalactosamine, which has the following structure:

[0035] [ka]

[0036] "HFIP" refers to hexafluoroisopropanol (C3H2F6O), "HPLC" refers to high performance liquid chromatography, "LPA" refers to the apolipoprotein(a) gene, and "meMOPs mU" refers to 4'-O-monomethylphosphonate-2'-O-methyluridine (sodium salt equivalent), which has the following structure:

[0037] [ka]

[0038] "MWCO" refers to molecular weight cut-off, "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, and "T m " refers to melting temperature, "UPLC" refers to ultra performance liquid chromatography, "UF / DF" refers to ultrafiltration and diafiltration, and "UV" refers to ultraviolet light.

[0039] Certain definitions used herein are defined as follows: As used herein, "about" means within a statistically significant range of a value, such as, for example, a stated concentration, length, molecular weight, pH, pressure, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0040] As used herein, "anneal," "annealing," and the like refer to hybridizing complementary oligonucleotides in a sequence-specific manner. Annealing conditions are determined by the melting temperature (T m ) and will be readily apparent to one skilled in the art. For example, the annealing temperature depends on the T m Alternatively, the annealing temperature may be lower than the T mThe annealing temperature may be close to (e.g., + / - about 1°C, 2°C, or 3°C). The annealing temperature is typically between the T m never exceeding about 10°C.

[0041] As used herein, "antisense strand" or "guide strand" refers to an ss oligonucleotide that is complementary to a region of a target sequence, such as a target sequence in an mRNA. Similarly, as used herein, "sense strand" or "passenger strand" refers to an ss oligonucleotide that is complementary to a region of the antisense strand.

[0042] As used herein, "asialoglycoprotein receptor" or "ASGPR" refers to a bipartite C-type lectin formed by a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2).

[0043] As used herein, a "chemically synthesized" oligonucleotide refers to an oligonucleotide that is produced, for example, by using a chemical reaction without the use of an enzyme. Methods for chemically synthesizing oligonucleotides, such as RNA molecules, are known in the art, in particular the chemical synthesis methods described in Verma & Eckstein (1998) or described herein. In general, ds RNA 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; 6,469,158; and Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441; also see Beaucage & Caruthers (1981) Tetrahedron Letters 22:1859-1862; McBride & Caruthers (1983) Tetrahedron Letters 24:245-248; Sinha et al. (1984) Nucleic Acids Res. 12:4539-4557 and Beaucage & Iyer (1992) Tetrahedron (48:2223-2311); and International Patent Application Publication Nos. 2005 / 070859 and 2012 / 157723.

[0044] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one oligonucleotide that are complementary to pyrimidine nucleotides of an opposing oligonucleotide may base pair with each other by forming hydrogen bonds. Complementary nucleotides may base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two oligonucleotides may have regions of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0045] As used herein, "deoxyribonucleotide" refers to a nucleotide that, when compared to a ribonucleotide, has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar. Modified deoxyribonucleotides have one or more modifications or substitutions of an atom other than a hydroxyl at the 2' position, including modifications or substitutions of the nucleobase, sugar, or phosphate group.

[0046] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide in duplex form. Complementary base pairing in the duplex region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently separated nucleic acids. Similarly, complementary base pairing in the duplex region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently linked nucleic acids. Furthermore, complementary base pairing in the duplex region(s) of a ds oligonucleotide can be formed from ss nucleic acids that are folded back (e.g., via a hairpin) to provide complementary antiparallel sequences of base-pairing nucleotides together. A ds oligonucleotide can contain two covalently separated nucleic acids that are completely duplex with each other. However, a ds oligonucleotide can also contain two covalently separated nucleic acids that are partially duplex (e.g., with overhangs at one or both ends). A ds oligonucleotide can contain antiparallel sequences of partially complementary nucleotides and therefore can have one or more mismatches, which can include internal or terminal mismatches.

[0047] As used herein, "duplex" and "duplex region" with respect to a nucleic acid (e.g., an oligonucleotide) refer to the structure formed through complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two covalently separated nucleic acid strands or by a single folded strand (e.g., via a hairpin).

[0048] As used herein, "enzymatic ligation," "enzymatically ligating," and the like, mean that a linkage between two adjacent nucleotides is enzymatically formed, and such 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.

[0049] As used herein, an "enzymatically synthesized" oligonucleotide refers to an oligonucleotide having a modification produced by the reaction of a nucleic acid with an enzyme, including naturally occurring and non-naturally occurring enzymes (e.g., kinases, ligases, methyltransferases, nicking enzymes, nucleases, phosphatases, sulfurylases, and recombinases). Correspondingly, as used herein, an "enzymatic" modification refers to a modification produced by the reaction of a nucleic acid with an enzyme, including naturally occurring and non-naturally occurring enzymes.

[0050] As used herein, "ligase" refers to an enzyme that catalyzes the joining (i.e., covalent bonding) of two oligonucleotides, for example, by forming a PO bond between the 3' end of one oligonucleotide (or fragment) and the 5' end of the same or another oligonucleotide (or fragment). These enzymes are often referred to as DNA ligases or RNA ligases and are typically members of the enzyme class EC6.5 (i.e., ligases used to form phosphoester bonds) as defined by the International Union of Biochemistry and Molecular Biology. Furthermore, the ligases herein are capable of joining an unmodified oligonucleotide to another unmodified oligonucleotide, an unmodified oligonucleotide to a modified oligonucleotide (i.e., a modified 5' oligonucleotide to an unmodified 3' oligonucleotide and / or an unmodified 5' oligonucleotide to a modified 3' oligonucleotide), and / or a modified oligonucleotide to another modified oligonucleotide.

[0051] As used herein, a "modified ligase" or "non-naturally occurring ligase" refers to a ligase that differs from a naturally occurring (i.e., wild-type) ligase by one or more amino acid residues.

[0052] As used herein, "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide may be a non-naturally occurring nucleotide. A modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, a modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0053] As used herein, "N-acetylgalactosamine" or "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactose or a derivative thereof, which may be directly or indirectly conjugated to the oligonucleotides herein to target the oligonucleotides to ASGPR.

[0054] As used herein, "nicked tetraloop structure" means a structure of an RNAi agent characterized by separate sense and antisense strands, where the sense strand has a region complementary to the antisense strand, and at least one of the strands, generally the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed within at least one strand.

[0055] As used herein, "non-naturally occurring" means an oligonucleotide, nucleic acid, peptide, polypeptide, or protein that does not occur in nature or that is identical to one that occurs in nature but that has been modified in a manner produced or derived by synthetic means (i.e., engineered, recombinant, or modified by human manipulation).

[0056] As used herein, "nucleotide" refers to an organic compound having a nucleoside (e.g., a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar such as ribose or 2'-deoxyribose) and a phosphate group. "Nucleotides" can function as monomer units of nucleic acid polymers, such as deoxyribonucleic acid (DNA) oligonucleotides and ribonucleic acid (RNA) oligonucleotides.

[0057] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide can 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 RNAs (DsiRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and small interfering RNAs (siRNAs).

[0058] As used herein, "overhang" refers to terminal unbase-paired nucleotide(s) originating from one strand or region that extends beyond the end of the complementary strand with which it forms a duplex. In some embodiments, the overhang comprises one or more unpaired nucleotides extending from the duplex region at the 5' or 3' end of the ds oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of the ds oligonucleotide.

[0059] As used herein, "pharmaceutically acceptable buffer" means any of the standard pharmaceutical buffers known to those of skill in the art.

[0060] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at the 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 of the nucleobase, sugar, or phosphate group.

[0061] As used herein, "iRNA," "iRNA agent," "RNAi," "RNAi agent," and "RNA interference agent" refer to an oligonucleotide that contains RNA and mediates targeted cleavage of an RNA transcript via RNA interference, e.g., through the RNA-induced silencing complex (RISC) pathway. An RNAi agent can have a sense strand and an antisense strand, where the sense strand and the antisense strand form a duplex. In some examples, the sense strand and the antisense strand of an RNAi agent can be 21-23 nucleotides in length. Alternatively, the sense strand and the antisense strand can be longer, e.g., 25-36 nucleotides in length, in which case the longer nucleotide sequence is processed first by the Dicer enzyme. An RNAi agent directs the sequence-specific degradation of mRNA via RNA interference. An RNAi agent attenuates, inhibits, modulates, or reduces gene expression (here, e.g., ANGPTL3 or LPA expression) in a cell, tissue, organ, system, or individual.

[0062] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together by internucleotide linkages (e.g., P-O or P-S linkages / bonds). A strand may have two free ends (e.g., a 5' end and a 3' end).

[0063] As used herein, a "targeting ligand" refers to a chemical moiety that facilitates the entry of an oligonucleotide, such as an RNAi agent herein, into a tissue or cell. It can be a compound (e.g., an amino sugar, carbohydrate, cholesterol, lipid, or polypeptide) that selectively binds to a cognate compound (e.g., a receptor) in a tissue or cell of interest and can be conjugated to another substance to target the other substance to the tissue or cell of interest. For example, a targeting ligand can be conjugated to an oligonucleotide herein for the purpose of targeting the oligonucleotide to a specific cell or tissue of interest. The targeting ligand can selectively bind to a cell surface receptor. Thus, when conjugated to an oligonucleotide, the targeting ligand facilitates delivery of the oligonucleotide to a specific cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, targeting ligand, and receptor. Furthermore, the targeting ligand can be conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, thereby releasing the oligonucleotide from the targeting ligand within the cell.

[0064] composition Oligonucleotide fragments The present disclosure describes oligonucleotide fragments (i.e., intermediate compounds) having exemplary sequences / structures according to the following:

[0065] Intermediate compound 1: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU 3' (SEQ ID NO: 5).

[0066] Intermediate compound 2: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG3' (SEQ ID NO: 6).

[0067] Intermediate compound 3: 5'p-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 7).

[0068] Intermediate compound 4: 5'p-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 8).

[0069] Intermediate compound 5: 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC-mC-fA 3' (SEQ ID NO: 9).

[0070] Intermediate compound 6: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC3' (SEQ ID NO: 10)

[0071] Intermediate compound 7: 5'p-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC3' (SEQ ID NO: 11).

[0072] Intermediate compound 8: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC3' (SEQ ID NO: 12).

[0073] Intermediate compound 9: 5'p-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' (SEQ ID NO: 13).

[0074] Intermediate compound 10: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU3' (SEQ ID NO: 14).

[0075] Intermediate compound 11: 5'p-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG3' (SEQ ID NO: 15).

[0076] Intermediate compound 12: 5'p-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 16).

[0077] Intermediate compound 13: 5'-[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG3' (SEQ ID NO: 17).

[0078] Intermediate compound 14: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC3' (SEQ ID NO: 18).

[0079] Intermediate compound 15: 5'p-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG3' (SEQ ID NO: 19).

[0080] Intermediate compound 16: 5'p-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 20).

[0081] Intermediate compound 17: 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA3' (SEQ ID NO: 21).

[0082] Intermediate compound 18: 5'mU S-mU-mG-mC-mC-mA-mA-fG3' (SEQ ID NO: 22).

[0083] Intermediate compound 19: 5'p-fC-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG3' (sequence number 23).

[0084] Intermediate compound 20: 5'p-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 24).

[0085] Intermediate compound 21: 5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC3' (SEQ ID NO: 25).

[0086] Intermediate compound 22: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG3' (sequence number 26).

[0087] Intermediate compound 23: 5'p-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC3' (SEQ ID NO: 27).

[0088] Intermediate compound 24: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA -GalNAc]-[ademA-GalNAc]-mG-mG3' (SEQ ID NO: 28).

[0089] Intermediate compound 25: 5'mC-mU-mG-mC3' (sequence number 29).

[0090] Intermediate compound 26: 5'p-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 30).

[0091] Intermediate compound 27: 5'p-mA-mG-mC-fU-mU-mG3' (SEQ ID NO: 31).

[0092] Intermediate compound 28: 5'mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU3' (SEQ ID NO: 32).

[0093] Intermediate compound 29: 5'p-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG3' (sequence number 33).

[0094] Intermediate compound 30: 5'p-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S -mG3' (SEQ ID NO: 34).

[0095] Intermediate compound 31: 5'-[MePhosphonate-4O-mU S ]-fG S -fU S -fA-fU-mA-fA-mC-mC-fU3' (SEQ ID NO: 35).

[0096] Intermediate compound 32: 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-mC3' (SEQ ID NO: 36).

[0097] Intermediate compound 33: 5'cyclo-(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: 37).

[0098] As detailed above and below, certain combinations of oligonucleotide fragments herein can be ligated together and are useful for creating 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 ligated together and are useful for creating a second RNAi agent having a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4.

[0099] RNAi agents As described above, particular combinations of oligonucleotide fragments (ie, intermediate compounds) herein may be ligated to form the RNAi agents herein.

[0100] A first RNAi agent that can be formed from a particular oligonucleotide fragment herein 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, as shown in Figure 1. The first RNAi agent is useful for attenuating, preventing, and / or treating diseases, disorders, and / or conditions associated with LPA expression.

[0101] Second RNAi agents that can be formed from certain of the oligonucleotide fragments herein include a sense strand having the nucleotide sequence of SEQ ID NO: 3 and an antisense strand having the nucleotide sequence of SEQ ID NO: 4, as shown in Figure 2. The second RNAi agents are useful for attenuating, preventing, and / or treating diseases, disorders, and / or conditions associated with ANGPTL3 expression.

[0102] method The method may include the steps described herein, which may be, but are not necessarily, performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping time, and / or individually or in multiple repeated steps. Furthermore, the method may include additional, unspecified steps.

[0103] Standard solid-phase oligonucleotide synthesis of intermediate compounds The oligonucleotide fragments (i.e., intermediate compounds) herein can be produced by standard oligonucleotide synthesis methods known in the art, such as SPOS. SPOS is achieved using standard amidite chemistry techniques with sequential coupling by 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, are commercially available from, for example, Applied Biosystems (Foster City, CA), BioAutomation (Irving, TX), and GE Healthcare Life Sciences (Pittsburgh, PA). See also WO 2005 / 070859 and WO 2012 / 157723.

[0104] As will be appreciated by those skilled in the art, other methods and / or techniques for synthesizing oligonucleotides may also be used. Additionally, the various synthetic steps may be performed in an alternating order or sequence to arrive at the desired compound. Other synthetic chemical transformations, protecting groups (e.g., present on bases for hydroxyl, amino, etc.), and protecting group methodologies (protection and deprotection) useful in synthesizing oligonucleotides are known in the art and are described, for example, in Larock, "Comprehensive Organic Transformations," VCH Publishers (1989); Greene & Wuts, "Protective Groups in Organic Synthesis," 2000; nd Ed., John Wiley & Sons (1991), Fieser & Fieser, "Fieser & Fieser's Reagents for Organic Synthesis", John Wiley & Sons (1994), and Paquette, ed., "Encyclopedia of Reagents for Organic Synthesis", John Wiley & Sons (1995).

[0105] Briefly, during SPOS, nucleoside phosphoramidite building blocks can be added to a solid support in successive cycles to prepare oligonucleotides of desired length and sequence. Each cycle consists of several chemical reactions: detritylation, coupling, oxidation or thiolation, and capping. After synthesis of a given oligonucleotide is complete, it can be released from the solid support and protecting groups can be removed in the same step.

[0106] For detritylation, the initial resin can be swelled with ACN and then treated with 10% DCA in toluene.

[0107] The crude oligonucleotide fragments produced can then be purified, generally using chromatographic purification, to isolate the full-length oligonucleotides from their associated impurities. Finally, isolation through desalting and further lyophilization yields the pure oligonucleotide solid material.

[0108] Hybrid solid-phase and enzymatic ligation to form RNAi agents Certain combinations of the oligonucleotide fragments (i.e., intermediate compounds) herein prepared via SPOS as described above can be combined according to methods known to those skilled in the art to obtain the RNAi agents of SEQ ID NOs: 1 and 2. Thus, the methods described herein can include synthesizing independent oligonucleotide fragments, followed by ligating such fragments, thereby forming the RNAi agents of SEQ ID NOs: 1 and 2.

[0109] For example, an exemplary method for making the oligonucleotide of SEQ ID NO:1 includes at least ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:5, 6, and 7. In some examples, the fragments may be ligated in the order SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:7 (i.e., from the 5' end to the 3' end).

[0110] Alternatively, the oligonucleotide of SEQ ID NO:1 can be made by ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:5, 10, and 11. In some examples, the fragments can be ligated in the order SEQ ID NO:5-SEQ ID NO:10-SEQ ID NO:11 (i.e., from the 5' end to the 3' end).

[0111] Alternatively, the oligonucleotide of SEQ ID NO:1 can be generated by ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:5, 12, and 13. In some examples, the fragments can be ligated in the order SEQ ID NO:5-SEQ ID NO:12-SEQ ID NO:13 (i.e., from the 5' end to the 3' end).

[0112] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences as set forth in SEQ ID NOs: 7, 14, and 15. In some examples, these fragments can be ligated in the order SEQ ID NO: 14-SEQ ID NO: 15-SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0113] Alternatively, the oligonucleotide of SEQ ID NO: 1 can be made by ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs: 7, 18, and 19. In some examples, these fragments can be ligated in the order SEQ ID NO: 18-SEQ ID NO: 19-SEQ ID NO: 7 (i.e., from the 5' end to the 3' end).

[0114] Alternatively, the oligonucleotide of SEQ ID NO:1 can be generated by ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:7, 22, and 23. In some examples, these fragments can be ligated in the order SEQ ID NO:22-SEQ ID NO:23-SEQ ID NO:7 (i.e., from the 5' end to the 3' end).

[0115] Alternatively, the oligonucleotide of SEQ ID NO:1 can be generated by ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:5, 26, and 27. In some examples, the fragments can be ligated in the order SEQ ID NO:5-SEQ ID NO:26-SEQ ID NO:27 (i.e., from the 5' end to the 3' end).

[0116] Similarly, an exemplary method for making the oligonucleotide of SEQ ID NO:2 includes ligating together at least the following two oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:8 and 9. In some examples, the fragments may be ligated in the order SEQ ID NO:9-SEQ ID NO:8 (i.e., from the 5' end to the 3' end).

[0117] Alternatively, the oligonucleotide of SEQ ID NO:2 can be made by ligating together the following two oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:16 and 17. In some instances, these fragments can be ligated in the order SEQ ID NO:17-SEQ ID NO:16 (i.e., from the 5' end to the 3' end).

[0118] Alternatively, the oligonucleotide of SEQ ID NO:2 can be made by ligating together the following two oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:20 and 21. In some examples, the fragments can be ligated in the order SEQ ID NO:21-SEQ ID NO:20 (i.e., from the 5' end to the 3' end).

[0119] Alternatively, the oligonucleotide of SEQ ID NO:2 can be made by ligating together the following two oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:24 and 25. In some examples, these fragments can be ligated in the order SEQ ID NO:25-SEQ ID NO:24 (i.e., from the 5' end to the 3' end).

[0120] Additionally, certain combinations of oligonucleotide fragments (i.e., intermediate compounds) herein prepared via SPOS as 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 involve synthesizing independent oligonucleotide fragments, followed by ligating such fragments, thereby forming the RNAi agents of SEQ ID NOs: 3 and 4.

[0121] For example, an exemplary method for making the oligonucleotide of SEQ ID NO:3 includes at least ligating together the following three oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:7, 32, and 33. In some examples, the fragments may be ligated in the order SEQ ID NO:32-SEQ ID NO:33-SEQ ID NO:7 (i.e., from the 5' end to the 3' end).

[0122] Similarly, an exemplary method for making the oligonucleotide of SEQ ID NO:4 includes at least ligating together the following two oligonucleotide fragments, such fragments having the nucleotide sequences set forth in SEQ ID NOs:34 and 35. In some examples, the fragments may be ligated in the order SEQ ID NO:35-SEQ ID NO:34 (i.e., from the 5' end to the 3' end).

[0123] In any of the above methods, the ligation can be carried out in an aqueous solution, such as a reaction buffer. In some examples, the solution can be an acetate buffer, a carbonate buffer, a citrate buffer, or a phosphate buffer, such as Tris buffer. Additionally, the solution can have a pH of about 5 to about 9, about 6 to about 8, or about 7. In some examples, the pH can be about 5, about 6, about 7, about 8, or about 9.

[0124] Additionally, the aqueous solution may contain cofactors (e.g., adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD)) and divalent metal salts (e.g., MgCl2).

[0125] Furthermore, the oligonucleotide fragments may be present in the aqueous solution at a concentration of about 1 μM to about 100,000 μM (100 mM). In some examples, the oligonucleotide fragment concentration may 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 examples, the oligonucleotide fragment concentration is 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 ,000μM, about 7,000μM to about 8,000μM, about 8,000μM to about 9,000μM, about 9,000μM to about 10,000μM, Approximately 10,000μM to approximately 20,000μM, approximately 20,000μM to approximately 30,000μM, approximately 30,000μM to approximately 40,000μM, approximately 40 The concentration may be about 1,000 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 another example, the oligonucleotide fragment concentration is 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, or about 3,000 μM. μM, approximately 2,500 μM, approximately 3,000 μM, approximately 3,500 μM, approximately 4,000 μM, approximately 4,500 μM, approximately 5,000 μM, approximately 5,500 μM, approximately 6,000 μM, approximately 6,500 μM, Approximately 7,000μM, approximately 7,500μM, approximately 8,000μM, approximately 8,500μM, approximately 9,000μM, approximately 9,500μM, approximately 10,000μM, approximately 10,500μM, approximately 11,000μM, approximately 11,500, approximately 12,000μM, approximately 12,500μM, approximately 13,000μM, approximately 13,500, approximately 14,000μM, approximately 14,500, approximately 15,000μM, approximately 15,500μM, approximately 16,000μM, approximately 16,500μM, approximately 17,000μM, approximately 17,500μM, approximately 18,000μM, approximately 18,500μM, approximately 19,000μM, approximately 19,500μM, approximately 20,00 The concentration of each oligonucleotide fragment may be about 0 μM, about 25,000 μM, about 30,000 μM, about 35,000 μM, about 40,000 μM, about 45,000 μM, about 50,000 μM, about 55,000 μM, about 60,000 μM, about 65,000 μM, about 70,000 μM, about 75,000 μM, about 80,000 μM, about 85,000 μM, about 90,000 μM, about 95,000 μM, or about 100,000 μM. Furthermore, in some examples, each oligonucleotide fragment may be at the same concentration as the other oligonucleotide fragments. In other examples, each oligonucleotide fragment may be at a different concentration than the other oligonucleotide fragments.

[0126] Furthermore, ligation can be performed at a reaction temperature sufficient to activate the enzyme, which can be about 2°C to about 50°C. In some examples, the reaction temperature is about 5°C to about 45°C, about 10°C to about 40°C, about 15°C to about 35°C, about 20°C to about 30°C, or about 25°C. In other examples, the reaction temperature is about 5°C to about 10°C, about 10°C to about 15°C, about 15°C to about 20°C, about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, about 35°C to about 40°C, about 40°C to about 45°C, or about 45°C to about 50°C. In yet other examples, the reaction temperature is 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.

[0127] Similarly, ligation can be performed for a reaction time sufficient to generate the target oligonucleotide, which can be from about 1 hour to about 72 hours. In some examples, the reaction time can be from about 2 hours to about 70 hours, from about 4 hours to about 68 hours, from about 6 hours to about 66 hours, from about 8 hours to about 64 hours, from about 10 hours to about 62 hours, from about 12 hours to about 60 hours, from about 14 hours to about 58 hours, from about 16 hours to about 56 hours, from about 18 hours to about 54 hours, from about 20 hours to about 52 hours, from about 22 hours to about 54 hours, from about 24 hours to about 52 hours, from about 26 hours to about 50 hours, from about 28 hours to about 48 hours, from about 30 hours to about 46 hours, from about 32 hours to about 44 hours, from about 34 hours to about 42 hours, from about 36 hours to about 40 hours, or about 38 hours. In other examples, the reaction time can be about 2 hours to about 10 hours, about 10 hours to about 20 hours, about 20 hours to about 30 hours, about 30 hours to about 40 hours, about 40 hours to about 50 hours, about 50 hours to about 60 hours, or about 60 hours to about 70 hours. In yet other examples, the reaction time can be about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, about 62 hours, about 64 hours, about 66 hours, about 68 hours, about 70 hours, or about 72 hours.

[0128] In the above methods, the ligation step may be mediated by an enzyme. In some examples, the enzyme is a ligase, such as a DNA ligase or an RNA ligase. In some examples, the ligase is a naturally occurring (i.e., wild-type) ligase. In other examples, the ligase is a non-naturally occurring (i.e., modified) ligase. Examples of ligases that may be used in the methods include, but are not limited to, T4 RNA ligase 1 or T4 RNA ligase 2. In some examples, the ligase is T4 RNA ligase 1. In other examples, the ligase is T4 RNA ligase 2.

[0129] RNA ligase is commercially available from sources such as Ajinomoto, Almac, Codexis, New England Biolabs, Takara, and ThermoFisher Scientific.

[0130] The enzyme activity can be from about 0.01 U / μL to about 1 U / μL, in some examples, the activity can 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 about 0.45 U / μL. In other examples, the activity is about 0.01U / μL, about 0.02U / μL, about 0.03U / μL, about 0.04U / μL, about 0.05U / μL, about 0.06U / μL, Approximately 0.07U / μL, approximately 0.08U / μL, approximately 0.09U / μL, approximately 0.1U / μL, approximately 0.15U / μL, approximately 0.2U / μL, approximately 0.25U / μL, approximately 0 .3U / μL, approximately 0.35U / μL, approximately 0.4U / μL, approximately 0.45U / μL, approximately 0.5U / μL, approximately 0.55U / μL, approximately 0.6U / μL, approximately 0.65U / μL, about 0.7U / μL, about 0.75U / μL, about 0.8U / μL, about 0.85U / μL, about 0.9U / μL, about 0.95, or about 1 U / μL.

[0131] Alternatively, the enzyme concentration can be from about 0.01 g / L to about 10 g / L. In some examples, the concentration can 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, from about 1.2 g / L to about 8.7 g / L, from about 1.3 g / L to about 8.6 g / L, from about 1.4 g / L to about 8.5 g / L, from about 1.5 g / L to about 8.4 g / L, from about 1.6 g / L to about 8.3 g / L, from about 1.7 g / L to about 8.2 g / L, from about 1.8 g / L to about 8.1 g / L, from about 1.9 g / L to about 8 g / L, from about 2 g / L to about 7.9 g / L, from about 2.1 g / L to about 7.8 g / L, from about 2.2 g / L to about 7.7 g / L, from about 2.3 g / L to about 7.6 g / L, from about 2.4 g / L to about 7.5 g / L, from about 2.5 g / L to about 7.4 g / L, from about 2.6 g / L to about 7.3 g / L, from about 2.7 g / L to about 7.2 g / L, from about 2.8 g / L to about 7.1 g / L, from about 2.9 g / L to about 7 g / L, from about 3 g / L to about 6.9 g / L, from about 3.1 g / L to about 6.8 g / L, from about 3.2 g / L to about 6.7, from about 3.3 g / L to about 6.6 g / L, from about 3.4 g / L to about 6.5 g / L, from about 3.5 g / L to about 6.4 g / L, from about 3.6 g / L to about 6.3 g / L, 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, from about 4.9 g / L to about 5 g / L.Other examples, concentrations, approximately 0.01 g / L, approximately 0.02 g / L, approximately 0.03 g / L, approximately 0.04 g / L, approximately 0.05 g / L, approximately 0.1 g / L, approximately 0.2 g / L, approximately 0.3 g / L, approximately 0.4 g / L, approximately 0.5 g / L, approximately 0.6 g / L, approximately 0.7 g / L, approximately 0.8 g / L, approximately 0.9 g / L, approximately 1 g / L, approximately 1.1 g / L, approximately 1.2 g / L, approximately 1.3 g / L, approximately 1.4 g / L, approximately 1.5 g / L, approximately 1.6 g / L, approximately 1.7 g / L, approximately 1.8 g / L, approximately 1.9 g / L, approximately 2 g / L Approximately 2.1 g / L, approximately 2.2 g / L, approximately 2.3 g / L, approximately 2.4 g / L, approximately 2.5 g / L, approximately 2.6 g / L, approximately 2.7 g / L, approximately 2.8 g / L, approximately 2.9 g / L, approximately 3 g / L, approximately 3.1 g / L, approximately 3.2 g / L, approximately 3.3 g / L, approximately 3.4 g / L, approximately 3.5 g / L, approximately 3.6 g / L, approximately 3.7 g / L, approximately 3.8 g / L, approximately 3.9 g / L, approximately 4 g / L, approximately 4.1 g / L, approximately 4.2 g / L, approximately 4.3 g / L, approximately 4.4 g / L, approximately 4.5 g / L, approximately 4.6 g / L, approximately 4.7 g / L. Approximately 4.8 g / L, approximately 4.9 g / L, approximately 5 g / L, approximately 5.1 g / L, approximately 5.2 g / L, approximately 5.3 g / L, approximately 5.4 g / L, approximately 5.5 g / L, approximately 5.6 g / L, approximately 5.7 g / L, approximately 5.8 g / L, approximately 5.9 g / L, approximately 6 g / L, approximately 6.1 g / L, approximately 6.2 g / L, approximately 6.3 g / L, approximately 6.4 g / L, approximately 6.5 g / L, approximately 6.6 g / L, approximately 6.7 g / L, approximately 6.8 g / L, approximately 6.9 g / L, approximately 7 g / L, approximately 7.1 g / L, approximately 7.2 g / L, approximately 7.3 g / L, approximately 7.4 g / L. Approximately 7.5g / L, approximately 7.6g / L, approximately 7.7g / L, approximately 7.8g / L, approximately 7.9g / L, approximately 8g / L, approximately 8.1g / L, approximately 8.2g / L, approximately 8.3g / L, approximately 8.4g / L, approximately 8.5g / L, approximately 8.6g / L, approximately 8.7g / L, approximately 8.8g / L, approximately 8.9g / L, approximately 9g / L, approximately 9.1g / L, approximately 9.2g / L, approximately 9.3g / L, approximately 9.4g / L, approximately 9.5g / L, approximately 9.6g / L, approximately 9.7g / L, approximately 9.8g / L, approximately 9.9g / L, and approximately 10g / L. Specific examples include: approximately 0.025g / L, approximately 0.1g / L, approximately 0.3g / L, and approximately 1g / L concentration.

[0132] The above-mentioned method can also comprise the step of annealing sense strand and antisense strand to form an RNAi agent.In some examples, SEQ ID NO:1 and SEQ ID NO:2 are annealed according to the method known to those skilled in the art, so that the complementary nucleotides in each strand hybridize / base pair with each other, to form the RNAi agent that regulates LPA expression.In other examples, SEQ ID NO:3 and SEQ ID NO:4 are annealed according to the method known to those skilled in the art, so that the complementary nucleotides in each strand hybridize / base pair with each other, to form the RNAi agent that regulates ANGPTL3 expression.

[0133] Other methods / uses The RNAi agent of this specification can be used in many therapeutic applications.For example, the RNAi agent of SEQ ID NO: 1 and 2 can be used in the method for attenuating, preventing and / or treating diseases, disorders and / or conditions related to LPA expression, such method comprises at least the step of administering to an individual who needs such treatment an effective amount of the RNAi agent of SEQ ID NO: 1 and 2 or its pharmaceutically acceptable salt.

[0134] Similarly, the RNAi agents of SEQ ID NOs: 3 and 4 may be used in methods for attenuating, preventing, and / or treating diseases, disorders, and / or conditions associated with ANGPTL3 expression, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of the RNAi agent of SEQ ID NOs: 3 and 4 or a pharmaceutically acceptable salt thereof. [Example]

[0135] The following non-limiting examples are offered by way of illustration and not limitation.

[0136] Development and synthesis of oligonucleotide fragments Example 1: SPOS of intermediate compound 1 Synthesis: 5'mU S-mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU3' (SEQ ID NO: 5), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL 2'-OMe U 250 polystyrene resin (246 μmol / g, approximately 750 mg, approximately 185 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed using the conditions outlined in Table 1.

[0137] [Table 1]

[0138] [Table 2]

[0139] Resin cleavage and isolation: After synthesis, nitrogen was passed through the resin-bound intermediate oligonucleotide fragment until a constant mass was achieved. Intermediate Compound 1 was cleaved from the resin, and the nucleobases were globally deprotected using concentrated NHOH in HO at 38 °C for 18 h. 20 mL of NHOH / g resin was loaded into a pressure-relief reaction vial containing dry resin (approximately 1.5 g / lot). The spent resin was filtered and rinsed with 2 x 20 mL of 1:1 EtOH:HO. The filtrate was collected in a round-bottom flask, and the NH was removed by rotary evaporation. Three lots of intermediate Compound 1 were combined for downstream processing.

[0140] Tangential flow filtration (TFF): Intermediate compound 1 was desalted and the NH salt was exchanged for Na salt using a PendoTECH TFF system. Omega PES membrane (0.1 m 2Two Pall T-series cassettes with a 1 kDa MWCO were used in series. The membrane was conditioned with HO (approximately 5 L) before processing. The intermediate compound 1 solution was concentrated to approximately 50 mL and then diafiltered 10 times using 0.5 M NaCl solution (500 mL) to convert it to sodium phosphate. The sodium salt of intermediate compound 1 was subjected to water diafiltration until the permeate conductivity was less than 50 μS / cm. The sodium salt retentate of intermediate compound 1 was collected with three water flushes of the membrane. Intermediate compound 1 was subjected to lyophilization and isolated as a crude oligo powder.

[0141] Analysis: A Water's Acquity UPLC system equipped with a tunable UV (TUV) detector was used to assess the purity of intermediate compound 1. The chromatographic mobile phase, column, gradient, and general parameters are outlined in Tables 3 and 4.

[0142] [Table 3]

[0143] [Table 4]

[0144] 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 the crude sodium salt.

[0145] Example 2: SPOS of intermediate compound 2 Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG3' (SEQ ID NO: 6), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL 2'-OMe G(iBu)250 polystyrene resin (249 μmol / g, approximately 850 mg, approximately 212 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0146] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0147] Analysis: Analysis was performed as described in Example 1.

[0148] TFF: TFF was performed as described in Example 1.

[0149] 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 the crude sodium salt.

[0150] Example 3: SPOS of intermediate compound 3 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. NittoPhase® HL 2'-OMe C(Ac)250 polystyrene resin (257 μmol / g, approximately 800 mg, approximately 206 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0151] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0152] TFF: TFF was performed as described in Example 1.

[0153] Analysis: The analysis was carried out as described in Example 1, except that the mobile phase, column, and gradient are outlined in Table 5.

[0154] [Table 5]

[0155] 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 the crude sodium salt.

[0156] Example 4: SPOS of intermediate compound 4 Synthesis:5'p-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 8), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL 2'-OMe G(iBu)250 polystyrene resin (249 μmol / g, approximately 800 mg, approximately 200 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0157] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0158] TFF: TFF was performed as described in Example 1.

[0159] Analysis: Analysis was performed as described in Example 3.

[0160] 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 the crude sodium salt.

[0161] Example 5: SPOS of intermediate compound 5 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. NittoPhase® HL 2'-Fluoro A(bz)250 polystyrene resin (229 μmol / g, approximately 850 mg, approximately 195 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0162] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0163] TFF: TFF was performed as described in Example 1.

[0164] Analysis: Analysis was performed as described in Example 3.

[0165] 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 the crude sodium salt.

[0166] Example 6: SPOS of intermediate compound 6 Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC3' (SEQ ID NO: 10), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8031 g, 277.9 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0167] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0168] TFF: The intermediate compound 6 was desalted and the NH salt was exchanged for the sodium salt using a Millipore Cogent μScale system. 2 The system was equipped with a 2 kDa MWCO membrane. The membrane was conditioned with HO (approximately 0.5 L) before processing. The intermediate compound 6 solution was concentrated to approximately 20 mL and then diafiltered 10 times using 0.5 M NaCl solution (200 mL) to convert it to sodium phosphate. The sodium salt of intermediate compound 6 was subjected to water diafiltration until the permeate conductivity was less than 50 μS / cm. The sodium salt retentate of intermediate compound 6 was collected with three water flushes of the membrane. Intermediate compound 6 was subjected to lyophilization, and the crude oligonucleotide powder was dissolved in Milli-Q water (13.22 mL, OD / mL=1678.7) to make a stock solution.

[0169] Analysis: Analysis was performed as described in Example 3.

[0170] Results: Intermediate compound 6 (0.783 g by optical density, 94.65% by UPLC, expected exact mass = 4000.708 Da, observed exact mass = 4000.719 Da) was prepared as the crude sodium salt.

[0171] Example 7: SPOS of intermediate compound 7 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. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7503 g, 259.6 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0172] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0173] TFF: TFF was performed as described in Example 6.

[0174] Analysis: Analysis was performed as described in Example 3.

[0175] Results: Intermediate compound 7 (0.819 g by optical density, 89.44% by UPLC, expected exact mass = 4660.208 Da, observed exact mass = 4660.209 Da) was prepared as the crude sodium salt.

[0176] Example 8: SPOS of intermediate compound 8 Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC3' (SEQ ID NO: 12), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8007 g, 277.0 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0177] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0178] TFF: TFF was performed as described in Example 6.

[0179] Analysis: Analysis was performed as described in Example 3.

[0180] Results: Intermediate compound 8 (0.719 g by optical density, 95.15% by UPLC, expected exact mass = 3681.651 Da, observed exact mass = 3681.655 Da) was prepared as the crude sodium salt.

[0181] Example 9: SPOS of intermediate compound 9 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. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7454 g, 257.9 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0182] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0183] TFF: TFF was performed as described in Example 6.

[0184] Analysis: Analysis was performed as described in Example 3.

[0185] Results: Intermediate compound 9 (0.896 g by optical density, 90.24% by UPLC, expected exact mass = 4979.265 Da, observed exact mass = 4979.264 Da) was prepared as the crude sodium salt.

[0186] Example 10: SPOS of intermediate compound 10 Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU3' (SEQ ID NO: 14), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8066 g, 279.1 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0187] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0188] TFF: Intermediate compound 10 was desalted and the NH salt was exchanged for Na salt using both PendoTECH and Millipore Cogent μScale TFF systems. Sartorius Hydrosart® membranes (0.02 m 2 The system was equipped with a 2 kDa MWCO membrane. The membrane was conditioned with HO (approximately 0.5 L) before processing. The intermediate compound 10 solution was concentrated to approximately 30 mL and then diafiltered 10 times using 0.5 M NaCl solution (300 mL) to convert it to sodium phosphate. The sodium salt of intermediate compound 10 was subjected to water diafiltration until the permeate conductivity was less than 50 μS / cm. The sodium salt retentate of intermediate compound 10 was collected with three water flushes of the membrane. Intermediate compound 10 was subjected to lyophilization, and the crude oligo powder was dissolved in Milli-Q water (14.05 mL, OD / mL=1368.3) to make a stock solution.

[0189] Analysis: Analysis was performed as described in Example 3.

[0190] Results: Intermediate compound 10 (0.655 g by optical density, 94.69% by UPLC, expected exact mass = 3239.518 Da, observed exact mass = 3239.522 Da) was prepared as the crude sodium salt.

[0191] Example 11: SPOS of intermediate compound 11 Synthesis: 5'p-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG3' (SEQ ID NO: 15), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8010 g, 277.1 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0192] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0193] TFF: TFF was performed as described in Example 10.

[0194] Analysis: Analysis was performed as described in Example 3.

[0195] Results: Intermediate compound 11 (0.930 g by optical density, 93.06% by UPLC, expected exact mass = 4708.782 Da, observed exact mass = 4708.791 Da) was prepared as the crude sodium salt.

[0196] Example 12: SPOS of intermediate compound 12 Synthesis: 5'p-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S-mG3' (SEQ ID NO: 16), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8027 g, 277.7 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0197] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0198] TFF: TFF was performed as described in Example 10.

[0199] Analysis: Analysis was performed as described in Example 3.

[0200] Results: Intermediate compound 12 (0.899 g by optical density, 82.82% by UPLC, expected exact mass = 5440.871 Da, observed exact mass = 5440.883 Da) was prepared as the crude sodium salt.

[0201] Example 13: SPOS of intermediate compound 13 Synthesis:5'-[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG3' (SEQ ID NO: 17) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8036 g, 278.0 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0202] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0203] TFF: TFF was performed as described in Example 10.

[0204] Analysis: Analysis was performed as described in Example 3.

[0205] Results: Intermediate compound 13 (0.388 g by optical density, 94.34% by UPLC, expected exact mass = 2076.222 Da, observed exact mass = 2076.226 Da) was prepared as the crude sodium salt.

[0206] Example 14: SPOS of intermediate compound 14 Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG-fC3' (SEQ ID NO: 18), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8043 g, 278.3 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0207] Resin cleavage and isolation: After synthesis, nitrogen was passed through the resin-bound intermediate oligonucleotide fragment (1.7125 g, 3.26 g / mmol mass gain) until a constant mass was achieved. Intermediate compound 14 was cleaved from the resin, and the nucleobases were globally deprotected using concentrated NH4OH in HO at 38 °C for 18 h. 20 mL of NH4OH / g resin was loaded into a pressure-release reaction vial containing the dry 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 concentrated to dryness using a Genevac® 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 use in enzymatic ligation reactions.

[0208] Analysis: Analysis was performed as described in Example 3.

[0209] Results: Intermediate compound 14 (0.388 g by optical density, 94.34% by UPLC, expected exact mass = 2076.222 Da, observed exact mass = 2076.226 Da) was prepared as the crude ammonium salt.

[0210] Example 15: SPOS of intermediate compound 15 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. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7019 g, 242.9 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0211] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0212] Analysis: Analysis was performed as described in Example 3.

[0213] Results: Intermediate compound 15 (0.824 g) (by optical density), 90.12% (by UPLC), expected exact mass = 5016.803 Da, observed exact mass = 5016.811 Da) was prepared as the crude ammonium salt.

[0214] Example 16: SPOS of intermediate compound 16 Synthesis: 5'p-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S-mG3' (SEQ ID NO: 20), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7036 g, 243.4 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0215] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0216] Analysis: Analysis was performed as described in Example 3.

[0217] Results: Intermediate compound 16 (0.698 g by optical density, 90.09% by UPLC, expected exact mass = 5109.823 Da, observed exact mass = 5109.830 Da) was prepared as the crude ammonium salt.

[0218] Example 17: SPOS of intermediate compound 17 Synthesis:5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA3' (SEQ ID NO: 21) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8041 g, 278.2 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0219] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0220] Analysis: Analysis was performed as described in Example 3.

[0221] Results: Intermediate compound 17 (0.414 g by optical density, 93.23% by UPLC, expected exact mass = 2407.270 Da, observed exact mass = 2407.274 Da) was prepared as the crude ammonium salt.

[0222] Example 18: SPOS of intermediate compound 18 Synthesis: 5'mU S -mU-mG-mC-mC-mA-mA-fG3' (SEQ ID NO: 22), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8010 g, 277.1 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0223] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0224] Analysis: Analysis was performed as described in Example 3.

[0225] Results: Intermediate compound 18 (0.536 g by optical density, 96.44% by UPLC, expected exact mass = 2624.460 Da, observed exact mass = 2624.463 Da) was prepared as the crude ammonium salt.

[0226] Example 19: SPOS of intermediate compound 19 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. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.6999 g, 242.2 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0227] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0228] Analysis: Analysis was performed as described in Example 3.

[0229] Results: Intermediate compound 19 (0.850 g by optical density, 88.94% by UPLC, expected exact mass = 5323.840 Da, observed exact mass = 5323.849 Da) was prepared as the crude ammonium salt.

[0230] Example 20: SPOS of intermediate compound 20 Synthesis: 5'p-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 24), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.7008 g, 242.5 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0231] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0232] Analysis: Analysis was performed as described in Example 3.

[0233] Results: Intermediate compound 20 (0.685 g by optical density, 89.06% by UPLC, expected exact mass = 4790.766 Da, observed exact mass = 4790.771 Da) was prepared as the crude ammonium salt.

[0234] Example 21: SPOS of intermediate compound 21 Synthesis:5'[MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC3' (SEQ ID NO: 25) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8031 g, 277.9 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0235] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 14.

[0236] Analysis: Analysis was performed as described in Example 3.

[0237] Results: Intermediate compound 21 (0.608 g by optical density, 92.67% by UPLC, expected exact mass = 2726.327 Da, observed exact mass = 2726.328 Da) was prepared as the crude ammonium salt.

[0238] Example 22: SPOS of intermediate compound 22 Synthesis: 5'p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG3' (SEQ ID NO: 26), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8081 g, 279.6 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0239] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0240] TFF: Intermediate compound 22 was desalted and the NH salt was exchanged for Na salt using a Millipore Cogent μScale TFF system. 2 The system was equipped with a 2 kDa MWCO membrane. The membrane was conditioned with HO (approximately 0.5 L) before processing. The intermediate compound 22 solution was concentrated to approximately 40 mL and then diafiltered 10 times using 0.5 M NaCl solution (400 mL) to convert it to sodium phosphate. The sodium salt of intermediate compound 22 was subjected to water diafiltration until the permeate conductivity was less than 60 μS / cm. The sodium salt retentate of intermediate compound 22 was collected with two water flushes of the membrane. Intermediate compound 22 was subjected to lyophilization, and the crude oligo powder was dissolved in Milli-Q water (60.24 mL, OD / mL=415.07) to make a stock solution.

[0241] Analysis: Analysis was performed as described in Example 3.

[0242] Results: Intermediate compound 22 (0.881 g by optical density, 94.11% by UPLC, expected exact mass = 4359.771 Da, observed exact mass = 4359.778 Da) was prepared as the crude sodium salt.

[0243] Example 23: SPOS of intermediate compound 23 Synthesis: 5'p-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC3' (SEQ ID NO: 27), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, 0.8043 g, 278.3 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0244] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0245] TFF: TFF was performed as described in Example 22.

[0246] Analysis: Analysis was performed as described in Example 3.

[0247] Results: Intermediate compound 23 (0.853 g by optical density, 86.25% by UPLC, expected exact mass = 4301.145 Da, observed exact mass = 4301.149 Da) was prepared as the crude sodium salt.

[0248] Example 24: SPOS of intermediate compound 24 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. NittoPhase® HL 2'-OMe G(Ac)250 polystyrene resin (247 μmol / g, approximately 650 mg, approximately 161 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0249] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1. In contrast to Example 1, in this example only two lots were prepared and combined prior to TFF.

[0250] TFF: TFF was performed as described in Example 22.

[0251] Analysis: Analysis was performed as described in Example 1.

[0252] 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 the crude sodium salt.

[0253] Example 25: SPOS of intermediate compound 25 Synthesis: 5'mC-mU-mG-mC3' (SEQ ID NO: 29), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. Briefly, SPOS was performed using pre-loaded resin (loading rate 0.250 mmol / g) under the conditions shown in Tables 6 and 7 below.

[0254] [Table 6]

[0255] [Table 7]

[0256] TFF: TFF was performed as described in Example 22.

[0257] Analysis: Analysis was performed as described in Example 1.

[0258] Results: Intermediate compound 25 (0.70 g, 96.7% by UPLC, expected accurate mass = 1335.228 Da) was isolated as the crude sodium salt.

[0259] Example 26: SPOS of intermediate compound 26 Synthesis:5'p-mG-mC-mA-mA S -mG S -mG3' (SEQ ID NO: 30), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS as described in Example 25.

[0260] TFF: TFF was performed as described in Example 22.

[0261] Analysis: Analysis was performed as described in Example 1.

[0262] 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 the crude sodium salt.

[0263] Example 27: SPOS of intermediate compound 27 Synthesis: 5'p-mA-mG-mC-fU-mU-mG3' (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS as described in Example 25.

[0264] TFF: TFF was performed as described in Example 22.

[0265] Analysis: Analysis was performed as described in Example 1.

[0266] 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 the crude sodium salt.

[0267] Example 28: SPOS of intermediate compound 28 Synthesis: 5'mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU3' (SEQ ID NO: 32), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, approximately 750 g, approximately 260 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1. In contrast to Example 1, two lots were produced, and the resin from each lot was divided into two separate isolation methods.

[0268] Resin cleavage and isolation: After 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 h. 20 mL of NH4OH / g resin was placed in a pressure-relief reaction vial containing the dry resin. 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 the NH3 was removed by rotary evaporation. Two lots of intermediate compound 28 were combined for downstream processing. Intermediate compound 28 was desalted and the ammonium salt was exchanged for the sodium salt using a PendoTECH® TFF system. Omega PES membrane (0.1 m 2Two Pall T-series cassettes with a 1 kDa MWCO were used in series. The membranes were conditioned with HO before processing. The intermediate compound 28 solution was concentrated to approximately 80 mL and then diafiltered 7.5 times using 0.5 M NaCl solution (600 mL) to convert it to sodium phosphate. The sodium salt of intermediate compound 28 was subjected to water diafiltration until the permeate conductivity was less than 50 μS / cm. The sodium salt retentate of intermediate compound 28 was collected along with several water flushes of the membrane (total of approximately 300 mL). Intermediate compound 28 was subjected to lyophilization and isolated as a crude oligo powder. Isolation Method B: The spent resin was filtered and rinsed with 2 × 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 use in enzymatic ligation reactions.

[0269] TFF: TFF was performed as described in Example 28 - Isolation Method A.

[0270] Analysis: Analysis was performed as described in Example 1.

[0271] 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 the crude sodium salt.

[0272] Example 29: SPOS of intermediate compound 29 Synthesis: 5'p-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG3' (SEQ ID NO: 33), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, approximately 850 mg, approximately 295 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1. In contrast to Example 1, two lots were made, and resin from each lot was divided into two separate isolation methods as described in Example 28.

[0273] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 28.

[0274] TFF: TFF was performed as described in Example 22.

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

[0276] 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 the crude sodium salt.

[0277] Example 30: SPOS of intermediate compound 30 Synthesis:5'p-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S-mG3' (SEQ ID NO: 34), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, approximately 800 mg, approximately 277 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1. In contrast to Example 1, two lots were made, and the resin from each lot was divided into two separate isolation methods as described in Example 28.

[0278] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 28.

[0279] TFF: TFF was performed as described in Example 22.

[0280] Analysis: Analysis was performed as described in Example 3.

[0281] 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 the crude sodium salt.

[0282] Example 31: SPOS of intermediate compound 31 Synthesis:5'[MePhosphonate-4O-mU S ]-fG S -fU S-fA-fU-mA-fA-mC-mC-fU3' (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, approximately 800 mg, approximately 277 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1. In contrast to Example 1, two lots were made, and resin from each lot was split into two separate isolation methods as described in Example 28.

[0283] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 28.

[0284] TFF: TFF was performed as described in Example 22.

[0285] Analysis: Analysis was performed as described in Example 3.

[0286] 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 the crude sodium salt.

[0287] Example 32: SPOS of intermediate compound 32 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-mC3' (SEQ ID NO: 36), or a pharmaceutically acceptable salt thereof, was synthesized by standard SPOS. NittoPhase® HL Unylinker 350 polystyrene resin (346 μmol / g, approximately 600 mg, approximately 208 μmol) was packed into a stainless steel column (6.3 CV, 20 mm diameter), which was then attached to an AKTA OligoPilot® Plus 100 synthesizer. SPOS was performed as described in Example 1.

[0288] Resin cleavage and isolation: Resin cleavage and isolation was performed as described in Example 1.

[0289] TFF: TFF was performed as described in Example 22.

[0290] Analysis: Analysis was performed as described in Example 1.

[0291] Results: Intermediate compound 32 (1.05 g by optical density, 71.55% by UPLC, expected exact mass = 8642.905 Da, observed exact mass = 8642.919 Da) was isolated as the crude sodium salt.

[0292] Enzymatic ligation of oligonucleotide fragments Example 33: Comparison of pure and crude oligonucleotide fragments in enzymatic ligation catalyzed by RNA ligase to form RNAi agents Method 1 (pure oligonucleotide fragment): A first RNAi agent with a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2 was synthesized by ligating purified intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) using 1 g / L of first RNA ligase (Almac) in the presence of 2 mM ATP and MgCl (10 mM). 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (295.3 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 35° C. for 3 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0293] Method 2 (crude oligonucleotide fragments): Alternatively, the first RNAi agent was synthesized by ligating crude intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) using 1 g / L of the first RNA ligase 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (312.3 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 35° C. for 3 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0294] Results: Ligation using Method 1 yielded 1.66 mg of first RNAi agent (81.87% by UPLC), and ligation using Method 2 yielded 1.66 mg of first RNAi agent (83.44% by UPLC).

[0295] Example 34: Comparison of pure and crude oligonucleotide fragments in enzymatic ligation catalyzed by RNA ligase to form RNAi agents Method 1 (pure oligonucleotide fragment): A first RNAi agent having a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2 was synthesized by ligating purified intermediate compounds 1, 2, 3, 4, and 5 (0.1 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (622.9 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 2 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0296] Method 2 (crude oligonucleotide fragments): Alternatively, the first RNAi agent was synthesized using 0.025 g / L of a second RNA ligase (0.1 mM) to catalyze the ligation of crude intermediate compounds 1, 2, 3, 4, and 5 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (622.9 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 2 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0297] Results: Ligation using Method 1 yielded 1.66 mg of the first RNAi agent (89.43% by UPLC), and ligation using Method 2 yielded 1.66 mg of the first RNAi agent (92.58% by UPLC).

[0298] Example 35: Standard conditions for enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) using 0.1 g / L of first RNA ligase (Almac) 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 Tris-HCl, pH 7.5 (1000 mM, 50 μL), MgCl (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) to nuclease-free water (181.8 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 35° C. for 24 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0299] Results: Ligation yielded 8.29 mg of the first RNAi agent (91.63% by UPLC).

[0300] Example 36: Standard conditions for enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 16, and 17 (0.4 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (171.0 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 24 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0301] Results: This ligation yielded 8.29 mg of the first RNAi agent (90.24% by UPLC).

[0302] Example 37: Enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 6, 7, 5, and 5 (0.4 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (298.7 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 23 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0303] Results: Ligation yielded 8.29 mg of the first RNAi agent (87.34% by UPLC).

[0304] Example 38: Enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 8, 9, 4, and 5 (0.4 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (307.9 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 23 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0305] Results: Ligation yielded 8.29 mg of the first RNAi agent (88.80% by UPLC).

[0306] Example 39: Enzymatic ligation using RNA ligase to form RNAi agents 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (298.7 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 24 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0307] Results: Ligation yielded 8.29 mg of the first RNAi agent (78.71% by UPLC).

[0308] Example 40: Enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 14, 15, 3, 16, and 17 (0.4 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (295.2 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 24 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0309] Results: Ligation yielded 8.29 mg of the first RNAi agent (90.44% by UPLC).

[0310] Example 41: Enzymatic ligation using RNA ligase to form RNAi agents 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 with 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (307.2 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 24 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0311] Results: Ligation yielded 8.29 mg of the first RNAi agent (89.18% by UPLC).

[0312] Example 42: Enzymatic ligation using RNA ligase to form RNAi agents 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 with 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (13.2 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA Ligase 2 (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 23 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0313] Result: The ligation reaction failed because ligation between intermediate compound 22 and intermediate compound 23 did not occur. Therefore, the ligation reaction was unable to produce the desired sense strand of SEQ ID NO: 1.

[0314] Example 43: Enzymatic ligation using RNA ligase to form RNAi agents Synthesis: A second RNAi agent with a sense strand of SEQ ID NO:3 and an antisense strand of SEQ ID NO:4 was synthesized by ligating crude intermediate compounds 28, 29, 3, 30, and 31 (0.4 mM) using 0.4 g / L of first RNA ligase (Almac) in the presence of ATP (2 mM) and MgCl (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 Tris-HCl, pH 7.5 (1000 mM, 50 μL), MgCl (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) to nuclease-free water (145.5 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 35° C. for 19 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0315] Results: Ligation yielded 8.29 mg of the second RNAi agent (84.93% by UPLC).

[0316] Example 44: Enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 28, 29, 3, 30, and 31 (0.4 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (154.7 μL). An enzyme treatment solution (0.125 g / L, 224 mL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 20 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0317] Results: Ligation yielded 8.29 mg of the second RNAi agent (82.91% by UPLC).

[0318] Example 45: Enzymatic ligation using RNA ligase to form intermediate compound 33 Synthesis: Intermediate compound 33 (SEQ ID NO: 37) was synthesized by ligating intermediate compounds 2 and 3 (0.08 mM) using 0.025 g / L of a second RNA ligase (Codexis) in the presence of ATP (0.4 mM) and MgCl (2.0 mM). In a 2 mL HPLC vial, reaction buffer (800 μL) was prepared by adding Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (655.6 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA Ligase 2 (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 2 hours. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0319] Results: The ligation gave 0.69 mg of intermediate compound 33 (94.58% by UPLC).

[0320] Example 46: Inhibition of the formation of intermediate compound 33 during enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 28, 29, 3, 30, and 31 (0.4 mM) using 0.025 g / L of a second RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 40 μL), MgCl (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) to nuclease-free water (154.7 μL). An enzyme treatment solution (0.125 g / L, 224 μL) was prepared by diluting RNA ligase (3.5 g / L, 8 μL) with enzyme storage buffer (216 μL). The enzyme treatment solution (200 μL) was added to reaction buffer (800 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37°C for 18 hours. The reaction was cooled to room temperature, and intermediate compound 3 (3.5 mM, 114.3 μL) was added to the 2 mL HPLC vial, which was then returned to the Eppendorf ThermoMixer® (500 rpm) at 37°C for 4 hours. The reaction was cooled to room temperature and quenched with EDTA (26.7 mM, 3 mL).

[0321] Results: Ligation yielded 8.29 mg of the second RNAi agent (93.97% by UPLC).

[0322] Example 47: Scaling up enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) using 0.025 g / L of 3 RNA ligase (Codexis) 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 Tris-HCl, pH 7.5 (1000 mM, 4.83 mL), MgCl (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) to nuclease-free water (21.52 mL). An enzyme treatment solution (0.125 g / L, 24.38 mL) was prepared by diluting RNA ligase (10.16 g / L, 300 μL) with enzyme storage buffer (24.08 mL). The enzyme treatment solution (24.13 mL) was added to reaction buffer (96.52 mL). The reaction mixture was thoroughly mixed by gentle inversion. A 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 for monitoring the internal reaction temperature. The reaction was heated to 37°C for 21 hours in a water bath with an immersed copper coil, temperature-controlled by a ThermoFisher Haake™ Phoenix II refrigerator / circulator. The reaction was quenched with EDTA (26.7 mM, 360 mL). After 21 hours, most of the intermediate compounds 1, 2, 3, 16, and 17 were consumed, yielding crude first RNAi agent (0.727 g, 92.72% by optical density (IM1)).

[0323] Results: The ligation yielded 0.587 g of purified second RNAi agent (89.46% (IM2) by UPLC).

[0324] Example 48: Scaling up enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) using 1 g / L of 4 RNA ligase (Codexis) 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 TrisHCl, pH 7.5 (12.56 mL). In a 200 mL pressure vial, reaction buffer (120.7 mL) was prepared by adding Tris-HCl, pH 7.5 (1000 mM, 6.0 mL), MgCl (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) to nuclease-free water (53.8 mL). The reaction mixture was thoroughly mixed by gentle inversion. A 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 for monitoring the internal reaction temperature. The reaction was heated to 37°C for 23.5 hours in a water bath with an immersed copper coil, temperature-controlled by a ThermoFisher Haake™ Phoenix II refrigerator / circulator. The reaction was quenched with EDTA (26.7 mM, 54.38 mL). After 21 hours, most of intermediate compounds 1, 2, 3, 4, and 5 had been consumed, yielding the crude first RNAi agent (1.01 g by optical density, 86.44% by UPLC (IM1)).

[0325] Results: The ligation yielded 0.682 g of purified second RNAi agent (90.39% (IM2) by UPLC).

[0326] Example 49: SPOS of intermediate compounds 1-5 on a 15 or 22 mmol scale Synthesis: Intermediate compounds 1, 2, 4, and 5, or pharmaceutically acceptable salts thereof, were synthesized on a 22 mmol scale by SPOS according to the process parameters outlined in Table 8. Similarly, intermediate compound 3, or a pharmaceutically acceptable salt thereof, was synthesized on a 15 mmol scale by SPOS according to the process parameters outlined in Table 8. All syntheses were carried out on an AKTA OligoPilot 400 synthesizer.

[0327] [Table 8-1]

[0328] [Table 8-2]

[0329] [Table 8-3]

[0330] After synthesis, the resin-bound crude oligonucleotide fragments were dried using nitrogen gas until a consistent mass was achieved. Ammonolysis was performed by adding concentrated NH4OH (100 mL / mmol) to the crude oligonucleotide fragments on the resin. This material was subjected to the cleavage and deprotection conditions shown in Table 9. After 16 hours, the spent resin was filtered from the ammonolysis solution to obtain the crude deprotected oligonucleotide fragments in solution.

[0331] [Table 9]

[0332] Results: The synthesis results after cleavage and deprotection (C&D) are shown in Table 10.

[0333] [Table 10]

[0334] The ammonolysis solution was further processed by ultrafiltration and diafiltration (UF / DF) using a Sartorius TFF system equipped with a Sartorius 2 kDa MCWO membrane. Salt exchange was performed using 0.5 M NaCl solution. The solution was desalted until the final conductivity value was achieved. Detailed parameters of the UF / DF process are listed in Table 11. After UF / DF, the product was isolated using lyophilization to obtain intermediate compounds 1-5 as sodium salts. The final yields and purities of intermediate compounds 1-5 are listed in Table 12.

[0335] [Table 11]

[0336] [Table 12]

[0337] Example 50: 7 gram scale-up of enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) using 0.1 g / L of No. 5 RNA ligase (Codexis, cell-free extract) 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 Tris-HCl, pH 7.5 (1 M, 42.30 mL), MgCl (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) to nuclease-free water (602.4 mL). RNA ligase (84.5 mg) was added to the reaction buffer. A 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 for monitoring the internal reaction temperature. The reaction was heated to 37 °C for 25 h in a water bath with an immersed copper coil, temperature-controlled by a ThermoFisher Haake™ Phoenix II refrigerator / circulator. After 25 h, 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 largely consumed to yield the crude first RNAi agent (83.04% (IM2) by UPLC).

[0338] Results: After purification, ligation yielded 6.62 g (93.82% yield) of potency-corrected RNAi agent (87.66% (IM2) by UPLC, 92.91% by non-denaturing UPLC method).

[0339] Example 51: 100 gram scale-up of enzymatic ligation using RNA ligase to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) using 0.1 g / L of No. 5 RNA ligase (Codexis, cell-free extract) in the presence of ATP (2.0 mM) and MgCl2 (10.0 mM). In a 22 L three-neck jacketed reactor, reaction buffer (12.066 L) was prepared by adding Tris-HCl, pH 7.5 (1 M, 603.275 mL), MgCl (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) to nuclease-free water (10.016 L). 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), baffles, a nitrogen gas line, and a thermocouple to monitor the internal reaction temperature. A ThermoFisher Haake™ Phoenix II chiller / circulator containing a 50:50 propylene glycol / water solution was plumbed to a fitting on the reactor jacket. The reaction was 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 cooled to room temperature. The crude reaction mixture was filtered through a Millipak 40 Gamma Gold Capsule with a sterile Durapore membrane (0.22 μm, PVDF) using a peristaltic pump. Intermediate compounds 1, 2, 3, 4, and 5 were largely consumed to yield the crude primary RNAi agent (84.1% (IM2) by UPLC).

[0340] Results: After purification, ligation yielded 92.20 g (76.16% yield, extrapolated and corrected for purity and water content) of the RNAi agent as the sodium salt (88.87% (IM2) by UPLC, 93.40% by native UPLC method).

[0341] Example 52: Standard conditions for enzymatic ligation using RNA ligase to form RNAi agents - four fragment approach 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 by ligating intermediate compounds 1, 3, 4, and 5 (0.4 mM) using 0.1 g / L of No. 5 RNA ligase (Codexis, cell-free extract) in the presence of ATP (2 mM) and MgCl (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 Tris-HCl, pH 7.5 (1000 mM, 50 μL), MgCl (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) to nuclease-free water (197.1 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vials were placed in an Eppendorf ThermoMixer® (500 rpm) for 23 hours at 37° C. The reaction was quenched with EDTA (26.7 mM, 3 mL).

[0342] Results: Ligation yielded 8.29 mg (theoretical) of RNAi agent (88.04% (IM1) by UPLC).

[0343] Example 53: Enzymatic ligation using commercially available RNA ligases to form RNAi agents 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 by ligating intermediate compounds 1, 2, 3, 4, and 5 (0.4 mM) using 0.025 g / L of No. 6 RNA ligase (New England Biolabs, M0239) 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 Tris-HCl, pH 7.5 (1000 mM, 25 μL), MgCl (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) to nuclease-free water (280.1 μL). The reaction mixture was thoroughly mixed by gently pipetting the solution up and down. The 2 mL HPLC vial was placed in an Eppendorf ThermoMixer® (500 rpm) at 37° C. for 20 hours. The reaction was quenched with EDTA (26.7 mM, 1.5 mL).

[0344] Results: Ligation yielded 4.15 mg (theoretical) of RNAi agent (84.71% (IM2) by UPLC).

[0345] array The following nucleotide and / or amino acid sequences are mentioned in the disclosure and are provided below for reference.

[0346] SEQ ID NO: 1 - Synthetic nucleic acid #1 (36 nt) 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

[0347] SEQ ID NO:2 - Synthetic nucleic acid #2 (22 nt) [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

[0348] SEQ ID NO:3 - Synthetic nucleic acid #3 (36 nt) 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

[0349] SEQ ID NO: 4 - Synthetic nucleic acid #4 (22 nt) [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

[0350] SEQ ID NO:5 - Intermediate compound 1 (14nt) mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU-fU-mG-mG-mU

[0351] SEQ ID NO: 6 - Intermediate compound 2 (10 nt) p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0352] SEQ ID NO: 7 - Intermediate compound 3 (12 nt) p-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0353] SEQ ID NO:8 - Intermediate compound 4 (12nt) p-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0354] SEQ ID NO: 9 - Intermediate compound 5 (10 nt) [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC-mC-fA

[0355] SEQ ID NO: 10 - Intermediate compound 6 (12nt) p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC

[0356] SEQ ID NO: 11 - Intermediate compound 7 (10 nt) p-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0357] SEQ ID NO: 12 - Intermediate compound 8 (11nt) p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC

[0358] SEQ ID NO: 13 - Intermediate compound 9 (11nt) p-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0359] SEQ ID NO: 14 - Intermediate compound 10 (10nt) mU S -mU-mG-mC-mC-mA-mA-fG-fC-fU

[0360] SEQ ID NO: 15 - Intermediate compound 11 (14nt) p-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0361] SEQ ID NO: 16 - Intermediate compound 12 (16nt) p-fA-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0362] SEQ ID NO: 17 - Intermediate compound 13 (6-nt) [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG

[0363] SEQ ID NO: 18 - Intermediate compound 14 (9 nt) mU S -mU-mG-mC-mC-mA-mA-fG-fC

[0364] SEQ ID NO: 19 - Intermediate compound 15 (15nt) p-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0365] SEQ ID NO:20 - Intermediate compound 16 (15nt) p-mC-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0366] SEQ ID NO: 21 - Intermediate compound 17 (7nt) [MePhosphonate-4O-mU S ]-fA S -fG S-fA-fU-mG-fA

[0367] SEQ ID NO: 22 - Intermediate compound 18 (8 nt) mU S -mU-mG-mC-mC-mA-mA-fG

[0368] SEQ ID NO: 23 - Intermediate compound 19 (16nt) p-fC-fU-fU-mG-mG-mU-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG

[0369] SEQ ID NO: 24 - Intermediate compound 20 (14nt) p-mC-fA-mA-mG-mC-fU-mU-mG-mG-mC-mA-mA S -mG S -mG

[0370] SEQ ID NO: 25 - Intermediate compound 21 (8 nt) [MePhosphonate-4O-mU S ]-fA S -fG S -fA-fU-mG-fA-mC

[0371] SEQ ID NO: 26 - Intermediate compound 22 (13nt) p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG

[0372] SEQ ID NO: 27 - Intermediate compound 23 (9 nt) p-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC

[0373] SEQ ID NO: 28 - Intermediate compound 24 (18nt) p-mC-mA-mU-mC-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA -GalNAc]-[ademA-GalNAc]-mG-mG

[0374] SEQ ID NO: 29 - Intermediate compound 25 (4nt) mC-mU-mG-mC

[0375] SEQ ID NO: 30 - Intermediate compound 26 (6nt) p-mG-mC-mA-mA S -mG S -mG

[0376] SEQ ID NO: 31 - Intermediate compound 27 (6nt) p-mA-mG-mC-fU-mU-mG

[0377] SEQ ID NO: 32 - Intermediate compound 28 (14 nt) mU S -mC-mA-mA-mA-mA-mU-fG-fG-fA-fA-mG-mG-mU

[0378] SEQ ID NO: 33 - Intermediate compound 29 (10 nt) p-mU-mA-mU-mA-mC-mA-mG-mC-mA-mG

[0379] SEQ ID NO: 34 - Intermediate compound 30 (12 nt) p-mU-mC-mC-fA-mU-mU-mU-mU-mG-mA S -mG S -mG

[0380] SEQ ID NO: 35 - Intermediate compound 31 (10 nt) [MePhosphonate-4O-mU S ]-fG S -fU S -fA-fU-mA-fA-mC-mC-fU

[0381] SEQ ID NO: 36 - Intermediate compound 32 (22nt) 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

[0382] SEQ ID NO: 37 - Intermediate compound 33 (22 nt) cyclo-(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 having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5 to 37, or a pharmaceutically acceptable salt thereof.

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

5.

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

6.

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

7.

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

8.

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

9.

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

10.

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

11.

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

12.

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

13.

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

14.

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

15.

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

16.

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

17.

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

18.

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

19.

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

20.

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

21.

19. The oligonucleotide of 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 of 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. 1. A method for making a nucleic acid of SEQ ID NO: 1, said method comprising: (a) SEQ ID NOs: 5, 6, and 7; (b) SEQ ID NOs: 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) ligating three oligonucleotide fragments selected from the group consisting of SEQ ID NOs: 5, 26, and 27.

36. 36. The method of claim 35, wherein the three oligonucleotide fragments are (a) SEQ ID NOs: 5, 6, and 7, and the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 5-SEQ ID NO: 6-SEQ ID NO:

7.

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

11.

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

13.

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

7.

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

7.

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

7.

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

27.

43. 1. A method of making a nucleic acid of SEQ ID NO: 2, said method comprising: (a') SEQ ID NOs: 8 and 9; (b') SEQ ID NOs: 16 and 17; (c') SEQ ID NOs: 20 and 21, and (d') ligating two oligonucleotide fragments selected from the group consisting of SEQ ID NOs: 24 and 25.

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

8.

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

16.

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

20.

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

24.

48. 1. A method of making a nucleic acid of SEQ ID NO: 3, said method comprising: (a) ligating three oligonucleotide fragments of SEQ ID NOs: 7, 32, and 33, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 32-SEQ ID NO: 33-SEQ ID NO:

7.

49. 1. A method of making a nucleic acid of SEQ ID NO: 4, said method comprising: (a') ligating two oligonucleotide fragments of SEQ ID NO:34 and 35, wherein the fragments are ligated from the 5' end to the 3' end as SEQ ID NO:35-SEQ ID NO:

34.

50. 1. A method of making an RNAi agent having a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2, said method comprising: forming the sense strand of SEQ ID NO: 1 by ligating three oligonucleotide fragments selected from the group consisting of: (a) SEQ ID NOs: 5, 6, and 7, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 5-SEQ ID NO: 6-SEQ ID NO: 7; (b) SEQ ID NOs: 5, 10, and 11, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 5-SEQ ID NO: 10-SEQ ID NO: 11; (c) SEQ ID NOs: 5, 12, and 13, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 5-SEQ ID NO: 12-SEQ ID NO: 13; (d) SEQ ID NOs: 7, 14, and 15, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 14-SEQ ID NO: 15-SEQ ID NO: 7; (e) SEQ ID NOs: 7, 18, and 19, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 18-SEQ ID NO: 19-SEQ ID NO: 7; (f) SEQ ID NOs: 7, 22, and 23, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 22-SEQ ID NO: 23-SEQ ID NO: 7; and (g) SEQ ID NOs: 5, 26, and 27, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO: 5-SEQ ID NO: 26-SEQ ID NO: 27; forming an antisense strand of SEQ ID NO:2 by ligating two oligonucleotide fragments selected from the group consisting of: (a') SEQ ID NOs: 8 and 9, wherein the fragments are ligated from the 5' end to the 3' end, such as SEQ ID NO: 9 - SEQ ID NO: 8; (b') SEQ ID NOs: 16 and 17, wherein the fragments are ligated from the 5' end to the 3' end, such as SEQ ID NO: 17-SEQ ID NO: 16; (c') SEQ ID NO: 20 and 21, wherein the fragments are ligated from the 5' end to the 3' end, such as SEQ ID NO: 21-SEQ ID NO: 20; (d') SEQ ID NOs: 24 and 25, wherein the fragments are ligated from the 5' end to the 3' end, such as SEQ ID NO: 25-SEQ ID NO: 24; forming the RNAi agent by annealing complementary nucleotides of the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO:

2.

51. 1. A method of making an RNAi agent having a sense strand of SEQ ID NO:3 and an antisense strand of SEQ ID NO:4, said method comprising: (a) forming a sense strand of SEQ ID NO:3 by ligating three oligonucleotide fragments of SEQ ID NOs:7, 32, and 33, wherein the fragments are ligated from the 5' end to the 3' end as follows: SEQ ID NO:32-SEQ ID NO:33-SEQ ID NO:7; (a') forming an antisense strand of SEQ ID NO: 4 by ligating two oligonucleotides of SEQ ID NO: 34 and 35, wherein the fragment is ligated from the 5' end to the 3' end as SEQ ID NO: 35-SEQ ID NO: 34; forming the RNAi agent by annealing complementary nucleotides of the sense strand of SEQ ID NO:3 and the antisense strand of SEQ ID NO:

4.

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

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

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

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