US20230139166A1 - RNAi Agents for Inhibiting Expression of Xanthine Dehydrogenase (XDH), Pharmaceutical Compositions Thereof, and Methods of Use - Google Patents

RNAi Agents for Inhibiting Expression of Xanthine Dehydrogenase (XDH), Pharmaceutical Compositions Thereof, and Methods of Use Download PDF

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US20230139166A1
US20230139166A1 US18/046,108 US202218046108A US2023139166A1 US 20230139166 A1 US20230139166 A1 US 20230139166A1 US 202218046108 A US202218046108 A US 202218046108A US 2023139166 A1 US2023139166 A1 US 2023139166A1
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xdh
rnai agent
nucleotides
aspects
sense strand
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Anthony Nicholas
Tao Pei
Zhao XU
Daniel BRAAS
Zhi-Ming Ding
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Arrowhead Pharmaceuticals Inc
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Arrowhead Pharmaceuticals Inc
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Assigned to Arrowhead Pharmaceuticals, Inc. reassignment Arrowhead Pharmaceuticals, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DING, Zhi-ming, PEI, TAO, BRAAS, Daniel, XU, Zhao, NICHOLAS, ANTHONY
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Definitions

  • RNA interference agents e.g., double stranded RNAi agents, for inhibition Xanthine Dehydrogenase (XDH; alternatively referred to as XO, XOR, xanthine dehydrogenase/oxidase, xanthine oxidoreductase, or XAN1)
  • XDH Xanthine Dehydrogenase
  • pharmaceutical compositions that include XDH RNAi agents, and methods of use thereof.
  • Gout is a progressive inflammatory arthritis caused by hyperuricemia (elevated serum uric acid levels) and deposition of monosodium urate crystals in joints and tendons. Gout is estimated to affect 0.6% of the world population with a substantially higher prevalence in certain geographical regions and ethnic groups. Gout patients without receiving a urate-lowering therapy suffer from recurrent episodes of gout flare (inflammation response) and ultimately can develop advanced gout, which is characterized by chronic joint pain and activity limitation.
  • Xanthine dehydrogenase is a molybdenum-containing hydroxylase that catalyzes the production of uric acid from xanthine.
  • XDH is highly expressed in liver and gastrointestinal tract. Hepatocyte-specific ablation of XDH or global inhibition of XDH activity reverses hyperuricemia phenotype in animal models.
  • XDH RNAi agents Small molecule inhibitors of XDH have been widely used for urate-lowering therapies. However, a large population of gout patients are intolerant of or refractory to these therapies, and some serious side effects include increased risk of death. There remains an unmet need for novel XDH inhibitors, such as XDH RNAi agents, to reduce hepatic XDH levels and treat hyperuricemia and gout.
  • RNAi agents for inhibiting expression of an XDH gene comprising an antisense strand comprising at least 17 contiguous nucleotides differing by 0 or 1 nucleotide from any one of the sequences of Table 2, Table 3, or Table 5C; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.
  • the antisense strand comprises nucleotides 2-18 of any one of the sequences of Table 2, Table 3, or Table 5C.
  • the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides differing by 0 or 1 nucleotide from 15 contiguous nucleotides of any one of the sense strand sequences of Table 2 or Table 4, and wherein the sense strand has a region of at least 85% complementarity over the 15 contiguous nucleotides to the antisense strand.
  • At least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.
  • nucleotides of the sense and/or antisense strand of the RNAi agent are modified nucleotides.
  • the modified nucleotide is selected from the group consisting of: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide, 2′,3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide, 2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxy nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3′-O-methyl nucleotide.
  • the all or substantially all of the modified nucleotides are 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.
  • the antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3.
  • the sense strand consists of, consists essentially of, or comprises the nucleotide sequence of any of the modified sense strand sequences of Table 4.
  • the antisense strand comprises the nucleotide sequence of any one of the modified sequences of Table 3 and the sense strand comprises the nucleotide sequence of any one of the modified sequences of Table 4.
  • the RNAi agents are linked to a targeting ligand.
  • the targeting ligand comprises N-acetyl-galactosamine.
  • the targeting ligand comprises the structure of (NAG37) or (NAG37)s.
  • the targeting ligand is linked to the sense strand.
  • the targeting ligand is linked to the 5′ terminal end of the sense strand.
  • the sense strand is between 15 and 30 nucleotides in length
  • the antisense strand is between 18 and 30 nucleotides in length.
  • the sense strand and the antisense strand are each between 18 and 27 nucleotides in length.
  • the sense strand and the antisense strand are each between 18 and 24 nucleotides in length.
  • sense strand and the antisense strand are each 21 nucleotides in length.
  • the RNAi agents have two blunt ends.
  • the sense strand comprises one or two terminal caps. In other aspects, the sense strand comprises one or two inverted abasic residues.
  • the RNAi agents are comprised of a sense strand and an antisense strand that form a duplex sequence of any one of the duplex structures shown in Table 5A, 5B or 5C.
  • the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
  • the sense strand of the RNAi agents is linked to a targeting ligand.
  • the targeting ligand has affinity for the asialoglycoprotein receptor.
  • the targeting ligand comprises N-acetyl-galactosamine.
  • the targeting ligand comprises.
  • compositions comprising the disclosed RNAi agents, wherein the compositions further comprise a pharmaceutically acceptable excipient.
  • Also provided herein are methods for inhibiting expression of an XDH gene in a cell the methods comprising introducing into a cell an effective amount of the disclosed RNAi agents or the disclosed compositions.
  • the cell is within a subject. In some aspects, the subject is a human subject.
  • the XDH gene expression is inhibited by at least about 30%. In some aspects, the XDH gene expression is inhibited by at least about 50% in the cytoplasm of hepatocytes.
  • compositions comprising administering to a human subject in need thereof a therapeutically effective amount of the disclosed compositions.
  • the disease is gout.
  • the symptom is hyperuricemia.
  • the RNAi agents are administered at a dose of about 0.05 mg/kg to about 5.0 mg/kg of body weight of the human subject.
  • the RNAi agent is administered in two or more doses.
  • RNAi agents for the treatment of a disease, disorder, or symptom that is mediated at least in part by XDH gene expression.
  • the disease is gout.
  • the symptom is hyperuricemia.
  • RNAi agents or the disclosed compositions for the preparation of a pharmaceutical compositions for treating a disease, disorder, or symptom that is mediated at least in part by XDH gene expression.
  • the RNAi agent is administered at a dose of about 0.05 mg/kg to about 5.0 mg/kg of body weight of the human subject.
  • RNAi agents compositions thereof, and methods of use may be understood more readily by reference to the following detailed description, which form a part of this disclosure. It is to be understood that the disclosure is not limited to what is specifically described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.
  • RNAi agent means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target gene in a sequence specific manner.
  • RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s).
  • RNAi agents While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action.
  • RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates.
  • the antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted (i.e. XDH mRNA).
  • RNAi agents can include one or more modified nucleotides and/or one or more non-phosphodiester linkages.
  • the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.
  • sequence and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature.
  • a nucleic acid molecule can comprise unmodified and/or modified nucleotides.
  • a nucleotide sequence can comprise unmodified and/or modified nucleotides.
  • a “base,” “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil.
  • a nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.
  • nucleotide has the same meaning as commonly understood in the art.
  • nucleotide refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleoside linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as nucleotide analogs herein.
  • a single nucleotide can be referred to as a monomer or unit.
  • first nucleobase or nucleotide sequence e.g., RNAi agent sense strand or targeted mRNA
  • second nucleobase or nucleotide sequence e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide
  • first nucleobase or nucleotide sequence e.g., RNAi agent sense strand or targeted mRNA
  • second nucleobase or nucleotide sequence e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide
  • oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or otherwise suitable in vivo or in vitro conditions) and form a duplex or double helical structure under certain standard conditions with an oligonucleotide that includes the second nucleotide sequence.
  • Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.
  • perfect complementary or “fully complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide.
  • the contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • partially complementary means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide.
  • the contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • substantially complementary means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide.
  • the contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used with respect to the nucleobase or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a sequence of an MUC5AC mRNA.
  • nucleic acid sequence means the nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity or more, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the subject matter disclosed herein encompass nucleotide sequences substantially identical to those disclosed herein.
  • the terms “individual”, “patient” and “subject”, are used interchangeably to refer to a member of any animal species including, but not limited to, birds, humans and other primates, and other mammals including commercially relevant mammals or animal models such as mice, rats, monkeys, cattle, pigs, horses, sheep, cats, and dogs.
  • the subject is a human.
  • treat means the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease in a subject.
  • “treat” and “treatment” may include the prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity, and/or frequency of one or more symptoms of a disease in a subject.
  • introducing into a cell when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell.
  • functional delivery means delivering the RNAi agent to the cell in a manner that enables the RNAi agent to have the expected biological activity, e.g., sequence-specific inhibition of gene expression.
  • isomers refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a “chiral center.”
  • each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms.
  • the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.
  • the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim.
  • the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending upon the environment in which the compound or composition is placed.
  • the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated.
  • the disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art.
  • compounds described herein with labile protons or basic atoms should also be understood to represent salt forms of the corresponding compound.
  • Compounds described herein may be in a free acid, free base, or salt form.
  • Pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.
  • the term “linked” or “conjugated” when referring to the connection between two compounds or molecules means that two compounds or molecules are joined by a covalent bond. Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.
  • the term “including” is used to herein mean, and is used interchangeably with, the phrase “including but not limited to.”
  • the term “or” is used herein to mean, and is used interchangeably with, the term “and/or.” unless the context clearly indicates otherwise.
  • RNAi agents for inhibiting expression of an XDH gene.
  • Each XDH RNAi agent comprises a sense strand and an antisense strand.
  • the sense strand can be 15 to 49 nucleotides in length.
  • the antisense strand can be 18 to 49 nucleotides in length.
  • the sense and antisense strands can be either the same length or they can be different lengths.
  • the sense and antisense strands are each independently 18 to 27 nucleotides in length.
  • both the sense and antisense strands are each 21-26 nucleotides in length.
  • the sense and antisense strands are each 21-24 nucleotides in length.
  • the sense and antisense strands are each independently 19-21 nucleotides in length. In some aspects, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some aspects, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some aspects, a sense strand is 23 nucleotides in length and an antisense strand is 21 nucleotides in length. In some aspects, both the sense and antisense strands are each 21 nucleotides in length.
  • the RNAi agent antisense strands are each 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the RNAi agent sense strands are each 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length.
  • the sense and antisense strands are annealed to form a duplex, and in some aspects, a double-stranded RNAi agent has a duplex length of about 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
  • RNAi agent duplexes that include the sense strand and antisense strand sequences in Tables 2, 3, 4 and 5C, are shown in Tables 5A, 5B and 5C.
  • the region of perfect, substantial, or partial complementarity between the sense strand and the antisense strand is 15-26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides in length and occurs at or near the 5′ end of the antisense strand (e.g., this region may be separated from the 5′ end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly, substantially, or partially complementary).
  • a sense strand of the XDH RNAi agents described herein includes at least 15 consecutive nucleotides that have at least 85% identity to a core stretch sequence (also referred to herein as a “core stretch” or “core sequence”) of the same number of nucleotides in an XDH mRNA.
  • a sense strand core stretch sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a core stretch sequence in the antisense strand, and thus the sense strand core stretch sequence is typically perfectly identical or at least about 85% identical to a nucleotide sequence of the same length (sometimes referred to, e.g., as a target sequence) present in the XDH mRNA target.
  • this sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some aspects, this sense strand core stretch is 17 nucleotides in length. In some aspects, this sense strand core stretch is 19 nucleotides in length.
  • An antisense strand of an XDH RNAi agent described herein includes at least 15 consecutive nucleotides that have at least 85% complementarity to a core stretch of the same number of nucleotides in an XDH mRNA and to a core stretch of the same number of nucleotides in the corresponding sense strand.
  • an antisense strand core stretch is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a nucleotide sequence (e.g., target sequence) of the same length present in the XDH mRNA target.
  • this antisense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • this antisense strand core stretch is 19 nucleotides in length. In some aspects, this antisense strand core stretch is 17 nucleotides in length.
  • a sense strand core stretch sequence can be the same length as a corresponding antisense core sequence or it can be a different length.
  • the XDH RNAi agent sense and antisense strands anneal to form a duplex.
  • a sense strand and an antisense strand of an XDH RNAi agent can be partially, substantially, or fully complementary to each other.
  • the sense strand core stretch sequence is at least 85% complementary or 100% complementary to the antisense core stretch sequence.
  • the sense strand core stretch sequence contains a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that is at least 85% or 100% complementary to a corresponding 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of an XDH RNAi agent have a region of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that is at least 85% base paired or 100% base paired.)
  • the antisense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2, Table 3, or Table 5C
  • the sense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2, Table 4, or Table 5C.
  • the sense strand and/or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of the core stretch sequences.
  • the antisense strand additional nucleotides may or may not be complementary to the corresponding sequence in the XDH mRNA.
  • the sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in the XDH mRNA.
  • the antisense strand additional nucleotides, if present may or may not be complementary to the corresponding sense strand's additional nucleotides, if present.
  • an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5′ and/or 3′ end of the sense strand core stretch sequence and/or antisense strand core stretch sequence.
  • the extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding antisense strand.
  • the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core stretch nucleotides or extension nucleotides, in the corresponding sense strand.
  • both the sense strand and the antisense strand of an RNAi agent contain 3′ and 5′ extensions.
  • one or more of the 3′ extension nucleotides of one strand base pairs with one or more 5′ extension nucleotides of the other strand. In other aspects, one or more of 3′ extension nucleotides of one strand do not base pair with one or more 5′ extension nucleotides of the other strand.
  • an XDH RNAi agent has an antisense strand having a 3′ extension and a sense strand having a 5′ extension. In some aspects, the extension nucleotide(s) are unpaired and form an overhang.
  • an “overhang” refers to a stretch of one or more unpaired nucleotides located at a terminal end of either the sense strand or the antisense strand that does not form part of the hybridized or duplexed portion of an RNAi agent disclosed herein.
  • an XDH RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other aspects, an XDH RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, or 3 nucleotides in length. In some aspects, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding XDH mRNA sequence. In some aspects, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding XDH mRNA sequence.
  • an XDH RNAi agent comprises a sense strand having a 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length.
  • one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides that correspond to or are the identical to nucleotides in the XDH mRNA sequence.
  • the 3′ sense strand extension includes or consists of one of the following sequences, but is not limited to: T, UT, TT, UU, UUT, TTT, or TIT (each listed 5′ to 3′).
  • a sense strand can have a 3′ extension and/or a 5′ extension.
  • an XDH RNAi agent comprises a sense strand having a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length.
  • one or more of the sense strand extension nucleotides comprise nucleotides that correspond to or are identical to nucleotides in the XDH mRNA sequence.
  • an XDH RNAi agent antisense strand includes a sequence of any of the sequences in Tables 2, 3, or 5C
  • an XDH RNAi agent antisense strand comprises or consists of any one of the modified sequences in Table 3.
  • an XDH RNAi agent antisense strand includes the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, of any of the sequences in Tables 2, 3, or 5C.
  • an XDH RNAi agent sense strand includes the sequence of any of the sequences in Tables 2, 4, or 5C. In some aspects, an XDH RNAi agent sense strand includes the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Tables 2, 4, or 5C. In certain aspects, an XDH RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4.
  • the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some aspects, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some aspects, the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a blunt end. In some aspects, the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a blunt end. In some aspects, both ends of an RNAi agent form blunt ends. In some aspects, neither end of an RNAi agent is blunt-ended. As used herein a “blunt end” refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base-pair).
  • the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a frayed end.
  • the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a frayed end.
  • both ends of an RNAi agent form a frayed end.
  • neither end of an RNAi agent is a frayed end.
  • a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands from a pair (i.e., do not form an overhang) but are not complementary (i.e. form a non-complementary pair).
  • one or more unpaired nucleotides at the end of one strand of a double stranded RNAi agent form an overhang.
  • the unpaired nucleotides may be on the sense strand or the antisense strand, creating either 3′ or 5′ overhangs.
  • the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′ overhang end, a frayed end and a 5′ overhang end, a frayed end and a 3′ overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhang end and a 3′ overhang end, two frayed ends, or two blunt ends.
  • overhangs are located at the 3′ terminal ends of the sense strand, the antisense strand, or both the sense strand and the antisense strand.
  • the XDH RNAi agents disclosed herein may also be comprised of one or more modified nucleotides. In some aspects, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the XDH RNAi agent are modified nucleotides.
  • the XDH RNAi agents disclosed herein may further be comprised of one or more modified internucleoside linkages, e.g., one or more phosphorothioate linkages.
  • an XDH RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some aspects, a 2′-modified nucleotide is combined with modified internucleoside linkage.
  • an XDH RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some aspects, an XDH RNAi agent is prepared as a pharmaceutically acceptable salt. In some aspects, an XDH RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms that are well known in the art are within the scope of the inventions disclosed herein.
  • Modified nucleotides when used in various oligonucleotide constructs, can preserve activity of the compound in cells while at the same time increasing the serum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans upon administering of the oligonucleotide construct.
  • an XDH RNAi agent contains one or more modified nucleotides.
  • a “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxyl nucleotide).
  • at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides.
  • modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2′-modified nucleotides, inverted nucleotides, modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2′,3′-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3′-O-methoxy (2′ internucleoside linked) nucleotides, 2′-F-Arabino nucleotides, 5′-Me, 2′-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides, 2′-modified
  • XDH RNAi agent sense strands and antisense strands can be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines. (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halour
  • the 5′ and/or 3′ end of the antisense strand can include abasic residues (Ab), which can also be referred to as an “abasic site” or “abasic nucleotide.”
  • An abasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1′ position of the sugar moiety.
  • an abasic residue can be placed internally in a nucleotide sequence.
  • Ab or AbAb can be added to the 3′ end of the antisense strand.
  • the 5′ end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)).
  • UUAb, UAb, or Ab are added to the 3′ end of the sense strand.
  • an abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.
  • RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified).
  • a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides.
  • an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides.
  • one or more nucleotides of an RNAi agent is an unmodified ribonucleotide. Chemical structures for certain modified nucleotides are set forth in Table 6 herein.
  • one or more nucleotides of an XDH RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones).
  • Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages,
  • a modified internucleoside linkage or backbone lacks a phosphorus atom.
  • Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages.
  • modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH 2 components.
  • a sense strand of an XDH RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • an antisense strand of an XDH RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.
  • a sense strand of an XDH RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • an antisense strand of an XDH RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
  • an XDH RNAi agent sense strand contains at least two phosphorothioate internucleoside linkages.
  • the phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3′ end of the sense strand.
  • one phosphorothioate internucleoside linkage is at the 5′ end of the sense strand nucleotide sequence, and another phosphorothioate linkage is at the 3′ end of the sense strand nucleotide sequence.
  • two phosphorothioate internucleoside linkages are located at the 5′ end of the sense strand, and another phosphorothioate linkage is at the 3′ end of the sense strand.
  • the sense strand does not include any phosphorothioate internucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5′ and 3′ ends and the optionally present inverted abasic residue terminal caps.
  • the targeting ligand is linked to the sense strand via a phosphorothioate linkage.
  • an XDH RNAi agent antisense strand contains four phosphorothioate internucleoside linkages.
  • the four phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5′ end of the antisense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5′ end.
  • three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5′ end of the antisense strand, and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5′ end of the antisense strand.
  • an XDH RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand.
  • the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a “cap,” a “terminal cap,” or a “capping residue.”
  • a “capping residue” is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of a nucleotide sequence of an RNAi agent disclosed herein.
  • a capping residue can provide the RNAi agent, in some instances, with certain beneficial properties, such as, for example, protection against exonuclease degradation.
  • inverted abasic residues inverted abasic residues (invAb) (also referred to in the art as “inverted abasic sites”) are added as capping residues.
  • Capping residues are generally known in the art, and include, for example, inverted abasic residues as well as carbon chains such as a terminal C 3 H 7 (propyl), C 6 H 13 (hexyl), or C 12 H 25 (dodecyl) groups.
  • a capping residue is present at either the 5′ terminal end, the 3′ terminal end, or both the 5′ and 3′ terminal ends of the sense strand.
  • the 5′ end and/or the 3′ end of the sense strand may include more than one inverted abasic deoxyribose moiety as a capping residue.
  • one or more inverted abasic residues are added to the 3′ end of the sense strand. In some aspects, one or more inverted abasic residues (invAb) are added to the 5′ end of the sense strand. In some aspects, one or more inverted abasic residues or inverted abasic sites are inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some aspects, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.
  • one or more inverted abasic residues are added to the 5′ end of the sense strand.
  • one or more inverted abasic residues can be inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent.
  • the inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)s), or other linkages.
  • the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent.
  • an inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue.
  • the 3′ end of the antisense strand core stretch sequence, or the 3′ end of the antisense strand sequence may include an inverted abasic residue.
  • the chemical structures for inverted abasic deoxyribose residues are shown in Table 6 below.
  • the XDH RNAi agents disclosed herein are designed to target specific positions on an XDH gene (e.g., SEQ ID NO:1).
  • XDH xanthine dehydrogenase
  • XDH xanthine dehydrogenase
  • an antisense strand sequence is designed to target an XDH gene at a given position on the gene when the 5′ terminal nucleobase of the antisense strand is aligned with a position that is 21 nucleotides downstream (towards the 3′ end) from the position on the gene when base pairing to the gene.
  • an antisense strand sequence designed to target an XDH gene at position 1322 requires that when base pairing to the gene, the 5′ terminal nucleobase of the antisense strand is aligned with position 1342 of the XDH gene.
  • an XDH RNAi agent does not require that the nucleobase at position 1 (5′ ⁇ 3′) of the antisense strand be complementary to the gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides.
  • complementarity e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity
  • the 5′ terminal nucleobase of the antisense strand of the of the XDH RNAi agent is aligned with position 1342 of the gene; however, the 5′ terminal nucleobase of the antisense strand may be, but is not required to be, complementary to position 1342 of an XDH gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides.
  • complementarity e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity
  • the specific site of binding of the gene by the antisense strand of the XDH RNAi agent is important to the level of inhibition achieved by the XDH RNAi agent.
  • the XDH RNAi agents disclosed herein target an XDH gene at or near the positions of the XDH gene sequence shown in Table 1.
  • the antisense strand of an XDH RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to a target XDH 19-mer sequence disclosed in Table 1.
  • an XDH RNAi agent includes an antisense strand wherein position 19 of the antisense strand (5′ ⁇ 3′) is capable of forming a base pair with position 1 of a 19-mer target sequence disclosed in Table 1. In some aspects, an XDH RNAi agent includes an antisense strand wherein position 1 of the antisense strand (5′ ⁇ 3′) is capable of forming a base pair with position 19 of the 19-mer target sequence disclosed in Table 1.
  • an XDH RNAi agent includes an antisense strand wherein position 2 of the antisense strand (5′ ⁇ 3′) is capable of forming a base pair with position 18 of the 19-mer target sequence disclosed in Table 1. In some aspects, an XDH RNAi agent includes an antisense strand wherein positions 2 through 18 of the antisense strand (5′ ⁇ 3′) are capable of forming base pairs with each of the respective complementary bases located at positions 18 through 2 of the 19-mer target sequence disclosed in Table 1.
  • the nucleotide at position 1 of the antisense strand can be perfectly complementary to the XDH gene, or can be non-complementary to the XDH gene.
  • the nucleotide at position 1 of the antisense strand is a U. A. or dT.
  • the nucleotide at position 1 of the antisense strand forms an A:U or U:A base pair with the sense strand.
  • an XDH RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences in Table 2, Table 3, or Table 5C.
  • an XDH RNAi sense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2, Table 4, or Table 5C.
  • an XDH RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences of Table 2, Table 3, or Table 5C.
  • an XDH RNAi sense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences of Table 2, Table 4, or Table 5C.
  • an XDH RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2 or Table 4.
  • the XDH RNAi agents include core 19-mer nucleotide sequences shown in the following Table 2.
  • the XDH RNAi agent sense strands and antisense strands that comprise or consist of the sequences in Table 2 can be modified nucleotides or unmodified nucleotides.
  • the XDH RNAi agents having the sense and antisense strand sequences that comprise or consist of the sequences in Table 2 are all or substantially all modified nucleotides.
  • the antisense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2.
  • the sense strand of an XDH RINAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2.
  • each N listed in a sequence disclosed in Table 2 may be independently selected from any and all nucleobases (including those found on both modified and unmodified nucleotides).
  • an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand.
  • an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand.
  • an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is the same as the N nucleotide at the corresponding position on the other strand. In some aspects, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is different from the N nucleotide at the corresponding position on the other strand.
  • modified XDH RNAi agent antisense strands are provided in Table 3.
  • Certain modified XDH RNAi agent sense strands, as well as their underlying unmodified nucleobase sequences are provided in Table 4.
  • each of the nucleotides in each of the underlying base sequences listed in Tables 3 and 4, as well as in Table 2, above can be a modified nucleotide.
  • the XDH RNAi agents described herein are formed by annealing an antisense strand with a sense strand.
  • a sense strand containing a sequence listed in Table 2 or Table 4 can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.
  • an XDH RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3.
  • an XDH RNAi agent comprises or consists of a duplex having the nucleobase sequences of the sense strand and the antisense strand of any of the sequences in Table 2, Table 3, or Table 4.
  • antisense strands containing modified nucleotides are provided in Table 3 and Table 5C.
  • sense strands containing modified nucleotides are provided in Table 4 and Table 5C.
  • nucleotide monomers when present in an oligonucleotide, are mutually linked by 5-3′-phosphodiester bonds.
  • a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides.
  • terminal nucleotide at the 3′ end of a given oligonucleotide sequence would typically have a hydroxyl (—OH) group at the respective 3′ position of the given monomer instead of a phosphate moiety ex vivo.
  • the inverted abasic residues are inserted such that the 3′ position of the deoxyribose is linked at the 3′ end of the preceding monomer on the respective strand (see, e.g., Table 6).
  • targeting ligands, targeting groups, and linking groups used with the XDH RNAi agents disclosed herein are provided below in Table 6. More specifically, targeting groups and linking groups (which together can form a targeting ligand) include (NAG37) and (NAG37)s, for which their chemical structures are provided below in Table 6. Each sense strand and/or antisense strand can have any targeting ligands, targeting groups, or linking groups listed herein, as well as other groups, conjugated to the 5′ and/or 3′ end of the sequence.
  • the XDH RNAi agents described herein are formed by annealing an antisense strand with a sense strand.
  • a sense strand containing a sequence listed in Table 2, Table 4, or Table 5C can be hybridized to any antisense strand containing a sequence listed in Table 2, Table 3, or Table 5C provided the two sequences have a region of at least 85% complementarity over a contiguous 15, 16, 17, 18, 19, 20, or 21 nucleotide sequence.
  • the antisense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3 or Table 5C. In some aspects, the sense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4 or Table 5C.
  • an XDH RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2, Table 3, or Table 5C. In some aspects, an XDH RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, of any of the sequences in Table 2, Table 3, or Table 5C. In certain aspects, an XDH RNAi agent antisense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 3 or Table 5C.
  • an XDH RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2, Table 4, or Table 5C. In some aspects, an XDH RNAi agent sense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) at positions 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, or 4-21, of any of the sequences in Table 2, Table 4, or Table 5C. In certain aspects, an XDH RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4 or Table 5C.
  • the nucleotide at position 1 of the antisense strand can be perfectly complementary to an XDH gene, or can be non-complementary to an XDH gene.
  • the nucleotide at position 1 of the antisense strand is a U, A, or dT (or a modified version thereof).
  • the nucleotide at position 1 of the antisense strand forms an A:U or U:A base pair with the sense strand.
  • a sense strand containing a sequence listed in Table 2, Table 4, or Table 5C can be hybridized to any antisense strand containing a sequence listed in Table 2.
  • Table 3, or Table 5C provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.
  • the XDH RNAi agent has a sense strand consisting of the modified sequence of any of the modified sequences in Table 4 or Table 5C, and an antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 5C.
  • Certain representative sequence pairings are exemplified by the Duplex ID Nos, shown in Tables 5A, 5B, and 5C.
  • an XDH RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the Duplex ID Nos. presented herein. In some aspects, an XDH RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the duplexes represented by any of the Duplex ID NOs. presented herein. In some aspects, an XDH RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the duplexes represented by any of the Duplex ID NOs.
  • an XDH RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID NOs. presented herein.
  • an XDH RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID NOs. presented herein and a targeting group and/or linking group, wherein the targeting group and/or linking group is covalently linked to the sense strand or the antisense strand.
  • an XDH RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Table 2 or Tables 5A, 5B, and 5C, and further comprises a targeting group or targeting ligand.
  • an XDH RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Table 2 or Tables 5A, 5B, and 5C, and further comprises an asialoglycoprotein receptor ligand targeting group.
  • a targeting group, with or without a linker can be linked to the 5′ or 3′ end of any of the sense and/or antisense strands disclosed in Tables 2, 3, 4, or 5C.
  • a linker, with or without a targeting group can be attached to the 5′ or 3′ end of any of the sense and/or antisense strands disclosed in Tables 2, 3, 4, and 5C.
  • an XDH RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Table 2 or Tables 5A, 5B and 5C, and further comprises a targeting ligand selected from the group consisting of: (NAG37) and (NAG37)s, each as defined in Table 6.
  • an XDH RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequence of any of the antisense strand and/or sense strand nucleotide sequences in Table 3 or Table 4.
  • an XDH RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand and/or sense strand nucleotide sequences of any of the duplexes Tables 5A, 5B, and 5C, and further comprises an asialoglycoprotein receptor ligand targeting group.
  • an XDH RNAi agent comprises, consists of, or consists essentially ofany of the duplexes of Tables 5A, 5B, and 5C.
  • AD09217 usUfsgsGfaAfgGfcAfuUfcUfcAfaUf 945 (NAG37)s(invAb)sgagauugaGfAfAfugccuuccaas(invAb) 1175 cUfsc
  • AD09218 usUfsggaAfgGfCfauucUfcAfaucusc 946 (NAG37)s(invAb)sgagauuGfaGfAfAfugccuuccaas(invAb) 1176 AD09219 asAfscsUfuGfaAfgAfaGfaAfgCfuGf 947 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguus(invAb) 1177 aGfsg AD09220 asGfsasAfcUfuGfaAfgAfaGfaAf
  • an XDH RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid.
  • the RNAi agents described herein upon delivery to a cell expressing an XDH gene, inhibit or knockdown expression of one or more XDH genes in vivo and/or in vitro.
  • an XDH RNAi agent is conjugated to one or more non-nucleotide groups including, but not limited to, a targeting group, a linking group, a targeting ligand, a delivery polymer, or a delivery vehicle.
  • the non-nucleotide group can enhance targeting, delivery or attachment of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6.
  • the non-nucleotide group can be covalently linked to the 3′ and/or 5′ end of either the sense strand and/or the antisense strand.
  • an XDH RNAi agent contains a non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand.
  • a non-nucleotide group is linked to the 5′ end of an XDH RNAi agent sense strand.
  • a non-nucleotide group may be linked directly or indirectly to the RNAi agent via a linker/linking group.
  • a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.
  • a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the RNAi agent or conjugate. In some aspects, a non-nucleotide group enhances endocytosis of the RNAi agent.
  • Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate or RNAi agent to which they are attached to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the conjugate or RNAi agent.
  • a targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency for the target to which it is directed.
  • Representative targeting groups include, without limitation, compounds with affinity to cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules.
  • a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) residues, which can in some instances serve as linkers.
  • a targeting ligand comprises a galactose-derivative cluster.
  • the XDH RNAi agents described herein can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine), at the 5′-terminus and/or the 3′-terminus.
  • a reactive group such as an amino group (also referred to herein as an amine)
  • the reactive group can be used subsequently to attach a targeting moiety using methods typical in the art.
  • a targeting group comprises an asialoghycoprotein receptor ligand.
  • an asialoglycoprotein receptor ligand is a ligand that contains a moiety having affinity for the asialoglycoprotein receptor. As noted herein, the asialoglycoprotein receptor is highly expressed on hepatocytes.
  • an asialoglycoprotein receptor ligand includes or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor that is equal to or greater than that of galactose.
  • Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosammne, and N-iso-butanoylgalactos-amine (see for example: S. T. Iobst and K. Drickamer, J. B. C., 1996, 271, 6686).
  • Galactose derivatives, and clusters of galactose derivatives, that are useful for in vivo targeting of oligonucleotides and other molecules to the liver are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).
  • Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to the asialoglycoprotein receptor(s) facilitates cell-specific targeting to hepatocytes and endocytosis of the molecule into hepatocytes.
  • Asialoglycoprotein receptor ligands can be monomeric (e.g., having a single galactose derivative, also referred to as monovalent or monodentate) or multimeric (e.g., having multiple galactose derivatives).
  • the galactose derivative or galactose derivative cluster can be attached to the 3′ or 5′ end of the sense or antisense strand of the RNAi agent using methods known in the art.
  • the preparation of targeting ligands, such as galactose derivative clusters, is described in, for example, International Patent Application Publication No. WO 2018/044350 to Arrowhead Pharmaceuticals, Inc., and International Patent Application Publication No. WO 2017/156012 to Arrowhead Pharmaceuticals, Inc., the contents of both of which are incorporated by reference herein in their entirety.
  • a galactose derivative cluster comprises a molecule having two to four terminal galactose derivatives. A terminal galactose derivative is attached to a molecule through its C-1 carbon.
  • the galactose derivative cluster is a galactose derivative trimer (also referred to as tri-antennary galactose derivative or tri-valent galactose derivative).
  • the galactose derivative cluster comprises N-acetyl-galactosamine moieties.
  • the galactose derivative cluster comprises three N-acetyl-galactosamine moieties.
  • the galactose derivative cluster is a galactose derivative tetramer (also referred to as tetra-antennary galactose derivative or tetra-valent galactose derivative). In some aspects, the galactose derivative cluster comprises four N-acetyl-galactosamine moieties.
  • a galactose derivative trimer contains three galactose derivatives, each linked to a central branch point.
  • a galactose derivative tetramer contains four galactose derivatives, each linked to a central branch point.
  • the galactose derivatives can be attached to the central branch point through the C-1 carbons of the saccharides.
  • the galactose derivatives are linked to the branch point via linkers or spacers.
  • the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, e.g., U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol.
  • the PEG spacer is a PEG3 spacer.
  • the branch point can be any small molecule which permits attachment of three galactose derivatives and further permits attachment of the branch point to the RNAi agent.
  • An example of branch point group is a di-lysine or di-glutamate. Attachment of the branch point to the RNAi agent can occur through a linker or spacer.
  • the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer.
  • the linker comprises a rigid linker, such as a cyclic group.
  • a galactose derivative comprises or consists of N-acetyl-galactosamine.
  • the galactose derivative cluster is comprised of a galactose derivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.
  • compositions for delivering an XDH RNAi agent to a liver cell in vivo can include, for example, an XDH RNAi agent conjugated to a galactose derivative cluster.
  • the galactose derivative cluster is comprised of a galactose derivative trimer, which can be, for example, an N-acetyl-galactosamine trimer, or galactose derivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.
  • a targeting ligand or targeting group can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of an XDH RNAi agent disclosed herein.
  • Targeting ligands include, but are not limited to (NAG37) and (NAG37)s as defined in Table 6.
  • Other targeting groups and targeting ligands, including galactose cluster targeting ligands, are known in the art.
  • a linking group is conjugated to the RNAi agent.
  • the linking group facilitates covalent linkage of the agent to a targeting group, delivery polymer, or delivery vehicle.
  • the linking group can be linked to the 3′ and/or the 5′ end of the RNAi agent sense strand or antisense strand.
  • the linking group is linked to the RNAi agent sense strand.
  • the linking group is conjugated to the 5′ or 3′ end of an RNAi agent sense strand.
  • a linking group is conjugated to the 5′ end of an RNAi agent sense strand.
  • linking groups can include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), and/or PEG groups.
  • a targeting group is linked internally to a nucleotide on the sense strand and/or the antisense strand of the RNAi agent. In some aspects, a targeting group is linked to the RNAi agent via a linker.
  • a linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds.
  • a labile linkage contains a labile bond.
  • a linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer can further add flexibility and/or length to the linkage.
  • Spacers include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.
  • each of the RNAi agents when two or more RNAi agents are included in a single composition, each of the RNAi agents may be linked to the same targeting group or two a different targeting groups (i.e., targeting groups having different chemical structure).
  • targeting groups are linked to the XDH RNAi agents disclosed herein without the use of an additional linker.
  • the targeting group itself is designed having a linker or other site to facilitate conjugation readily present.
  • each of the RNAi agents may utilize the same linker or different linkers (i.e., linkers having different chemical structures).
  • Any of the XDH RNAi agent nucleotide sequences listed in Tables 2, 3, 4, or 5C, whether modified or unmodified, can contain 3′ and/or 5′ targeting group(s) or linking group(s).
  • Any of the XDH RNAi agent sequences listed in Table 3 or 4, or are otherwise described herein, which contain a 3′ or 5′ targeting group or linking group, can alternatively contain no 3′ or 5′ targeting group or linking group, or can contain a different 3′ or 5′ targeting group or linking group including, but not limited to, those depicted in Table 6.
  • any of the XDH RNAi agent duplexes listed in Tables 5A, 5B and 5C, whether modified or unmodified, can further comprise a targeting group or linking group, including, but not limited to, those depicted in Table 6, and the targeting group or linking group can be attached to the 3′ or 5′ terminus of either the sense strand or the antisense strand of the XDH RNAi agent duplex.
  • Targets and linking groups (which when combined can form targeting ligands) are provided in Table 6.
  • Table 4 and Table 5C provide several aspects of XDH RNAi agent sense strands having a targeting group or linking group linked to the 5′ or 3′ end.
  • NAG comprises an N-acetyl-galactosamine or another galactose derivative, as would be understood by a person of ordinary skill in the art to be attached in view of the structures above and description provided herein.
  • Other linking groups known in the art may be used.
  • a delivery vehicle can be used to deliver an RNAi agent to a cell or tissue.
  • a delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue.
  • a delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.
  • the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art.
  • the RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO 2008/0022309.
  • WO 2011/104169 and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference
  • hydrogels cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors, or other delivery systems suitable for nucleic acid or oligonucleotide delivery as known and available in the art.
  • XDH RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “medicaments”).
  • pharmaceutical compositions include at least one XDH RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism.
  • the pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from reduction in the level of the target XDH mRNA, or inhibition in expression of the target gene.
  • the pharmaceutical compositions can be used to treat a subject at risk of developing a disease, disorder, symptom, or condition that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene.
  • the method includes administering an XDH RNAi agent linked to a targeting ligand as described herein, to a subject to be treated.
  • one or more pharmaceutically acceptable excipients are added to the pharmaceutical compositions that include an XDH RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.
  • compositions that include an XDH RNAi agent and methods disclosed herein decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, organ, or subject, including by administering to the subject a therapeutically effective amount of a herein described XDH RNAi agent, thereby inhibiting the expression of XDH mRNA in the subject.
  • the subject has been previously identified as having a pathogenic upregulation of the target gene in hepatocytes.
  • the subject has been previously identified or diagnosed as having gout or hyperuricemia.
  • the subject has been suffering from symptoms associated with gout or hyperuricemia.
  • the subject would benefit from a reduction of XDH gene expression in the subject's liver.
  • the described pharmaceutical compositions including an XDH RNAi agent are used for treating or managing clinical presentations associated with gout or hyperuricemia.
  • a therapeutically (including prophylactically) effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment.
  • administration of any of the disclosed XDH RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
  • compositions that include an XDH RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of XDH mRNA and/or a reduction in serum uric acid levels. Measuring serum uric acid levels can be conducted in accordance with established methods known in the art.
  • the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions that include an XDH RNAi agent thereby treating the symptom.
  • the subject is administered a prophylactically effective amount of one or more XDH RNAi agents, thereby preventing or inhibiting the at least one symptom.
  • the route of administration is the path by which an XDH RNAi agent is brought into contact with the body.
  • methods of administering drugs and oligonucleotides and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein.
  • the XDH RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route.
  • herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some aspects, the herein described pharmaceutical compositions are administered via subcutaneous injection.
  • compositions including an XDH RNAi agent described herein can be delivered to a cell, group of cells, tissue, or subject using oligonucleotide delivery technologies known in the art.
  • any suitable method recognized in the art for delivering a nucleic acid molecule in vitro or in vivo can be adapted for use with the compositions described herein.
  • delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • the compositions are administered by subcutaneous or intravenous infusion or injection.
  • the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions described herein are formulated for administration to a subject.
  • a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients are substances other than the Active Pharmaceutical Ingredient (API, therapeutic product, e.g., XDH RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage.
  • Excipients can act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use.
  • a pharmaceutically acceptable excipient may or may not be an inert substance.
  • Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
  • compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water. Cremophor® ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Suitable carriers should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutical formulations that include the XDH RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in an aqueous sodium phosphate buffer (e.g., the XDH RNAi agent formulated in 0.5 mM sodium phosphate monobasic, 0.5 mM sodium phosphate dibasic, in water).
  • pharmaceutical formulations that include the XDH RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in water for injection (sterile water).
  • XDH RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in isotonic saline (0.9%).
  • Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension.
  • Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
  • Formulations suitable for oral administration of the XDH RNAi agents disclosed herein can also be prepared.
  • the XDH RNAi agents disclosed herein are administered orally.
  • the XDH RNAi agents disclosed herein are formulated in a capsule for oral administration.
  • the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • the XDH RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions.
  • additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, analgesics, antihistamines, or anti-inflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine, etc.).
  • RNAi agents e.g., acetaminophen, NSAIDs, diphenhydramine, etc.
  • pharmaceutical compositions may be used as “pharmaceutical compositions.”
  • “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.
  • the methods disclosed herein further comprise the step of administering a second therapeutic or treatment in addition to administering an RNAi agent disclosed herein.
  • the second therapeutic is another XDH RNAi agent (e.g., an XDH RNAi agent that targets a different sequence within the XDH target).
  • the second therapeutic can be a small molecule drug, an antibody, an antibody fragment, or an aptamer.
  • the described XDH RNAi agent(s) are optionally combined with one or more additional therapeutics.
  • the XDH RNAi agent and additional therapeutic(s) can be administered in a single composition or they can be administered separately.
  • the one or more additional therapeutics is administered separately in separate dosage forms from the RNAi agent (e.g., the XDH RNAi agent is administered by subcutaneous injection, while the additional therapeutic involved in the method of treatment dosing regimen is administered orally)
  • the described XDH RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered orally, which together provide for a treatment regimen for diseases and conditions associated with gout or hyperuricemia.
  • the described XDH RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered via a separate subcutaneous injection.
  • the XDH RNAi agent and one or more additional therapeutics are combined into a single dosage form (e.g., a “cocktail” formulated into a single composition for subcutaneous injection).
  • the XDH RNAi agents, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.
  • an effective amount of an XDH RNAi agent will be in the range of from about 0.1 to about 100 mg/kg of body weight/dose, e.g., from about 1.0 to about 50 mg/kg of body weight/dose.
  • an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose.
  • an effective amount of an active ingredient will be in the range of from about 0.5 to about 4 mg/kg of body weight per dose.
  • an effective amount of an XDH RNAi agent may be a fixed dose. In some aspects, the fixed dose is in the range of from about 5 mg to about 1,000 mg of XDH RNAi agent.
  • the fixed does is in the range of 50 to 400 mg of XDH RNAi agent. Dosing may be weekly, bi-weekly, monthly, quarterly, or at any other interval depending on the dose of XDH RNAi agent administered, the activity level of the particular XDH RNAi agent, and the desired level of inhibition for the particular subject. The Examples herein show suitable levels for inhibition in certain animal species.
  • the amount administered will depend on such variables as the overall health status of the patient or subject, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
  • compositions described herein including an XDH RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, peptide and/or an aptamer.
  • the described XDH RNAi agents when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.
  • the pharmaceutical compositions described herein may be packaged in pre-filled syringes, pen injectors, autoinjectors, infusion bags/devices, or vials.
  • the XDH RNAi agents disclosed herein can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of the RNAi agent.
  • the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from reduction and/or inhibition in expression of XDH mRNA and/or XDH protein levels, which can lead to a reduction in serum uric acid levels in, for example, a subject that has been diagnosed with or is suffering from symptoms related to gout or hyperuricemia.
  • the subject is administered a therapeutically effective amount of any one or more XDH RNAi agents.
  • Treatment of a subject can include therapeutic and/or prophylactic treatment.
  • the subject is administered a therapeutically effective amount of any one or more XDH RNAi agents described herein.
  • the subject can be a human, patient, or human patient.
  • the subject may be an adult, adolescent, child, or infant.
  • Administration of a pharmaceutical composition described herein can be to a human being or animal.
  • the XDH RNAi agents described herein can be used to treat at least one symptom in a subject having an XDH-related disease or disorder, or having a disease or disorder that is mediated at least in part by XDH gene expression.
  • the XDH RNAi agents are used to treat or manage a clinical presentation of a subject with a disease or disorder that would benefit from or be mediated at least in part by a reduction in XDH mRNA.
  • the subject is administered a therapeutically effective amount of one or more of the XDH RNAi agents or XDH RNAi agent-containing compositions described herein.
  • the methods disclosed herein comprise administering a composition comprising an XDH RNAi agent described herein to a subject to be treated.
  • the subject is administered a prophylactically effective amount of any one or more of the described XDH RNAi agents, thereby treating the subject by preventing or inhibiting the at least one symptom.
  • the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by XDH gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the XDH RNAi agents described herein.
  • the RNAi agent comprises an antisense strand comprising an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882. AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • the XDH RNAi agent comprises an antisense strand comprising a modified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising a modified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of UUCCAUAAUACUCUGAGAGAG (SEQ ID NO:1448) and a sense strand comprising a nucleic acid sequence of CUCUCUCAGAGUAUUAUGGAA (SEQ ID NO:1603).
  • a nucleic acid sequence of the antisense strand comprises a nucleic acid sequence of cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146) and a nucleic acid sequence of the sense strand comprises a nucleic acid sequence of cucucucaGfaGfuAfuuauggaa (SEQ ID NO: 1663) or (invAb)scucucucaGfaGfuAfuuauggaas(invAb) (SEQ ID NO:1680).
  • the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of AUGACAAUAUCUGUGCGGAGG (SEQ ID NO:1468) and a sense strand comprising a nucleic acid sequence of CCUCCGCACAGAUAUUGUCAU (SEQ ID NO:1623).
  • a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and a nucleic acid sequence of the sense strand comprises a nucleic acid sequence of ccuccgcaCfAfGfauauugucau (SEQ ID NO:1664) or (invAb)sccuccgcaCfAfGfauauugucaus(invAb) (SEQ ID NO:1681).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UGCAUAUUCACCAUUUAGGCA (SEQ ID NO:1397) and a sense strand comprising a nucleic acid sequence of UGCCUAAAUGGUGAAUAUGCA (SEQ ID NO:1551).
  • a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO: 1155) and a nucleic acid sequence of the sense strand comprises ugccuaaaUfgGfuGfaauaugca (SEQ ID NO:1665) or (invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) (SEQ ID NO:1682).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AUGAAACAAACAAACCCUGGA (SEQ ID NO:1440) and a sense strand comprising a nucleic acid sequence of UCCAGGGUUUGUUUGUUUCAU (SEQ ID NO:1595).
  • a nucleic acid sequence of the antisense strand comprises asUfsgsAfaAfcaaacAfaAfcCfcUfggsa (SEQ ID NO:1048) and a nucleic acid sequence of the sense strand comprises uccaggguUfUfGfuuuguuucau (SEQ ID NO:1666) or (invAb)succaggguUfUfGfuuuguuucaus(invAb) (SEQ ID NO: 1683).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AGACGAUCAUACUUGGAGAGC (SEQ ID NO:1454) and a sense strand comprising a nucleic acid sequence of GCUCUCCAAGUAUGAUCIUCU (SEQ ID NO:1609).
  • a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises gcucuccaAfGfUfaugauciucu (SEQ ID NO:1667) or (invAb)sgcucuccaAfGfUfaugauciucus(invAb) (SEQ ID NO:1684).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUGAAUGCUGAGAAAUACUC (SEQ ID NO:1438) and a sense strand comprising a nucleic acid sequence of GAGUAUUUCUCAGCAUUCAAA (SEQ ID NO:1593).
  • a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and a nucleic acid sequence of the sense strand comprises gaguauuuCfUfCfagcauucaaa (SEQ ID NO:1668) or (invAb)sgaguauuuCfUfCfagcauucaaas(invAb) (SEQ ID NO:1685).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUCCAACAAUUCUCCUUGUC (SEQ ID NO:1466) and a sense strand comprising a nucleic acid sequence of GACAAGGAGAAUUGUUGGAAA (SEQ ID NO:1621).
  • a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and a nucleic acid sequence of the sense strand comprises gacaaggaGfAfAfuuguuggaaa (SEQ ID NO:1669) or (invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) (SEQ ID NO:1686).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUGUCAACCUCACUCUUCCGA (SEQ ID NO:1465) and a sense strand comprising a nucleic acid sequence of UCGGAAGAGUGAGGUUGACAA (SEQ ID NO:1620).
  • a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and a nucleic acid sequence of the sense strand comprises ucggaagaGfUfGfagguugacaa (SEQ ID NO:1670) or (invAb)sucggaagaGfUfGfagguugacaas(invAb) (SEQ ID NO:1687).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UCAUGAUACUGAGAGCUUGCU (SEQ ID NO:1464) and a sense strand comprising a nucleic acid sequence of AGCAAGCUCUCAGUAUCAUGA (SEQ ID NO:1619).
  • a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and a nucleic acid sequence of the sense strand comprises agcaagcuCfUfCfaguaucauga (SEQ ID NO:1671) or (invAb)sagcaagcuCfUfCfaguaucaugas(invAb) (SEQ ID NO: 1688).
  • the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37)s.
  • the gene expression level and/or mRNA level of an XDH gene in a subject to whom a described XDH RNAi agent is administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the XDH RNAi agent or to a subject not receiving the XDH RNAi agent.
  • the gene expression level and/or mRNA level in the subject may be reduced in a cell, group of cells, and/or tissue of the subject.
  • the XDH gene expression is inhibited by at least about 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, or greater than 65% in the cytoplasm of hepatocytes relative to the subject prior to being administered the XDH RNAi agent or to a subject not receiving the XDH RNAi agent.
  • the XDH protein expression level in a subject to whom a described XDH RNAi agent has been administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the XDH RNAi agent or to a subject not receiving the XDH RNAi agent.
  • the protein expression level in the subject may be reduced in a cell, group of cells, tissue, blood, and/or other fluid of the subject.
  • a reduction in XDH mRNA expression levels and XDH protein expression levels can be assessed by any methods known in the art.
  • a reduction or decrease in XDH mRNA level and/or protein level are collectively referred to herein as a reduction or decrease in XDH or inhibiting or reducing the gene expression of XDH.
  • the Examples set forth herein illustrate known methods for assessing inhibition of XDH gene expression. The person of ordinary skill in the art would further know suitable methods for assessing inhibition of XDH gene expression in vivo and/or in vitro.
  • XDH RNAi agent that includes an antisense strand that is at least partially complementary to the portion of the XDH mRNA having the sequence in Table 1.
  • RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Tables 2, 3 or 5C, and a sense strand that comprises any of the sequences in Tables 2, 4, or 5C that is at least partially complementary to the antisense strand.
  • RNAi agent that includes a sense strand that comprises any of the sequences in Tables 2, 4, or 5C, and an antisense strand comprising the sequence of any of the sequences in Tables 2, 3, or 5C that is at least partially complementary to the sense strand.
  • the RNAi agent comprises an antisense strand comprising an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • the RNAi agent comprises an antisense strand comprising a modified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216. AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising a modified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of UUCCAUAAUACUCUGAGAGAG (SEQ ID NO:1448) and a sense strand comprising a nucleic acid sequence of CUCUCUCAGAGUAUUAUGGAA (SEQ ID NO:1603).
  • a nucleic acid sequence of the antisense strand comprises cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146) and a nucleic acid sequence of the sense strand comprises cucucucaGfaGfuAfuuauggaa (SEQ ID NO:1663) or (invAb)scucucucaGfaGfuAfuuauggaas(invAb) (SEQ ID NO:1680).
  • the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of AUGACAAUAUCUGUGCGGAGG (SEQ ID NO:1468) and a sense strand comprising a nucleic acid sequence of CCUCCGCACAGAUAUUGUCAU (SEQ ID NO:1623).
  • a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and a nucleic acid sequence of the sense strand comprises ccuccgcaCfAfGfauauugucau (SEQ ID NO:1664) or (invAb)sccuccgcaCfAfGfauauugucaus(invAb) (SEQ ID NO:1681).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UGCAUAUUCACCAUUUAGGCA (SEQ ID NO:1397) and a sense strand comprising a nucleic acid sequence of UGCCUAAAUGGUGAAUAUGCA (SEQ ID NO:1551).
  • a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO:1155) and a nucleic acid sequence of the sense strand comprises ugccuaaaUfgGfuGfaauaugca (SEQ ID NO:1665) or (invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) (SEQ ID NO:1682).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AUGAAACAAACAAACCCUGGA (SEQ ID NO:1440) and a sense strand comprising a nucleic acid sequence of UCCAGGGUUUGUUUGUUUCAU (SEQ ID NO:1595).
  • a nucleic acid sequence of the antisense strand comprises asUfsgsAfaAfcaaacAfaAfcCfcUfggsa (SEQ ID NO:1048) and a nucleic acid sequence of the sense strand comprises uccaggguUfUfGfuuuguuucau (SEQ ID NO: 1666) or (invAb)succaggguUfUfGfuuuguuucaus(invAb) (SEQ ID NO:1683).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AGACGAUCAUACUUGGAGAGC (SEQ ID NO:1454) and a sense strand comprising a nucleic acid sequence of GCUCUCCAAGUAUGAUCIUCU (SEQ ID NO:1609).
  • a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises gcucuccaAfGfUfaugauciucu (SEQ ID NO:1667) or (invAb)sgcucuccaAfGfUfaugauciucus(invAb) (SEQ ID NO: 1684).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUGAAUGCUGAGAAAUACUC (SEQ ID NO:1438) and a sense strand comprising a nucleic acid sequence of GAGUAUUUCUCAGCAUUCAAA (SEQ ID NO:1593).
  • a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and a nucleic acid sequence of the sense strand comprises gaguauuuCfUffagcauucaaa (SEQ ID NO:1668) or (invAb)sgaguauuuCfUfCfagcauucaaas(invAb) (SEQ ID NO:1685).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUCCAACAAUUCUCCUUGUC (SEQ ID NO:1466) and a sense strand comprising a nucleic acid sequence of GACAAGGAGAAUUGUUGGAAA (SEQ ID NO:1621).
  • a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and a nucleic acid sequence of the sense strand comprises gacaaggaGfAfAfuuguuggaaa (SEQ ID NO:1669) or (invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) (SEQ ID NO:1686).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUGUCAACCUCACUCUUCCGA (SEQ ID NO:1465) and a sense strand comprising a nucleic acid sequence of UCGGAAGAGUGAGGUUGACAA (SEQ ID NO:1620).
  • a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and a nucleic acid sequence of the sense strand comprises ucggaagaGfUfGfagguugacaa (SEQ ID NO:1670) or (invAb)sucggaagaGfUfGfagguugacaas(invAb) (SEQ ID NO:1687).
  • the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UCAUGAUACUGAGAGCUUGCU (SEQ ID NO:1464) and a sense strand comprising a nucleic acid sequence of AGCAAGCUCUCAGUAUCAUGA (SEQ ID NO:1619).
  • a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and a nucleic acid sequence of the sense strand comprises agcaagcuCfUfCfaguaucauga (SEQ ID NO:1671) or (invAb)sagcaagcuCfUfCfaguaucaugas(invAb) (SEQ ID NO:1688).
  • the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37)s.
  • methods for inhibiting expression of an XDH gene in a cell include administering to the cell an XDH RNAi agent that includes an antisense strand that is at least partially complementary to the portion of the XDH mRNA having the sequence in Table 1.
  • methods of inhibiting expression of an XDH gene in a cell include administering to a cell an XDH RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Tables 2, 3, or 5C and a sense strand that comprises any of the sequences in Tables 2, 4, or 5C that is at least partially complementary to the antisense strand.
  • RNAi agent that includes a sense strand that comprises any of the sequences in Tables 2, 4, or 5C, and an antisense strand that includes the sequence of any of the sequences in Tables 2, 3, or 5C that is at least partially complementary to the sense strand.
  • the XDH RNAi agents are administered to a subject in need thereof as a first line therapy. In some aspects, the XDH RNAi agents are administered to a subject in need thereof as a second line therapy. In certain aspects, the XDH RNAi agents are administered as a second line therapy to patients who have failed one or more first line standard of care therapies. In certain aspects, the XDH RNAi agents are administered as a maintenance therapy following the administration of one or more prior therapies. In certain aspects, the XDH RNAi agents administered as a maintenance therapy following the administration of one or more standard of care therapies. In some aspects, the XDH RNAi agents administered in combination with one or more additional therapies. In some aspects, the one or more additional therapies is a standard of care therapy. In some aspects, the one or more additional therapies is an oral therapy.
  • RNAi agent comprising an antisense strand comprising an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • the gout is uncontrolled gout.
  • the oligonucleotide, composition, or pharmaceutical composition described herein is administered as a second line therapy to patients who have failed allopurinol and/or febuxostat. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered prior to KRYSTEXXA. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered as a maintenance therapy following the administration of KRYSTEXXA.
  • XDH RNAi agents provides methods for therapeutic (including prophylactic) treatment of diseases/disorders associated with gout, hyperuricemia, elevated serum uric acid levels, or elevated XDH gene expression.
  • the described XDH RNAi agents mediate RNA interference to inhibit the expression of one or more genes necessary for production of XDH protein.
  • XDH RNAi agents can also be used to treat or prevent various diseases, disorders, or conditions, including gout.
  • compositions for delivery of XDH RNAi agents to liver cells, and specifically to hepatocytes, in vivo are described.
  • Cells, tissues, organs, and non-human organisms that include at least one of the XDH RNAi agents described herein are contemplated.
  • the cell, tissue, organ, or non-human organism is made by delivering the RNAi agent to the cell, tissue, organ or non-human organism.
  • Embodiment 1 An RNAi agent for inhibiting expression of an XDH gene, comprising:
  • an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3, nucleotides from any one of the sequences antisense strand sequences disclosed in Table 2, Table 3, or Table 5C; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.
  • Embodiment 2 An RNAi agent for inhibiting expression of an XDH gene, comprising:
  • a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from a stretch of the same length of nucleotides of SEQ ID NO: 1; and an antisense strand comprising a nucleotide sequences that is at least partially complementary to the sense strand.
  • Embodiment 3 The RNAi agent of embodiment 1, wherein the antisense strand comprises nucleotides at positions 2-18 of any one of the antisense strand sequences of Table 2, Table 3, or Table 5C.
  • Embodiment 4 The RNAi agent of embodiment 1 or embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides differing by 0 or 1 nucleotide from any one of the sense strand sequences of Table 2, Table 4, or Table 5C, and wherein the sense strand has a region of at least 85% complementarity over the 15 contiguous nucleotides to the antisense strand.
  • Embodiment 5 The RNAi agent of any one of embodiments 1-4, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.
  • Embodiment 6 The RNAi agent of any one of aspects 1-5, wherein all or substantially all of the nucleotides of the sense and/or antisense strand of the RNAi agent are modified nucleotides.
  • Embodiment 7 The RNAi agent of any one of aspects 5-6, wherein the modified nucleotide is selected from the group consisting of: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide, 2′,3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide, 2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxy nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3′-O-methyl nucle
  • Embodiment 8 The RNAi agent of embodiment 7, wherein all or substantially all of the modified nucleotides are 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.
  • Embodiment 9 The RNAi agent of any one of aspects 1-8, wherein the antisense strand comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3 or Table 5C.
  • Embodiment 10 The RNAi agent of any one of aspects 1-9, wherein the sense strand comprises the nucleotide sequence of any of the modified sense strand sequences of Table 4 or Table 5C.
  • Embodiment 11 The RNAi agent of embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences of Table 5C and the sense strand comprises the nucleotide sequence of any one of the modified sequences of Table 5C.
  • Embodiment 12 The RNAi agent of any one of aspects 1-11, wherein the RNAi agent is linked to a targeting ligand.
  • Embodiment 13 The RNAi agent of embodiment 12, wherein the targeting ligand comprises n-acetyl-galactosamine.
  • Embodiment 14 The RNAi agent of embodiment 12 or 13, wherein the targeting ligand comprises the structure of (NAG37) or (NAG37)s.
  • Embodiment 15 The RNAi agent of any one of aspects 11-14, wherein the targeting ligand is linked to the sense strand.
  • Embodiment 16 The RNAi agent of embodiment 15, wherein the targeting ligand is linked to the 5′ terminal end of the sense strand.
  • Embodiment 17 The RNAi agent of any one of aspects 1-16, wherein the sense strand is between 15 and 30 nucleotides in length, and the antisense strand is between 18 and 30 nucleotides in length.
  • Embodiment 18 The RNAi agent of embodiment 17, wherein the sense strand and the antisense strand are each between 18 and 27 nucleotides in length.
  • Embodiment 19 The RNAi agent of embodiment 18, wherein the sense strand and the antisense strand are each between 18 and 24 nucleotides in length.
  • Embodiment 20 The RNAi agent of embodiment 19, wherein the sense strand and the antisense strand are each 21 nucleotides in length.
  • Embodiment 21 The RNAi agent of any one of aspects 17-20, wherein the RNAi agent has two blunt ends.
  • Embodiment 22 The RNAi agent of any one of aspects 1-21, wherein the sense strand comprises one or two terminal caps.
  • Embodiment 23 The RNAi agent of any one of aspects 1-22, wherein the sense strand comprises one or two inverted abasic residues.
  • Embodiment 24 The RNAi agent of embodiment 1, wherein the RNAi agent is comprised of a sense strand and an antisense strand that form a duplex sequence of any one of the duplexes as listed in Table 5A, Table 5B, or Table 5C.
  • Embodiment 25 The RNAi agent of any one of aspects 1-23, wherein the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
  • Embodiment 26 The RNAi agent of embodiment 1, comprising an antisense strand that comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the antisense strand nucleotide sequences of Table 3 or Table 5C, wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, cytidine, guanosine, and uridine, respectively; cPrpa and cPrpu represent 5′-cyclopropyl phosphonate-2′-O-methyl adenosine and 5′-cyclopropyl phosphonate-2′-O-methyl uridine, respectively; CUNA and UUNA represent 2′,3′-seco-cyt
  • Embodiment 27 The RNAi agent of embodiment 1, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the nucleotide sequences of Table 4 or Table 5C, wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, cytidine, guanosine, and uridine, respectively, a_2N represents 2′-O-methyl-2-aminoadenosine; s represents a phosphorothioate linkage; and wherein all or substantially all of the nucleotides on the antisense strand are modified nucleotides.
  • Embodiment 28 The RNAi agent of any one of aspects 24-27, wherein the sense strand includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
  • Embodiment 29 The RNAi agent of any one of aspects 24-28, wherein the sense strand of the RNAi agent is linked to a targeting ligand.
  • Embodiment 30 The RNAi agent of embodiment 29, wherein the targeting ligand has affinity for the asialoglycoprotein receptor.
  • Embodiment 31 The RNAi agent of embodiment 30, wherein the targeting ligand comprises N-acetyl-galactosamine.
  • Embodiment 32 The RNAi agent of embodiment 1, wherein the targeting ligand comprises:
  • Embodiment 33 The RNAi agent of embodiment 1, wherein the antisense strand consists of a modified nucleotide sequence of Table 3 or Table 5C and the sense strand consists of a modified nucleotide sequence of Table 4 or Table 5C, wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, cytidine, guanosine, and uridine, respectively; cPrpa and cPrpu represent 5′-cyclopropyl phosphonate-2′-O-methyl adenosine and 5′-cyclopropyl phosphonate-2′-O-methyl uridine, respectively; a_2N represents 2′-O-methyl-2-aminoadenosine; CUNA and
  • Embodiment 34 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a nucleic acid sequence of the sense strand comprises an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • Embodiment 35 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises a modified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a nucleic acid sequence of the sense strand comprises a modified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877. AD14239, AD14237, or AD14235.
  • Embodiment 36 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUCCAUAAUACUCUGAGAGAG (SEQ ID NO:1448) and a nucleic acid sequence of the sense strand comprises CUCUCUCAGAGUAUUAUGGAA (SEQ ID NO:1603).
  • a nucleic acid sequence of the antisense strand comprises cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146)
  • Embodiment 39 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises AUGACAAUAUCUGUGCGGAGG (SEQ ID NO:1468) and a nucleic acid sequence of the sense strand comprises CCUCCGCACAGAUAUUGUCAU (SEQ ID NO:1623).
  • a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and the sense strand comprises (invAb)sccuccgcaCfAfGfauauugucaus(invAb) (SEQ ID NO:168
  • Embodiment 42 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UGCAUAUUCACCAUUUAGGCA (SEQ ID NO:1397) and a nucleic acid sequence of the sense strand comprises UGCCUAAAUGGUGAAUAUGCA (SEQ ID NO:1551).
  • a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO: 1155) and the sense strand comprises (invAb)sugccuaaaUfgGfu
  • Embodiment 45 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises AUGAAACAAACAAACCCUGGA (SEQ ID NO:1440) and a nucleic acid sequence of the sense strand comprises UCCAGGGUUUGUUUGUUUCAU (SEQ ID NO:1595).
  • Embodiment 48 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises AGACGAUCAUACUUGGAGAGC (SEQ ID NO:1454) and a nucleic acid sequence of the sense strand comprises GCUCUCCAAGUAUGAUCIUCU (SEQ ID NO:1609).
  • a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and the sense strand comprises (invAb)sgcucuccaAfGfUfaugauciucus(invAb) (SEQ ID NO:1684), wherein lower
  • Embodiment 51 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUUGAAUGCUGAGAAAUACUC (SEQ ID NO:1438) and a nucleic acid sequence of the sense strand comprises GAGUAUUUCUCAGCAUUCAAA (SEQ ID NO:1593).
  • a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and the sense strand
  • Embodiment 54 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUUCCAACAAUUCUCCUUGUC (SEQ ID NO:1466) and a nucleic acid sequence of the sense strand comprises GACAAGGAGAAUUGUUGGAAA (SEQ ID NO:1621).
  • a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and the sense strand comprises (invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) (S
  • Embodiment 57 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUGUCAACCUCACUCUUCCGA (SEQ ID NO:1465) and a nucleic acid sequence of the sense strand comprises UCGGAAGAGUGAGGUUGACAA (SEQ ID NO:1620).
  • a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and the sense strand comprises (invAb)sucggaagaGfUfGfagguugacaas(invAb) (SEQ ID NO
  • Embodiment 60 The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UCAUGAUACUGAGAGCUUGCU (SEQ ID NO:14(4) and a nucleic acid sequence of the sense strand comprises AGCAAGCUCUCAGUAUCAUGA (SEQ ID NO:1619).
  • a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and the sense strand comprises (invAb)sagcaagcuCfUfCfaguaucaugas(invAb) (
  • Embodiment 63 The RNAi agent of any one of embodiments 31-62, wherein the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37) or (NAG37)s.
  • Embodiment 64 The RNAi agent of any one of embodiments 31-62, wherein the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37)s.
  • Embodiment 65 The RNAi agent of any one of embodiments 31-64, wherein RNAi agent is a pharmaceutically acceptable salt.
  • Embodiment 66 A composition comprising the RNAi agent of any one of embodiments 1-65, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • Embodiment 67 A method for inhibiting expression of an XDH gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of embodiments 1-66 or the composition of embodiment 66.
  • Embodiment 68 The method of embodiment 67, wherein the cell is within a subject.
  • Embodiment 69 The method of embodiment 68, wherein the subject is a human subject.
  • Embodiment 70 The method of any one of embodiments 67-69, wherein the XDH gene expression is inhibited by at least about 30%.
  • Embodiment 71 The method of any one of embodiments 67-70, wherein the XDH activity is reduced by at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.
  • Embodiment 72 A method of treating an XDH-related disease, disorder, or symptom, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the composition of embodiment 66.
  • Embodiment 73 The method of embodiment 72, wherein the disease is gout.
  • Embodiment 74 The method of any one of embodiments 67-73, wherein the RNAi agent is administered at a dose of about 0.05 mg/kg to about 5.0 mg/kg of body weight of the human subject.
  • Embodiment 75 The method of any one of embodiments 67-74, wherein the RNAi agent is administered in two or more doses.
  • Embodiment 76 A single-stranded antisense compound for inhibiting an XDH gene, comprising an antisense nucleotide sequence having at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides, wherein the nucleotides are complementary to any of the target nucleotide sequences of Table 1.
  • Embodiment 77 A single-stranded antisense compound for inhibiting an XDH gene, comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides of any of the antisense strand sequences disclosed in Table 2, Table 3, or Table 5C.
  • XDH RNAi agent duplexes shown in Tables 5A, 5B, and 5C, above, were synthesized in accordance with the following general procedures:
  • RNAi agents The sense and antisense strands of the RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Such standard synthesis is generally known in the art. Depending on the scale, either a MerMade96E® (Bioautomation), a MerMade121, (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 ⁇ or 600 ⁇ , obtained from Prime Synthesis, Aston. Pa., USA). The monomer positioned at the 3′ end of the respective strand was attached to the solid support as a starting point for synthesis.
  • CPG controlled pore glass
  • RNA and 2′-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, Wis., USA) or Hongene Biotech (Shanghai, PRC).
  • the 2′-O-methyl phosphoramidites included the following: (5′-O-dimethoxytrityl-N-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphommidite, 5′-O-dimethoxy-trityl-N 4 -(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5′-O-dimethoxytrityl-N 2 -(isobutyryl)-2′-O-methyl-guanosine-3′-O-
  • the 2′-deoxy-2′-fluoro-phosphoramidites carried the same protecting groups as the 2′-O-methyl amidites, 5′-(4,4′-Dimethoxytrityl)-2′,3′-seco-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was also purchased from Thermo Fisher Scientific or Hongene Biotech.
  • 5′-dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia) or Hongene Biotech.
  • the cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017/214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).
  • the inverted abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, Mass., USA) or SAFC (St Louis, Mo., USA).
  • 5′-O-dimethoxytrityl-N 2 ,N 6 -(phenoxvacetate)-2′-O-methyl-diaminopurine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were obtained from ChemGenes or Hongene Biotech.
  • Targeting ligand-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), or anhydrous dimethylformamide and molecular sieves (3 ⁇ ) were added.
  • 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 12 min (RNA), 15 min (targeting ligand), 90 sec (2′-OMe), and 60 sec (2′-F).
  • the dried solid support was treated with a 1:1 volume solution of 40 wt. % methylamine in water and 28% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below).
  • Crude oligomers were purified by anionic exchange HPLC using a TSKgel SuperQ-5PW 13 ⁇ m column and Shimadzu LC-8 system.
  • Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride UV traces at 260 nm were recorded.
  • Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 26/40 column packed with Sephadex G-25 fine with a running buffer of filtered DI water or 100 mM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile.
  • RNAi agents were lyophilized and stored at ⁇ 15 to ⁇ 25° C.
  • Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 Phosphate-Buffered Saline. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the duplex concentration. The conversion factor used was either 0.050 mg/(mL-cm) or was calculated from an experimentally determined extinction coefficient.
  • XDH-GLuc Gaussia Luciferase
  • AAV Addeno-associated virus
  • Six- to eight-week-old male C57BL/6 mice were transduced with XDH-GLuc AAV serotype 8, administered at least 14 days prior to administration of an XDH RNAi agent or control.
  • Two types of XDH-GLuc AAV were used.
  • the genome of the first XDH-GLuc AAV contains the 80-2899 region of the human XDH cDNA sequence (GenBank NM_000379.4 (SEQ ID NO:1)) inserted into the 3′ UTR of the GLuc reporter gene sequence.
  • the genome of the second XDH-GLuc AAV contains the 2820-5715 region of the human XDH cDNA sequence (GenBank NM_000379.4 (SEQ ID NO:1)) inserted into the 3′ UTR of the GLuc reporter gene sequence.
  • 5E12 to 1E13 GC/kg of the respective virus in PBS in a total volume of 10 mL/kg animal's body weight was injected into mice via the tail vein to create XDH-GLuc AAV model mice.
  • Inhibition of expression of XDH by an XDH RNAi agent results in concomitant inhibition of GLuc expression, which is measured.
  • GLuc expression levels in serum were measured by the PierceTM Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific), and the mice were grouped according to average GLuc levels.
  • mice were anesthetized with 2-3% isoflurane and blood samples were collected from the submandibular area into serum separation tubes (Sarstedt AG & Co., Nümbrecht, Germany). Blood was allowed to coagulate at ambient temperature for 20 min. The tubes were centrifuged at 8,000 ⁇ g for 3 min to separate the serum and stored at 4° C. Serum was collected and measured by the PierceTM Gaussia Luciferase Glow Assay Kit according to the manufacturer's instructions. Serum GLuc levels for each animal can be normalized to the control group of mice injected with vehicle control in order to account for the non-treatment related shift in XDH expression with this model.
  • the GLuc level for each animal at a time point was divided by the pre-treatment level of expression in that animal (Day 1) in order to determine the ratio of expression “normalized to pre-treatment”.
  • Expression at a specific time point was then normalized to the control group by dividing the “normalized to pre-treatment” ratio for an individual animal by the average “normalized to pre-treatment” ratio of all mice in the normal vehicle control group.
  • the serum GLuc levels for each animal was assessed by normalizing to pre-treatment levels only.
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 7.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agent AD09218 (Group 2) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 488 of the gene; the XDH RNAi agent AD09724 (Group 3) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 122 of the gene; the XDH RNAi agent AD09599 (Group 4) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 249 of the gene; the XDH RNAi agent AD09600 (Group 5) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 252 of the gene; the XDH RNAi agent AD09733 (Group 6) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1285 of the gene; the XDH RNAi agent AD09740 (Group 7) included nucleotide sequences that were designed to inhibit expression of an XDH gene
  • RNAi agent targeting position 488 of the XDH gene can be active in vitro and in vivo in mice and in rats, the nucleotide sequence of an RNAi agent targeting this position is compromised and unsuitable for therapeutic use.
  • the seed region (2 to 7 nt) of the RNAi agent targeting position 488 matches perfectly with that of a known human microRNA (miRNA), thus this agent is expected to result in undesired silencing of hundreds of potential off-targets mimicking the known miRNA (See, e.g., Kamola et al., The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects, 11(12) PLoS Comput Biol (2015)).
  • the core 17-mer sequence (nucleotides located at positions 2-18 of the antisense strand (5′ ⁇ 3′)) of the RNAi agent targeting position 488 is complementary to transcripts of four human genes with only one mismatch, hence bearing an additional risk of reducing the expression of these four genes through a different off-target mechanism.
  • the RNAi agent of Group 2 is not a viable candidate for human therapeutic treatment.
  • RNAi agents of Group 8 (AD09736) and Group 9 (AD09937), both of which target the XDH gene at position 1963, showed generally comparable reductions of XDH (reductions of 47.9% and 40.7% on day 15, respectively) with Group 2.
  • the RNAi agents of Group 10 (AD09744) and Group 11 (AD09938), both of which target the XDH gene at position 2696, showed generally comparable reductions of XDH (showing reductions of 30.6% and 52.2%) with Group 2.
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 9.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents AD09736 (Group 2), AD09965 (Group 3), AD09937 (Group 4), AD09966 (Group 5), AD09967 (Group 6), AD09%8 (Group 7), AD09969 (Group 8), and AD09970 (Group 9) all included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene;
  • the XDH RNAi agent AD09962 (Group 10) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1964 of the gene;
  • the XDH RNAi agent AD09963 (Group 11) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1965 of the gene;
  • the XDH RNAi agent AD0964 (Group 12) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1967 of the gene.
  • RNAi agents of Groups 2-9 which all included nucleotide sequences targeting position 1963 of the XDH gene, reported substantial inhibitory activity, with certain XDH RNAi agents achieving greater than 50% inhibition in vivo.
  • the XDH RNAi agents of each of Groups 2-9 all of which target position 1963 of the XDH gene, generally showed an increase in inhibition of XDH gene expression of approximately 20-35% compared to sequences targeting neighboring positions of an XDH gene, shown in Groups 10-12 (Compare, for example, Group 5 (AD09600) at day 15 showing 53.1% inhibition (0.469) with Groups 10-12 at day 15 showing 18.2% inhibition (0.818); 25.7% inhibition (0.743); and 21.7% inhibition (0.783), respectively).
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 11.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents of Groups 2-11 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2696 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 13.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agent AD09218 included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 488 of the gene; the XDH RNAi agent AD10016 (Group 3) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 231 of the gene; the XDH RNAi agent AD10017 (Group 4) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 242 of the gene; and the XDH RNAi agents AD09734 (Group 5), AD10091 (Group 6), AD10092 (Group 7), AD10093 (Group 8), AD10094 (Group 9), AD10095 (Group 10), AD10096 (Group 11), and AD10097 (Group 12) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1322 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • RNAi agent targeting position 488 of the XDH gene (Group 2), while previously reported to be active in vivo in mice and rats, includes a compromised nucleotide sequence and is unsuitable for therapeutic use due to toxicity concerns.
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 15.
  • Example 7 Targeted Gene Position (within SEQ RNAi Agent Group ID NO: 1) and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 3083 2.0 mg/kg AD09325 Single injection on day 1 3 2995 2.0 mg/kg AD09981 Single injection on day 1 4 3016 2.0 mg/kg AD09982 Single injection on day 1 5 3041 2.0 mg/kg AD09983 Single injection on day 1 6 3498 2.0 mg/kg AD09984 Single injection on day 1 7 3598 2.0 mg/kg AD09985 Single injection on day 1 8 3877 2.0 mg/kg AD09987 Single injection on day 1 9 4394 2.0 mg/kg AD09989 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agent AD09325 (Group 2) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3083 of the gene; the XDH RNAi agent AD09981 (Group 3) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2995 of the gene; the XDH RNAi agent AD09982 (Group 4) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3016 of the gene; the XDH RNAi agent AD09983 (Group 5) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3041 of the gene; the XDH RNAi agent AD09984 (Group 6) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3498 of the gene; the XDH RNAi agent AD09985 (Group 7) included nucleotide sequences that were designed to inhibit expression of an XDH
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 17.
  • Example 8 Targeted Gene Position (within SEQ RNAi Agent Group ID NO: 1) and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 3083 2.0 mg/kg AD09325 Single injection on day 1 3 3600 2.0 mg/kg AD09986 Single injection on day 1 4 3930 2.0 mg/kg AD09988 Single injection on day 1 5 4513 2.0 mg/kg AD09990 Single injection on day 1 6 4531 2.0 mg/kg AD09991 Single injection on day 1 7 4666 2.0 mg/kg AD09992 Single injection on day 1 8 4843 2.0 mg/kg AD09993 Single injection on day 1 9 5234 2.0 mg/kg AD09994 Single injection on day 1 10 5411 2.0 mg/kg AD09995 Single injection on day 1 11 4136 2.0 mg/kg AD09608 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-11 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at the specific positions of the gene as set forth in Table 17, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • XDH RNAi agents have sufficient homology with the mouse XDH gene transcript that they are suitable to be examined for XDH gene expression inhibitory activity in wild-type mice.
  • day 1 six- to eight-week-old female C57bl/6 mice were given a single subcutaneous administration of 200 ⁇ l/20 g animal weight containing 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 19.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 51B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-14 each included nucleotide sequences that, while also being homologous to the mouse XDH gene transcript, were designed to inhibit expression of an XDH gene at the specific positions of the human XDH gene as set forth in Table 19, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • mice were euthanized on study day 10, and total RNA was isolated from both livers following collection and homogenization.
  • Mouse XDH mRNA expression was quantitated by probe-based quantitative PCR, normalized to mouse beta-actin expression, and expressed as fraction of vehicle control group (geometric mean, +/ ⁇ 95% confidence interval).
  • RNAi agent targeting position 488 of the XDH gene of Group 2 (AD09217) and Group 3 (AD09218) while being previously identified as having activity in mice and rats in vivo, includes a compromised nucleotide sequence and is unsuitable for therapeutic use due to toxicity concerns.
  • the XDH RNAi agent AD09219 (Group 4), which targets position 1612 of the XDH gene transcript, showed mRNA reductions of approximately 35.1% (0.649) in mice, which was generally comparable to the reductions exhibited by the XDH RNAi agents of Group 2 (40% inhibition (0.600)) and Group 3 (37.2% inhibition; (0.628)), which both included RNAi agents having sequences targeting position 488 of the XDH gene which as noted above has toxicity concerns.
  • XDH RNAi agents have sufficient homology with the mouse XDH gene transcript that they are suitable to be examined for XDH gene expression inhibitory activity in wild-type mice.
  • At day 1 six- to eight-week-old male C57bl/6 mice were given a single subcutaneous administration of 200 ⁇ l/20 g animal weight containing 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 21.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-14 each included nucleotide sequences that, while also being homologous to the mouse XDH gene transcript, were designed to inhibit expression of an XDH gene at positions 1612 of the human XDH gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • mice were euthanized on study day 8, and total RNA was isolated from both livers following collection and homogenization.
  • Mouse XDH mRNA expression was quantitated by probe-based quantitative PCR, normalized to mouse beta-actin expression, and expressed as fraction of vehicle control group (geometric mean, +/ ⁇ 95% confidence interval).
  • XDH RNAi agents have sufficient homology with the rat XDH gene transcript that they are suitable to be examined for XDH gene expression inhibitory activity in wild-type rats.
  • male Sprague Dawley rats were given a single subcutaneous administration of 4 mL/1 kg animal weight containing a dose of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 23.
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agent in Groups 2-5 included nucleotide sequences that, while also being homologous to the rat XDH gene transcript, were designed to inhibit expression of an XDH gene at position 488 of the human XDH gene;
  • the XDH RNAi agent in Groups 6-9 included nucleotide sequences that, while also being homologous to the rat XDH gene transcript, were designed to inhibit expression of an XDH gene at position 2612 of the human XDH gene;
  • the XDH RNAi agents in Groups 10-13 included nucleotide sequences that, while also being homologous to the rat XDH gene transcript, were designed to inhibit expression of an XDH gene at position 2616 of the human XDH gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • Rat XDH mRNA expression was quantitated by probe-based quantitative PCR, normalized to rat beta-actin expression, and expressed as fraction of vehicle control group (geometric mean, +/ ⁇ 95% confidence interval).
  • the RNAi agent targeting position 488 of the XDH gene of Groups 2-5 includes a compromised nucleotide sequence and is unsuitable for therapeutic use due to toxicity concerns.
  • the XDH RNAi agent AD09651 (Groups 6-9), which targets position 2612 of the XDH gene transcript, and the XDH RNAi agent AD09663 (Groups 10-13), which targets position 2616, both showed dose-dependent mRNA reductions that were comparable to AD09218 (targeting position 488 of the XDH gene).
  • Example 12 Targeted Gene Position (within SEQ RNAi Agent Dosing Regimen Group ID NO: 1) and Dose (on day 1) A 3083 3.0 mg/kg AD09325 Single subcutaneous injection B 4725 3.0 mg/kg AD09307 Single subcutaneous injection
  • the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agent in Groups A included nucleotide sequences that were designed to inhibit expression of a human XDH gene at position 3083; and the XDH RNAi agent in Group B (AD09307) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4725. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • AD09663, and AD09611 were evaluated in cynomolgus monkeys (cynos).
  • cynos cynomolgus monkeys
  • On days 1 and 31, three male cynos for each group (n 3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3.0 mg/kg (10 mg/mL) of the respective XDH RNAi agent, formulated in isotonic saline.
  • Example 13 Targeted Gene Position (within SEQ RNAi Agent Dosing Regimen Group ID NO: 1) and Dose (on days 1 and 31) 1 1322 3.0 mg/kg AD09734 Two subcutaneous injections 2 2612 3.0 mg/kg AD09651 Two subcutaneous injections 3 2616 3.0 mg/kg AD09663 Two subcutaneous injections 4 4289 3.0 mg/kg AD09611 Two subcutaneous injections
  • the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 27, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • liver biopsies On days ⁇ 14 or ⁇ 7 (pre-dose), 15, 43, and 80 (for Group 4 only) liver biopsies were taken. On the date of each biopsy collection, cynos were anesthetized and laparoscopy was used to extract two liver tissue samples approximately 80 mg to 120 mg each, and aliquots of approximately 50 mg were snap-frozen and stored at ⁇ 70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day ⁇ 14 or ⁇ 7, depending on the animals) cXDH mRNA measurements. Batch analysis of samples across timepoints was performed where applicable. The resulting mRNA data are reflected in the following Table 28:
  • XDH Activity Assay Using RNAScope (see, e.g., RNAscope, A Novel in Situ RNA Analysis Platform for Formalin-Fixed, Paraffin-Embedded Tissues, J Mol Diagn. 2012 January; 14(1): 22-29), it was determined that XDH mRNA transcripts are partitioned between both nuclear and cytosolic compartments. As translation to XDH protein only occurs in the cytoplasm, inhibition of cytoplasmic mRNA transcripts is considered therapeutically relevant.
  • XDH mRNA transcripts using q-PCR from whole liver homogenate is therefore not necessarily reflective of determining therapeutically relevant XDH inhibition as it measures the presence of XDH mRNA in both the cytosolic and nucleic compartments.
  • an XDH activity assay was developed capable of indirectly measuring the amount of XDH protein inhibited by the XDH RNAi agents through the RNA interference mechanism.
  • XDH activity was assessed using the following method: frozen cyno liver biopsy samples were homogenized in buffer containing 100 mM oxonic acid to inhibit endogenous uricase activity which is known to degrade uric acid. Liver homogenates were purified using Sephadex G25 spin columns, and protein concentrations adjusted to 0.5 ⁇ g/ ⁇ l total protein (lysate). XDH activity was measured by liquid-chromatography mass spectrometry (LCMS) as the conversion of xanthine to uric acid at 37° C. within a 30-minute timeframe.
  • LCMS liquid-chromatography mass spectrometry
  • the amount of uric acid generated over time is an indirect measure of the amount of cXDH protein present in the lysate; accordingly, the less uric acid identified, the less cXDH protein was present in lysate, thereby indicating a more potent XDH RNAi agent for reducing XDH protein.
  • the resulting XDH activity data (normalized to pre-dose) are shown in Table 29.
  • RNAi agent AD09611 showed substantial reductions of XDH activity that were maintained for seven weeks post the last dose (day 31).
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 30.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 4289 2.0 mg/kg AD09611 Single injection on day 1 3 4289 2.0 mg/kg AD10183 Single injection on day 1 4 4289 2.0 mg/kg AD10629 Single injection on day 1 5 4289 2.0 mg/kg AD10630 Single injection on day 1 6 4289 2.0 mg/kg AD10631 Single injection on day 1 7 4289 2.0 mg/kg AD10632 Single injection on day 1 8 4289 2.0 mg/kg AD10184 Single injection on day 1 9 4289 2.0 mg/kg AD10633 Single injection on day 1 10 4289 2.0 mg/kg AD10634 Single injection on day 1 11 4289 2.0 mg/kg AD10635 Single injection on day 1 12 4289 2.0 mg/kg AD10636 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-12 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4289 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • the XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 1.0 mg/kg (mpk) or 3.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 32.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 1963 3.0 mg/kg AD09736 Single injection on day 1 3 1963 1.0 mg/kg AD09736 Single injection on day 1 4 1963 3.0 mg/kg AD09937 Single injection on day 1 5 1963 1.0 mg/kg AD09937 Single injection on day 1 6 1963 3.0 mg/kg AD09967 Single injection on day 1 7 1963 1.0 mg/kg AD09967 Single injection on day 1 8 1963 3.0 mg/kg AD10278 Single injection on day 1 9 1963 1.0 mg/kg AD10278 Single injection on day 1 10 1963 3.0 mg/kg AD10281 Single injection on day 1 11 1963 1.0 mg/kg AD10281 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents of Groups 2-11 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 34.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 4289 4.0 mg/kg AD09611 Single injection on day 1 3 4289 2.0 mg/kg AD09611 Single injection on day 1 4 4289 1.0 mg/kg AD09611 Single injection on day 1 5 4289 4.0 mg/kg AD10183 Single injection on day 1 6 4289 2.0 mg/kg AD10183 Single injection on day 1 7 4289 1.0 mg/kg AD10183 Single injection on day 1 8 4289 4.0 mg/kg AD10631 Single injection on day 1 9 4289 2.0 mg/kg AD10631 Single injection on day 1 10 4289 1.0 mg/kg AD10631 Single injection on day 1 11 4289 4.0 mg/kg AD10184 Single injection on day 1 12 4289 2.0 mg/kg AD10184 Single injection on day 1 13 4289 1.0 mg/kg AD10184 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-13 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4289 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • the XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 200 ⁇ l/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 36.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 3598 4.0 mg/kg AD09985 Single injection on day 1 3 3598 2.0 mg/kg AD09985 Single injection on day 1 4 3598 1.0 mg/kg AD09985 Single injection on day 1 5 3598 4.0 mg/kg AD10729 Single injection on day 1 6 3598 2.0 mg/kg AD10729 Single injection on day 1 7 3598 1.0 mg/kg AD10729 Single injection on day 1 8 3598 4.0 mg/kg AD10730 Single injection on day 1 9 3598 2.0 mg/kg AD10730 Single injection on day 1 10 3598 1.0 mg/kg AD10730 Single injection on day 1 11 3598 4.0 mg/kg AD10734 Single injection on day 1 12 3598 2.0 mg/kg AD10734 Single injection on day 1 13 3598 1.0 mg/kg AD10734 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C. and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-13 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3598 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • the XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 and 2820-5715 regions of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 200 ⁇ l/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 38.
  • RNAi Agent and Dose Dosing Regimen 1 2696 Saline (no RNAi agent) Single injection on day 1 2 2696 4.0 mg/kg AD09744 Single injection on day 1 3 2696 2.0 mg/kg AD09744 Single injection on day 1 4 2696 1.0 mg/kg AD09744 Single injection on day 1 5 2696 4.0 mg/kg AD10621 Single injection on day 1 6 2696 2.0 mg/kg AD10621 Single injection on day 1 7 2696 1.0 mg/kg AD10621 Single injection on day 1 8 1963 4.0 mg/kg AD09736 Single injection on day 1 9 1963 2.0 mg/kg AD09736 Single injection on day 1 10 1963 1.0 mg/kg AD09736 Single injection on day 1 11 1963 4.0 mg/kg AD09937 Single injection on day 1 12 1963 2.0 mg/kg AD09937 Single injection on day 1 13 1963 1.0 mg/kg AD09937 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C. and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-13 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at positions 2696 and 1963 of the gene. (See, e.g. SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • the XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 200 ⁇ l/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 40.
  • RNAi Agent and Dose Dosing Regimen 1 1963 Saline (no RNAi agent) Single injection on day 1 2 1963 4.0 mg/kg AD09736 Single injection on day 1 3 1963 2.0 mg/kg AD09736 Single injection on day 1 4 1963 4.0 mg/kg AD10967 Single injection on day 1 5 1963 2.0 mg/kg AD10967 Single injection on day 1 6 1963 4.0 mg/kg AD10968 Single injection on day 1 7 1963 2.0 mg/kg AD10968 Single injection on day 1 8 1963 4.0 mg/kg AD10969 Single injection on day 1 9 1963 2.0 mg/kg AD10969 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-9 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • the XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 200 ⁇ l/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 42.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 4289 4.0 mg/kg AD09611 Single injection on day 1 3 4289 2.0 mg/kg AD09611 Single injection on day 1 4 4289 1.0 mg/kg AD09611 Single injection on day 1 5 4289 4.0 mg/kg AD10631 Single injection on day 1 6 4289 2.0 mg/kg AD10631 Single injection on day 1 7 4289 1.0 mg/kg AD10631 Single injection on day 1 8 3598 4.0 mg/kg AD09985 Single injection on day 1 9 3598 2.0 mg/kg AD09985 Single injection on day 1 10 3598 1.0 mg/kg AD09985 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-10 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at positions 4289 and 3598 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • XDH RNAi agent AD09611 which was previously evaluated in cynomolgus monkeys (cynos) in the study described in Example 13, was further evaluated in cynomolgus monkeys (cynos).
  • On days 1, 15, and 29, three male cynos for each group (n 3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3.0 mg/kg (10 mg/mL) of the respective XDH RNAi agent, formulated in isotonic saline.
  • Example 21 Targeted Gene Position (within SEQ RNAi Agent Dosing Regimen Group ID NO: 1) and Dose (on days 1, 15, and 29) 1 4289 3.0 mg/kg AD09611 Three subcutaneous injections 2 4289 3.0 mg/kg AD09611 Three subcutaneous injections
  • the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 43, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • liver biopsies were taken from Group 1 animals. On days ⁇ 7, 43, 71, and 99, liver biopsies were taken from Group 2 animals. On the date of each biopsy collection, cynos were sedated and Menghini technique was used to extract two liver tissue samples, and aliquots of approximately 10 mg were snap-frozen and stored at ⁇ 70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day ⁇ 14 or ⁇ 7, depending on the animals) cXDH mRNA measurements. The resulting mRNA data are reflected in the following Table 45:
  • Example 22 Targeted Gene Position (within SEQ RNAi Agent Dosing Regimen Group ID NO: 1) and Dose (on days 1, 15, and 29) 1 4289 3.0 mg/kg AD10631 Three subcutaneous injections 2 1963 3.0 mg/kg AD09736 Three subcutaneous injections 3 2696 3.0 mg/kg AD10621 Three subcutaneous injections 4 3598 3.0 mg/kg AD09985 Three subcutaneous injections
  • the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 47. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • AD10631 was designed to target position 4289 and was comprised of a chemically modified nucleotide sequence similar to AD09611, but included a 5′-cyclopropyl-phosphonate modified nucleotide at the 5′ terminal end of the antisense strand.
  • liver biopsies were taken.
  • cynos were sedated and 3.5 mm ⁇ 310 mm clamshell biopsy forceps were used to extract one liver tissue sample approximately 160 mg to 240 mg, and aliquots of approximately 50 mg were snap-frozen and stored at ⁇ 70° C. until analysis.
  • the biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day ⁇ 7) cXDH mRNA measurements.
  • the resulting mRNA data are reflected in Table 48:
  • AD09736 Group 2
  • AD010621 Group 3
  • AD09985 Group 4
  • AD10631 was reported to have a 74% reduction in XDH activity, which is similar to what was seen with the XDH activity assay performed on cyno liver biopsy samples administered AD09611 (which targeted the same position on the XDH gene as AD10631) as reported in Example 13 and Example 21.
  • the XDH RNAi agents were prepared in accordance with the procedures set forth in Example 1.
  • the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • RNAi Agent SEQ ID NO: 1 AD09218 488 AD09744 2696 AD10012 2696 AD10621 2696 AD09736 1963 AD09937 1963 AD10278 1963 AD09218 488 AD09985 3598 AD10731 3598 AD09611 4289 AD10184 4289 AD10631 4289
  • XDH RNAi agents in vitro was performed by seeding primary human or cynomolgus monkey hepatocytes cells. Cells were seeded at 25,000 cells per well in 50 ⁇ L culture medium in 96-well collagen coated plate. Cells were treated with each of the XDH RNAi agent duplexes shown in Table 50 immediately after cells were seeded by adding 50 ⁇ L per well at 2 ⁇ the final concentration, followed by gentle mixing and incubation at 37° C., 5% CO 2 , for 48 hours without disturbing the cells. Isolation and purification of RNA was completed using a commercially available kit according to the manufacturer's instructions (Zymo Quick-RNA Miniprep Kit (Zymo Research, Irvine, Calif.)). Relative expression of each of the XDH RNAi agents was determined by qRT-PCR by comparing the expression levels of XDH mRNA to an endogenous control (PPIA).
  • PPIA endogenous control
  • RNAi agent AD10012 in primary human hepatocytes, at 1 nM, results in 0.2485 residual XDH gene relative expression, or 75.15% XDH gene knockdown.
  • AD10012 was reported to have an EC50 of 0.012 nM (6 point repeat with free uptake in primary human hepatocytes), meaning AD10012 achieves 50% XDH gene knockdown at 0.012 nM concentration.
  • RNAi 0.01 nM EC50 Agent Avg SD 0.1 nM SD 1 nM SD 10 nM SD 100 nM SD 1000 nM SD (nM) AD09218 1.1908 0.4415 0.7427 0.2296 0.3515 0.1042 0.3459 0.1794 0.2624 0.0653 0.3672 0.1138 0.073 AD09744 1.1048 0.2004 0.6711 0.0780 0.6122 0.0581 0.1599 0.0769 0.3055 0.0624 0.3551 0.1225 0.098 AD10012 0.8735 0.0804 0.3435 0.0463 0.2485 0.0293 0.1707 0.1403 0.1840 0.0354 0.2882 0.1552 0.012 AD10621 0.6962 0.1486 0.3373 0.0537 0.2388 0.0516 0.1614 0.0148 0.1714 0.0338 0.1947 0.0297 0.033 AD097
  • the XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 53.
  • RNAi Agent and Dose Dosing Regimen 1 2696 Saline (no RNAi agent) Single injection on day 1 2 2696 2.0 mg/kg AD09744 Single injection on day 1 3 2696 2.0 mg/kg AD10012 Single injection on day 1 4 2696 2.0 mg/kg AD10619 Single injection on day 1 5 2696 2.0 mg/kg AD10620 Single injection on day 1 6 2696 2.0 mg/kg AD10621 Single injection on day 1 7 2696 2.0 mg/kg AD10622 Single injection on day 1 8 2696 2.0 mg/kg AD10623 Single injection on day 1 9 2696 2.0 mg/kg AD10624 Single injection on day 1 10 2696 2.0 mg/kg AD10625 Single injection on day 1 11 2696 2.0 mg/kg AD10626 Single injection on day 1 12 2696 2.0 mg/kg AD10627 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents in Groups 2-12 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 26% of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • Example 22 Targeted Gene Position (within RNAi Agent Group SEQ ID NO: 1) and Dose Dosing Regimen (on day 1) 1 2696 3.0 mg/kg AD10621 Single subcutaneous injection 2 3598 3.0 mg/kg AD09985 Single subcutaneous injection 3 2696 1.0 mg/kg AD10621 Single subcutaneous injection 4 3598 1.0 mg/kg AD09985 Single subcutaneous injection
  • the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 55. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • liver biopsies were taken on days ⁇ 6 (day ⁇ 3 for one of the animals), 29, 55, and 99 (day 100 for one of the animals).
  • cynos were sedated and needles were used to extract two liver tissue samples approximately 20 mg each. Samples were weighed, snap-frozen and stored at ⁇ 70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day ⁇ 6 or day ⁇ 3) cXDH mRNA measurements. The resulting mRNA data are reflected in Table 56:
  • XDH activity was assessed using the XDH Activity Assay method described in Example 13. The resulting XDH activity data are shown in Table 57.
  • AD10621 Group 1
  • AD09985 Group 2
  • XDH RNAi agents capable of reducing XDH protein expression by ⁇ 85% in liver cells (hepatocytes).
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 58.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 488 2.0 mg/kg AD09218 Single injection on day 1 3 139 2.0 mg/kg AD09725 Single injection on day 1 4 235 2.0 mg/kg AD09598 Single injection on day 1 5 239 2.0 mg/kg AD09726 Single injection on day 1 6 332 2.0 mg/kg AD09727 Single injection on day 1 7 2320 2.0 mg/kg AD09741 Single injection on day 1 8 2357 2.0 mg/kg AD09742 Single injection on day 1 9 2361 2.0 mg/kg AD09743 Single injection on day 1 10 2696 2.0 mg/kg AD09744 Single injection on day 1 11 2701 2.0 mg/kg AD09745 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents of Groups 2-11 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at the positions of the gene listed on Table 58. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used.
  • each mouse was given a single subcutaneous administration of 250 ⁇ l/25 g animal weight containing either 2.0 mg/kg (mpk) or 4.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 60.
  • RNAi Agent and Dose Dosing Regimen 1 N/A Saline (no RNAi agent) Single injection on day 1 2 2696 2.0 mg/kg AD10621 Single injection on day 1 3 2696 4.0 mg/kg AD10621 Single injection on day 1 4 2701 2.0 mg/kg AD09745 Single injection on day 1 5 2701 4.0 mg/kg AD09745 Single injection on day 1 6 2701 2.0 mg/kg AD12167 Single injection on day 1 7 2701 4.0 mg/kg AD12167 Single injection on day 1 8 2696 2.0 mg/kg AD12168 Single injection on day 1 9 2696 4.0 mg/kg AD12168 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • the XDH RNAi agents of Groups 2-9 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at positions 2696 and 2701 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • the XDH RNAi agent of Group 2 and 3 showed superior XHD inhibition compared to each of the RNAi agents in Groups 4-9 in vivo.
  • a single dose of 2.0 mg/kg of AD10621 reported approximately 59% inhibition of XDH (0.410) and a single 4.0 mg/kg dose reported approximately 77% inhibition (0.232) on day 22.

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Abstract

The present disclosure relates to RNAi agents, e.g., double stranded RNAi agents, able to inhibit xanthine dehydrogenase (XDH) gene expression. Also disclosed are pharmaceutical compositions that include XDH RNAi agents and methods of use thereof. The XDH RNAi agents disclosed herein may be conjugated to targeting ligands to facilitate the delivery to cells, including to hepatocytes. Delivery of the XDH RNAi agents in vivo provides for inhibition of XDH gene expression. The RNAi agents can be used in methods of treatment of diseases, disorders, or symptoms mediated in part by XDH gene expression, such as gout and hyperuricemia.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a Continuation of U.S. patent application Ser. No. 17/748,779, filed on May 19, 2022, which claims priority from U.S. Provisional Patent Application Ser. No. 63/213,097, filed on Jun. 21, 2021, and U.S. Provisional Patent Application Ser. No. 63/323,430, filed on Mar. 24, 2022, the contents of each of which are incorporated herein by reference in their entirety.
    • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Oct. 12, 2022, is named 58651-713_302_SL.xml and is 2,552,829 bytes in size.
    • FIELD OF THE INVENTION
  • The present disclosure relates to RNA interference (RNAi) agents, e.g., double stranded RNAi agents, for inhibition Xanthine Dehydrogenase (XDH; alternatively referred to as XO, XOR, xanthine dehydrogenase/oxidase, xanthine oxidoreductase, or XAN1), pharmaceutical compositions that include XDH RNAi agents, and methods of use thereof.
    • BACKGROUND
  • Gout is a progressive inflammatory arthritis caused by hyperuricemia (elevated serum uric acid levels) and deposition of monosodium urate crystals in joints and tendons. Gout is estimated to affect 0.6% of the world population with a substantially higher prevalence in certain geographical regions and ethnic groups. Gout patients without receiving a urate-lowering therapy suffer from recurrent episodes of gout flare (inflammation response) and ultimately can develop advanced gout, which is characterized by chronic joint pain and activity limitation.
  • Xanthine dehydrogenase is a molybdenum-containing hydroxylase that catalyzes the production of uric acid from xanthine. XDH is highly expressed in liver and gastrointestinal tract. Hepatocyte-specific ablation of XDH or global inhibition of XDH activity reverses hyperuricemia phenotype in animal models.
  • Small molecule inhibitors of XDH have been widely used for urate-lowering therapies. However, a large population of gout patients are intolerant of or refractory to these therapies, and some serious side effects include increased risk of death. There remains an unmet need for novel XDH inhibitors, such as XDH RNAi agents, to reduce hepatic XDH levels and treat hyperuricemia and gout.
    • SUMMARY
  • Disclosed herein are RNAi agents for inhibiting expression of an XDH gene, comprising an antisense strand comprising at least 17 contiguous nucleotides differing by 0 or 1 nucleotide from any one of the sequences of Table 2, Table 3, or Table 5C; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.
  • In some aspects, the antisense strand comprises nucleotides 2-18 of any one of the sequences of Table 2, Table 3, or Table 5C.
  • In some aspects, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides differing by 0 or 1 nucleotide from 15 contiguous nucleotides of any one of the sense strand sequences of Table 2 or Table 4, and wherein the sense strand has a region of at least 85% complementarity over the 15 contiguous nucleotides to the antisense strand.
  • In some aspects, at least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.
  • According to some aspects, all or substantially all of the nucleotides of the sense and/or antisense strand of the RNAi agent are modified nucleotides.
  • In some aspects, the modified nucleotide is selected from the group consisting of: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide, 2′,3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide, 2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxy nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3′-O-methyl nucleotide.
  • In certain aspects, the all or substantially all of the modified nucleotides are 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.
  • In some aspects, the antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3.
  • In some aspects, the sense strand consists of, consists essentially of, or comprises the nucleotide sequence of any of the modified sense strand sequences of Table 4.
  • In some aspects, the antisense strand comprises the nucleotide sequence of any one of the modified sequences of Table 3 and the sense strand comprises the nucleotide sequence of any one of the modified sequences of Table 4.
  • In certain aspects, the RNAi agents are linked to a targeting ligand. In some aspects, the targeting ligand comprises N-acetyl-galactosamine. In certain aspects, the targeting ligand comprises the structure of (NAG37) or (NAG37)s. In certain aspects, the targeting ligand is linked to the sense strand. In some aspects, the targeting ligand is linked to the 5′ terminal end of the sense strand.
  • In some aspects, the sense strand is between 15 and 30 nucleotides in length, and the antisense strand is between 18 and 30 nucleotides in length. In other aspects, the sense strand and the antisense strand are each between 18 and 27 nucleotides in length. In other aspects, the sense strand and the antisense strand are each between 18 and 24 nucleotides in length. In still other aspects, sense strand and the antisense strand are each 21 nucleotides in length.
  • In some aspects, the RNAi agents have two blunt ends.
  • In some aspects, the sense strand comprises one or two terminal caps. In other aspects, the sense strand comprises one or two inverted abasic residues.
  • In some aspects, the RNAi agents are comprised of a sense strand and an antisense strand that form a duplex sequence of any one of the duplex structures shown in Table 5A, 5B or 5C.
  • In some aspects, the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
  • In some aspects, the sense strand of the RNAi agents is linked to a targeting ligand. In some aspects, the targeting ligand has affinity for the asialoglycoprotein receptor. In some aspects, the targeting ligand comprises N-acetyl-galactosamine.
  • In further aspects, the targeting ligand comprises.
  • Figure US20230139166A1-20230504-C00001
  • Also disclosed herein are compositions comprising the disclosed RNAi agents, wherein the compositions further comprise a pharmaceutically acceptable excipient.
  • Also provided herein are methods for inhibiting expression of an XDH gene in a cell, the methods comprising introducing into a cell an effective amount of the disclosed RNAi agents or the disclosed compositions.
  • In some aspects, the cell is within a subject. In some aspects, the subject is a human subject.
  • In some aspects, the XDH gene expression is inhibited by at least about 30%. In some aspects, the XDH gene expression is inhibited by at least about 50% in the cytoplasm of hepatocytes.
  • Further provided herein are methods of treating an XDH-related disease, disorder, or symptom, the methods comprising administering to a human subject in need thereof a therapeutically effective amount of the disclosed compositions.
  • In some aspects, the disease is gout.
  • In some aspects, the symptom is hyperuricemia.
  • In some aspects, the RNAi agents are administered at a dose of about 0.05 mg/kg to about 5.0 mg/kg of body weight of the human subject.
  • In other aspects, the RNAi agent is administered in two or more doses.
  • Also provided herein are usages of the disclosed RNAi agents or the disclosed compositions, for the treatment of a disease, disorder, or symptom that is mediated at least in part by XDH gene expression.
  • In some aspects, the disease is gout.
  • In some aspects, the symptom is hyperuricemia.
  • Further provided herein are usages of the disclosed RNAi agents or the disclosed compositions, for the preparation of a pharmaceutical compositions for treating a disease, disorder, or symptom that is mediated at least in part by XDH gene expression.
  • In some aspects, the RNAi agent is administered at a dose of about 0.05 mg/kg to about 5.0 mg/kg of body weight of the human subject.
    • DETAILED DESCRIPTION
  • The disclosed RNAi agents, compositions thereof, and methods of use may be understood more readily by reference to the following detailed description, which form a part of this disclosure. It is to be understood that the disclosure is not limited to what is specifically described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.
  • It is to be appreciated that while certain features of the disclosures included herein are, for clarity, described herein in the context of separate embodiments, they may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.
    • Definitions
  • As used herein, an “RNAi agent” means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target gene in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted (i.e. XDH mRNA). RNAi agents can include one or more modified nucleotides and/or one or more non-phosphodiester linkages.
  • As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.
  • As used herein, the terms “sequence” and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature. A nucleic acid molecule can comprise unmodified and/or modified nucleotides. A nucleotide sequence can comprise unmodified and/or modified nucleotides.
  • As used herein, a “base,” “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.
  • As used herein, the term “nucleotide” has the same meaning as commonly understood in the art. Thus, the term “nucleotide” as used herein, refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleoside linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as nucleotide analogs herein. Herein, a single nucleotide can be referred to as a monomer or unit.
  • As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleobase or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or otherwise suitable in vivo or in vitro conditions) and form a duplex or double helical structure under certain standard conditions with an oligonucleotide that includes the second nucleotide sequence. The person of ordinary skill in the art would be able to select the set of conditions most appropriate for a hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.
  • As used herein, “perfectly complementary” or “fully complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • As used herein, “partially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • As used herein, “substantially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
  • As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used with respect to the nucleobase or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a sequence of an MUC5AC mRNA.
  • As used herein, the term “substantially identical” or “substantial identity,” as applied to a nucleic acid sequence means the nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity or more, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The subject matter disclosed herein encompass nucleotide sequences substantially identical to those disclosed herein.
  • As used herein, the terms “individual”, “patient” and “subject”, are used interchangeably to refer to a member of any animal species including, but not limited to, birds, humans and other primates, and other mammals including commercially relevant mammals or animal models such as mice, rats, monkeys, cattle, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.
  • As used herein, the terms “treat.” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include the prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity, and/or frequency of one or more symptoms of a disease in a subject.
  • As used herein, the phrase “introducing into a cell,” when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell. The phrase “functional delivery,” means delivering the RNAi agent to the cell in a manner that enables the RNAi agent to have the expected biological activity, e.g., sequence-specific inhibition of gene expression.
  • Unless stated otherwise, use of the symbol
    Figure US20230139166A1-20230504-P00001
    as used herein means that any group or groups may be linked thereto that is in accordance with the scope of the subject matters described herein.
  • As used herein, the term “isomers” refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a “chiral center.”
  • As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are present and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.
  • As used in a claim herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • The person of ordinary skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art. Correspondingly, compounds described herein with labile protons or basic atoms should also be understood to represent salt forms of the corresponding compound. Compounds described herein may be in a free acid, free base, or salt form. Pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.
  • As used herein, the term “linked” or “conjugated” when referring to the connection between two compounds or molecules means that two compounds or molecules are joined by a covalent bond. Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.
  • As used herein, the term “including” is used to herein mean, and is used interchangeably with, the phrase “including but not limited to.” The term “or” is used herein to mean, and is used interchangeably with, the term “and/or.” unless the context clearly indicates otherwise.
  • 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each sub-combination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.
  • The term “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−20% or less, +/−10% or less, +/−5% or less, or +/−1% or less of and from the specified value, insofar such variations are appropriate to perform in the present disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself. For example, “about 4” includes 4.
  • Other objects, features, aspects, and advantages of the invention will be apparent from the following detailed description, accompanying figures, and from the claims.
    • DETAILED DESCRIPTION RNAi Agents
  • Described herein are RNAi agents for inhibiting expression of an XDH gene. Each XDH RNAi agent comprises a sense strand and an antisense strand. The sense strand can be 15 to 49 nucleotides in length. The antisense strand can be 18 to 49 nucleotides in length. The sense and antisense strands can be either the same length or they can be different lengths. In some aspects, the sense and antisense strands are each independently 18 to 27 nucleotides in length. In some aspects, both the sense and antisense strands are each 21-26 nucleotides in length. In some aspects, the sense and antisense strands are each 21-24 nucleotides in length. In some aspects, the sense and antisense strands are each independently 19-21 nucleotides in length. In some aspects, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some aspects, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some aspects, a sense strand is 23 nucleotides in length and an antisense strand is 21 nucleotides in length. In some aspects, both the sense and antisense strands are each 21 nucleotides in length. In some aspects, the RNAi agent antisense strands are each 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the RNAi agent sense strands are each 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. The sense and antisense strands are annealed to form a duplex, and in some aspects, a double-stranded RNAi agent has a duplex length of about 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
  • Examples of nucleotide sequences used in forming XDH RNAi agents are provided in Tables 2, 3, 4, and 5C. Examples of RNAi agent duplexes, that include the sense strand and antisense strand sequences in Tables 2, 3, 4 and 5C, are shown in Tables 5A, 5B and 5C.
  • In some aspects, the region of perfect, substantial, or partial complementarity between the sense strand and the antisense strand is 15-26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides in length and occurs at or near the 5′ end of the antisense strand (e.g., this region may be separated from the 5′ end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly, substantially, or partially complementary).
  • A sense strand of the XDH RNAi agents described herein includes at least 15 consecutive nucleotides that have at least 85% identity to a core stretch sequence (also referred to herein as a “core stretch” or “core sequence”) of the same number of nucleotides in an XDH mRNA. In some aspects, a sense strand core stretch sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a core stretch sequence in the antisense strand, and thus the sense strand core stretch sequence is typically perfectly identical or at least about 85% identical to a nucleotide sequence of the same length (sometimes referred to, e.g., as a target sequence) present in the XDH mRNA target. In some aspects, this sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some aspects, this sense strand core stretch is 17 nucleotides in length. In some aspects, this sense strand core stretch is 19 nucleotides in length.
  • An antisense strand of an XDH RNAi agent described herein includes at least 15 consecutive nucleotides that have at least 85% complementarity to a core stretch of the same number of nucleotides in an XDH mRNA and to a core stretch of the same number of nucleotides in the corresponding sense strand. In some aspects, an antisense strand core stretch is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a nucleotide sequence (e.g., target sequence) of the same length present in the XDH mRNA target. In some aspects, this antisense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some aspects, this antisense strand core stretch is 19 nucleotides in length. In some aspects, this antisense strand core stretch is 17 nucleotides in length. A sense strand core stretch sequence can be the same length as a corresponding antisense core sequence or it can be a different length.
  • The XDH RNAi agent sense and antisense strands anneal to form a duplex. A sense strand and an antisense strand of an XDH RNAi agent can be partially, substantially, or fully complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least 85% complementary or 100% complementary to the antisense core stretch sequence. In some aspects, the sense strand core stretch sequence contains a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that is at least 85% or 100% complementary to a corresponding 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of an XDH RNAi agent have a region of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides that is at least 85% base paired or 100% base paired.)
  • In some aspects, the antisense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2, Table 3, or Table 5C In some aspects, the sense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2, Table 4, or Table 5C.
  • In some aspects, the sense strand and/or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of the core stretch sequences. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sequence in the XDH mRNA. The sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in the XDH mRNA. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sense strand's additional nucleotides, if present.
  • As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5′ and/or 3′ end of the sense strand core stretch sequence and/or antisense strand core stretch sequence. The extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding antisense strand. Conversely, the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core stretch nucleotides or extension nucleotides, in the corresponding sense strand. In some aspects, both the sense strand and the antisense strand of an RNAi agent contain 3′ and 5′ extensions. In some aspects, one or more of the 3′ extension nucleotides of one strand base pairs with one or more 5′ extension nucleotides of the other strand. In other aspects, one or more of 3′ extension nucleotides of one strand do not base pair with one or more 5′ extension nucleotides of the other strand. In some aspects, an XDH RNAi agent has an antisense strand having a 3′ extension and a sense strand having a 5′ extension. In some aspects, the extension nucleotide(s) are unpaired and form an overhang. As used herein, an “overhang” refers to a stretch of one or more unpaired nucleotides located at a terminal end of either the sense strand or the antisense strand that does not form part of the hybridized or duplexed portion of an RNAi agent disclosed herein.
  • In some aspects, an XDH RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other aspects, an XDH RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, or 3 nucleotides in length. In some aspects, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding XDH mRNA sequence. In some aspects, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding XDH mRNA sequence.
  • In some aspects, an XDH RNAi agent comprises a sense strand having a 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In some aspects, one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides that correspond to or are the identical to nucleotides in the XDH mRNA sequence. In some aspects, the 3′ sense strand extension includes or consists of one of the following sequences, but is not limited to: T, UT, TT, UU, UUT, TTT, or TIT (each listed 5′ to 3′).
  • A sense strand can have a 3′ extension and/or a 5′ extension. In some aspects, an XDH RNAi agent comprises a sense strand having a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some aspects, one or more of the sense strand extension nucleotides comprise nucleotides that correspond to or are identical to nucleotides in the XDH mRNA sequence.
  • Examples of sequences used in forming XDH RNAi agents are provided in Tables 2, 3, 4, and 5C. In some aspects, an XDH RNAi agent antisense strand includes a sequence of any of the sequences in Tables 2, 3, or 5C In certain aspects, an XDH RNAi agent antisense strand comprises or consists of any one of the modified sequences in Table 3. In some aspects, an XDH RNAi agent antisense strand includes the sequence of nucleotides (from 5′ end→3′ end) at positions 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, of any of the sequences in Tables 2, 3, or 5C. In some aspects, an XDH RNAi agent sense strand includes the sequence of any of the sequences in Tables 2, 4, or 5C. In some aspects, an XDH RNAi agent sense strand includes the sequence of nucleotides (from 5′ end→3′ end) at positions 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Tables 2, 4, or 5C. In certain aspects, an XDH RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4.
  • In some aspects, the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some aspects, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some aspects, the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a blunt end. In some aspects, the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a blunt end. In some aspects, both ends of an RNAi agent form blunt ends. In some aspects, neither end of an RNAi agent is blunt-ended. As used herein a “blunt end” refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base-pair).
  • In some aspects, the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a frayed end. In some aspects, the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a frayed end. In some aspects, both ends of an RNAi agent form a frayed end. In some aspects, neither end of an RNAi agent is a frayed end. As used herein a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands from a pair (i.e., do not form an overhang) but are not complementary (i.e. form a non-complementary pair). In some aspects, one or more unpaired nucleotides at the end of one strand of a double stranded RNAi agent form an overhang. The unpaired nucleotides may be on the sense strand or the antisense strand, creating either 3′ or 5′ overhangs. In some aspects, the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′ overhang end, a frayed end and a 5′ overhang end, a frayed end and a 3′ overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhang end and a 3′ overhang end, two frayed ends, or two blunt ends. Typically, when present, overhangs are located at the 3′ terminal ends of the sense strand, the antisense strand, or both the sense strand and the antisense strand.
  • The XDH RNAi agents disclosed herein may also be comprised of one or more modified nucleotides. In some aspects, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the XDH RNAi agent are modified nucleotides. The XDH RNAi agents disclosed herein may further be comprised of one or more modified internucleoside linkages, e.g., one or more phosphorothioate linkages. In some aspects, an XDH RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some aspects, a 2′-modified nucleotide is combined with modified internucleoside linkage.
  • In some aspects, an XDH RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some aspects, an XDH RNAi agent is prepared as a pharmaceutically acceptable salt. In some aspects, an XDH RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms that are well known in the art are within the scope of the inventions disclosed herein.
    • Modified Nucleotides
  • Modified nucleotides, when used in various oligonucleotide constructs, can preserve activity of the compound in cells while at the same time increasing the serum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans upon administering of the oligonucleotide construct.
  • In some aspects, an XDH RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxyl nucleotide). In some aspects, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2′-modified nucleotides, inverted nucleotides, modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2′,3′-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3′-O-methoxy (2′ internucleoside linked) nucleotides, 2′-F-Arabino nucleotides, 5′-Me, 2′-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides, 2′-modified nucleotides (i.e., a nucleotide with a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring) include, but are not limited to, 2′-O-methyl nucleotides, 2′-fluoro nucleotides (also referred to herein as 2′-deoxy-2′-fluoro nucleotides), 2′-deoxy nucleotides, 2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides (also referred to as 2′-MOE), 2′-amino nucleotides, and 2′-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in a single XDH RNAi agent or even in a single nucleotide thereof. The XDH RNAi agent sense strands and antisense strands can be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines. (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.
  • In some aspects, the 5′ and/or 3′ end of the antisense strand can include abasic residues (Ab), which can also be referred to as an “abasic site” or “abasic nucleotide.” An abasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1′ position of the sugar moiety. In some aspects, an abasic residue can be placed internally in a nucleotide sequence. In some aspects, Ab or AbAb can be added to the 3′ end of the antisense strand. In some aspects, the 5′ end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some aspects, UUAb, UAb, or Ab are added to the 3′ end of the sense strand. In some aspects, an abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.
  • In some aspects, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. As used herein, an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. In some aspects, one or more nucleotides of an RNAi agent is an unmodified ribonucleotide. Chemical structures for certain modified nucleotides are set forth in Table 6 herein.
    • Modified Internucleoside Linkages
  • In some aspects, one or more nucleotides of an XDH RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. In some aspects, a modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some aspects, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.
  • In some aspects, a sense strand of an XDH RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of an XDH RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some aspects, a sense strand of an XDH RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of an XDH RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
  • In some aspects, an XDH RNAi agent sense strand contains at least two phosphorothioate internucleoside linkages. In some aspects, the phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3′ end of the sense strand. In some aspects, one phosphorothioate internucleoside linkage is at the 5′ end of the sense strand nucleotide sequence, and another phosphorothioate linkage is at the 3′ end of the sense strand nucleotide sequence. In some aspects, two phosphorothioate internucleoside linkages are located at the 5′ end of the sense strand, and another phosphorothioate linkage is at the 3′ end of the sense strand. In some aspects, the sense strand does not include any phosphorothioate internucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5′ and 3′ ends and the optionally present inverted abasic residue terminal caps. In some aspects, the targeting ligand is linked to the sense strand via a phosphorothioate linkage.
  • In some aspects, an XDH RNAi agent antisense strand contains four phosphorothioate internucleoside linkages. In some aspects, the four phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5′ end of the antisense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5′ end. In some aspects, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5′ end of the antisense strand, and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5′ end of the antisense strand. In some aspects, an XDH RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand.
    • Capping Residues or Moieties
  • In some aspects, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a “cap,” a “terminal cap,” or a “capping residue.” As used herein, a “capping residue” is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of a nucleotide sequence of an RNAi agent disclosed herein. A capping residue can provide the RNAi agent, in some instances, with certain beneficial properties, such as, for example, protection against exonuclease degradation. In some aspects, inverted abasic residues (invAb) (also referred to in the art as “inverted abasic sites”) are added as capping residues. (See, e.g., F. Czaudema, Nucleic Acids Res., 2003, 31(11), 2705-16; U.S. Pat. No. 5,998,203). Capping residues are generally known in the art, and include, for example, inverted abasic residues as well as carbon chains such as a terminal C3H7 (propyl), C6H13 (hexyl), or C12H25 (dodecyl) groups. In some aspects, a capping residue is present at either the 5′ terminal end, the 3′ terminal end, or both the 5′ and 3′ terminal ends of the sense strand. In some aspects, the 5′ end and/or the 3′ end of the sense strand may include more than one inverted abasic deoxyribose moiety as a capping residue.
  • In some aspects, one or more inverted abasic residues (invAb) are added to the 3′ end of the sense strand. In some aspects, one or more inverted abasic residues (invAb) are added to the 5′ end of the sense strand. In some aspects, one or more inverted abasic residues or inverted abasic sites are inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some aspects, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.
  • In some aspects, one or more inverted abasic residues (invAb) are added to the 5′ end of the sense strand. In some aspects, one or more inverted abasic residues can be inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)s), or other linkages. In some aspects, the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent. In some aspects, an inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some aspects, the 3′ end of the antisense strand core stretch sequence, or the 3′ end of the antisense strand sequence, may include an inverted abasic residue. The chemical structures for inverted abasic deoxyribose residues are shown in Table 6 below.
    • XDH RNAi Agents
  • The XDH RNAi agents disclosed herein are designed to target specific positions on an XDH gene (e.g., SEQ ID NO:1).
  • NM_000379.4 Homo sapiens xanthine dehydrogenase (XDH),  
    mRNA transcript (SEQ ID NO: 1):
    1 acagagcagt gataactacc tgccagtgtc tcttaggagt gaggtacctg gagttcgggg
    61 accccaacct gtgacaatga cagcagacaa attggttttc tttgtgaatg gcagaaaggt
    121 ggtggagaaa aatgcagatc cagagacaac ccittiggcc tacctgagaa gaaagttggg
    181 gctgagtgga accaagctcg gctgtggaga ggggggctgc ggggcttgca cagtgatgct
    241 ctccaagtat gatcgtctgc agaacaagat cgtccacttt tctgccaatg cctgcctggc
    301 ccccatctgc tccttgcacc atgttgcagt gacaactgtg gaaggaatag gaagcaccaa
    361 gacgaggctg catcctgtgc aggagagaat tgccaaaagc cacggctccc agtgcgggtt
    421 ctgcacccct ggcatcgtca tgagtatgta cacactgctc cggaatcagc ccgagcccac
    481 catggaggag attgagaatg ccttccaagg aaatctgtgc cgctgcacag gctacagacc
    541 catcctccag ggcttccgga cctttgccag ggatggtgga tgctgtggag gagatgggaa
    601 taatccaaat tgctgcatga accagaagaa agaccactca gtcagcctct cgccatcttt
    661 attcaaacca gaggagttca cgcccctgga tccaacccag gagcccattt ttcccccaga
    721 gttgctgagg ctgaaagaca ctcctcggaa gcagctgcga tttgaagggg agcgtgtgac
    781 gtggatacag gcctcaaccc tcaaggagct gctggacctc aaggctcagc accctgacgc
    841 caagctggtc gtggggaaca cggagattgg cattgagatg aagttcaaga atatgctgtt
    901 tcctatgatt gtctgcccag cctggatccc tgagctgaat tcggtagaac atggacccga
    961 cggtatctcc tttggagctg cttgccccct gagcattgtg gaaaaaaccc tggtggatgc
    1021 tgttgctaag cttcctgccc aaaagacaga ggtgttcaga ggggtcctgg agcagctgcg
    1081 ctggtttgct gggaagcaag tcaagtctgt ggcgtccgtt ggagggaaca tcatcactgc
    1141 cagccccatc tccgacctca accccgtgtt catggccagt ggggccaagc tgacacttgt
    1201 gtccagaggc accaggagaa ctgtccagat ggaccacacc ttcttccctg gctacagaaa
    1261 gaccctgctg agcccggagg agatactgct ctccatagag atcccctaca gcagggaggg
    1321 ggagtatttc tcagcattca agcaggcctc ccggagagaa gatgacattg ccaaggtaac
    1381 cagtggcatg agagattat tcaagccagg aaccacagag gtacaggagc tggccctttg
    1441 ctatggtgga atggccaaca gaaccatctc agccctcaag accactcaga ggcagctttc
    1501 caagctctgg aaggaggagc tgctgcagga cgtgtgtgca ggactggcag aggagctgca
    1561 tctgcctccc gatgcccctg gtggcatggt ggacttccgg tgcaccctca ccctcagctt
    1621 cttcttcaag ttctacctga cagtccttca gaagctgggc caagagaacc tggaagacaa
    1681 gtgtggtaaa ctggacccca ctttcgccag tgcaacttta ctgtttcaga aagacccccc
    1741 agccgatgtc cagctcttcc aagaggtgcc caagggtcag tctgaggagg acatggtggg
    1801 ccggcccctg ccccacctgg cagcggacat gcaggcctct ggtgaggccg tgtactgtga
    1861 cgacattcct cgctacgaga atgagctgtc tctccggctg gtcaccagca cccgggccca
    1921 cgccaagatc aagtccatag atacatcaga agctaagaag gttccagggt ttgtttgttt
    1981 catttccgct gatgatgttc ctgggagtaa cataactgga atttgtaatg atgagacagt
    2041 ctttgcgaag gataaggtta cttgtgttgg gcatatcatt ggtgctgtgg ttgctgacac
    2101 cccggaacac acacagagag ctgcccaagg ggtgaaaatc acctatgaag aactaccagc
    2161 cattatcaca attgaggatg ctataaagaa caactccttt tatggacctg agctgaagat
    2221 cgagaaaggg gacctaaaga agggglatc cgaagcagat aatgttgtgt caggggagat
    2281 atacatcggt ggccaagagc acttctacct ggagactcac tgcaccattg ctgttccaaa
    2341 aggcgaggca ggggagatgg agctctttgt gtctacacag aacaccatga agacccagag
    2401 ctttgttgca aaaatgttgg gggttccagc aaaccggatt gtggttcgag tgaagagaat
    2461 gggaggaggc tttggaggca aggagacccg gagcactgtg gtgtccacgg cagtggccct
    2521 ggctgcatat aagaccggcc gccctgtgcg atgcatgctg gaccgtgatg aggacatgct
    2581 gataactggt ggcagacatc ccttcctggc cagatacaag gttggcttca tgaagactgg
    2641 gacagttgtg gctcttgagg tggaccactt cagcaatgtg gggaacaccc aggatctctc
    2701 tcagagtatt atggaacgag ctttattcca catggacaac tgctataaaa tccccaacat
    2761 ccggggcact gggcggctgt gcaaaaccaa ccttccctcc aacacggcct tccggggctt
    2821 tggggggccc caggggatgc tcattgccga gtgctggatg agtgaagttg cagtgacctg
    2881 tgggatgcct gcagaggagg tgcggagaaa aaacctgtac aaagaagggg acctgacaca
    2941 cttcaaccag aagcttgagg gtttcacctt gcccagatgc tgggaagaat gcctagcaag
    3001 ctctcagtat catgctcgga agagtgaggt tgacaagttc aacaaggaga attgttggaa
    3061 aaagagagga ttgtgcataa ttcccaccaa gtttggaata agctttacag ttccatict
    3121 gaatcaggca ggagccctac ttcatgtgta cacagatggc tctgtgctgc tgacccacgg
    3181 ggggactgag atgggccaag gccttcatac caaaatggtc caggtggcca gtagagctct
    3241 gaaaatcccc acctctaaga tttatatcag cgagacaagc actaacactg tgcccaacac
    3301 ctctcccacg gctgcctctg tcagcgctga cctcaatgga caggccgtct atgcggcttg
    3361 tcagaccatc ttgaaaaggc tggaacccta caagaagaag aatcccagtg gctcctggga
    3421 agactgggtc acagctgcct acatggacac agtgagcttg tctgccactg ggttttatag
    3481 aacacccaat ctgggctaca gctttgagac taactcaggg aaccccttcc actacttcag
    3541 ctatggggtg gcttgctctg aagtagaaat cgactgccta acaggagatc ataagaacct
    3601 ccgcacagat attgtcatgg atgttggctc cagtctaaac cctgccattg atattggaca
    3661 ggtggaaggg gcatttgtcc agggccttgg cctcttcacc ctagaggagc tacactattc
    3721 ccccgagggg agcctgcaca cccgtggccc tagcacctac aagatcccgg catttggcag
    3781 catccccatt gagttcaggg tgtccctgct ccgcgactgc cccaacaaga aggccatcta
    3841 tgcatcgaag gctgttggag agccgcccct cttcctggct gcttctatct tctttgccat
    3901 caaagatgcc atccgtgcag ctcgagctca gcacacaggt aataacgtga aggaactctt
    3961 ccggctagac agccctgcca ccccggagaa gatccgcaat gcctgcgtgg acaagttcac
    4021 caccctgtgt gtcactggtg tcccagaaaa ctgcaaaccc tggtctgtga gggtctaaag
    4081 agagagtcct cagcagagtc ttcttgtgct gcctttgggc ttccatggag caggaggaac
    4141 ataccacaga acatggatct attaaagtca cagaatgaca gacctgtgat ttgtcaagat
    4201 gggatttgga agacaagtga atgcaatgga agattagat caaaaatgta atttgtaaac
    4261 acaatgataa gcaaattcaa aactgttatg cctaaatggt gaatatgcaa ttaggatcat
    4321 tttctgtctg ttttaatcat gtatctggaa tagggtcggg aagggtttgt gctattcccc
    4381 acttactgga cagcctgtat aacctcaagt tctgatggtg tctgtccttt gaagaggatt
    4441 cccacaaacc tctagaagct taaaccgaag ttactttaaa tcgtgtgcct tcctgtgaaa
    4501 gcctggcctt caaaccaatg aacagcaaag cataaccttg aatctatact caaattagc
    4561 aatgaggcag tggggtaagg ttaaatcctc taaccatctt tgaatcattg gaaagaataa
    4621 agaatgaaac aaattcaagg ttaattggat ctgattagt gaagctgcat aaagcaagat
    4681 tactctataa tacaaaaatc caaccaactc aattattgag cacgtacaat gttctagatt
    4741 tctttccctt cctctttgaa gagaatattt gtattccaaa tactctttga gtatttacaa
    4801 aaaagattat gtttaatctt tacatttgaa gccaaagtaa tttccaccta gaaatgatgc
    4861 tatcagtcct ggcatggtgg ctcaccccta taatcccagc actttgggag gctaaggcag
    4921 gagaattgct tgagcccagc agtttgagac cagcctgggc aacatagaga gctcctgtct
    4981 ttaaaaaaaa ttlattaat tagttggtct tgatagtgca tgcctgtagt cccaactact
    5041 tgaaaggctg aggtggagag atcatttgag ctcaggaggt tgaggctgca gtgagctatg
    5101 attgcgccac tgcactcctg cctgagcgac tgagcaagat cttgtctctg aagaaaaaaa
    5161 aagaaataaa aatgctgcta tcaaaatcaa gcccaaccag aggtagaaga gccaagaagc
    5221 ctgggttctc atcctagctc tgtctcttct gtctctatct ttgtgatctt ggactgtcaa
    5281 ttccccttcc tgtgatccat tttactgcaa acataagggt tgcagtaaag ggttgtctca
    5341 cgtcttctgc tttaaaagcc tataaatata tgacctgaaa actccagtta cataaaggat
    5401 ctgcagctat ctaaggcttg gttttcttac tgtcatatga tacctgggtc taatgaactc
    5461 tgctgagatc acctcaagtt tctgcggttg gtaaagagaa caagggaaga acaaacatcc
    5521 callattgc tccaaatggt gatttaatcc ctacatggtg ctgggtggac aatgtgtcac
    5581 tgtcacatgc cttcactgta taaatccaac cttctgccag agagaatctg tggttctggc
    5641 catggaggga ggatagtgga aatgatatag ttggactggt gcttgatgtc actaataaat
    5701 gaaactgtca gctgg
  • As defined herein, an antisense strand sequence is designed to target an XDH gene at a given position on the gene when the 5′ terminal nucleobase of the antisense strand is aligned with a position that is 21 nucleotides downstream (towards the 3′ end) from the position on the gene when base pairing to the gene. For example, as illustrated in Tables 1 and 2 herein, an antisense strand sequence designed to target an XDH gene at position 1322 requires that when base pairing to the gene, the 5′ terminal nucleobase of the antisense strand is aligned with position 1342 of the XDH gene.
  • As provided herein, an XDH RNAi agent does not require that the nucleobase at position 1 (5′→3′) of the antisense strand be complementary to the gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides. For example, for an XDH RNAi agent disclosed herein that is designed to target position 1322 of an XDH gene, the 5′ terminal nucleobase of the antisense strand of the of the XDH RNAi agent is aligned with position 1342 of the gene; however, the 5′ terminal nucleobase of the antisense strand may be, but is not required to be, complementary to position 1342 of an XDH gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides. As shown by, among other things, the various examples disclosed herein, the specific site of binding of the gene by the antisense strand of the XDH RNAi agent (e.g., whether the XDH RNAi agent is designed to target an XDH gene at position 238, at position 1322, at position 1963, at position 2696, at position 2995, at position 3041, at position 3016, at position 3598, at position 4289, at position 2612, or at some other position) is important to the level of inhibition achieved by the XDH RNAi agent.
  • In some aspects, the XDH RNAi agents disclosed herein target an XDH gene at or near the positions of the XDH gene sequence shown in Table 1. In some aspects, the antisense strand of an XDH RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to a target XDH 19-mer sequence disclosed in Table 1.
  • TABLE 1
    XDH 19-mer mRNA Target Sequences (taken from homo sapiens xanthine
    dehydrogenase (XDH), mRNA, GenBank NM_000379.4 (SEQ ID NO: 1))
    XDH 19-mer Corresponding Targeted Gene
    SEQ  Target Sequences Positions of Sequence Position (as
    ID No. (5′ → 3′) on SEQ ID NO: 1  referred to herein)
    2 UCAGCUUCUUCUUCAAGUU 1614-1632 1612
    3 AGCUUCUUCUUCAAGUUCU 1616-1634 1614
    4 UUCUUCUUCAAGUUCUACC 1619-1637 1617
    5 GGGUGAAAAUCACCUAUGA 2130-2148 2128
    6 GUGAAAAUCACCUAUGAAG 2132-2150 2130
    7 UGAAAAUCACCUAUGAAGA 2133-2151 2131
    8 GAAAAUCACCUAUGAAGAA 2134-2152 2132
    9 ACCAGCCAUUAUCACAAUU 2155-2173 2153
    10 AGAACAACUCCUUUUAUGG 2187-2205 2185
    11 GAACAACUCCUUUUAUGGA 2188-2206 2186
    12 GACAAGCACUAACACUGUG 3274-3292 3272
    13 GUCAUGAGUAUGUACACAC 437-455 435
    14 GACAUGCUGAUAACUGGUG 2573-2591 2571
    15 AUACAAGGUUGGCUUCAUG 2614-2632 2612
    16 AAGGUUGGCUUCAUGAAGA 2618-2636 2616
    17 AGGUUGGCUUCAUGAAGAC 2619-2637 2617
    18 GUUGGCUUCAUGAAGACUG 2621-2639 2619
    19 GAGAAUUGUUGGAAAAAGA 3047-3065 3045
    20 GGCUUGCUCUGAAGUAGAA 3550-3568 3548
    21 UUGCUCUGAAGUAGAAAUC 3553-3571 3551
    22 CUGCCAUUGAUAUUGGACA 3642-3660 3640
    23 AGAUCGUCCACUUUUCUGC 267-285 265
    24 CCGAAGCAGAUAAUGUUGU 2250-2268 2248
    25 CUCUCUCAGAGUAUUAUGG 2696-2714 2694
    26 CACCAAGUUUGGAAUAAGC 3085-3103 3083
    27 GCAUAAAGCAAGAUUACUC 4667-4685 4665
    28 CAAUGUUCUAGAUUUCUUU 4727-4745 4725
    29 UGCUGGAUGAGUGAAGUUG 2852-2870 2850
    30 GCUGGAUGAGUGAAGUUGC 2853-2871 2851
    31 CUGGAUGAGUGAAGUUGCA 2854-2872 2852
    32 UGCUCUCCAAGUAUGAUCG 237-255 235
    33 GAUCGUCUGCAGAACAAGA 251-269 249
    34 CGUCUGCAGAACAAGAUCG 254-272 252
    35 CGCCAGUGCAACUUUACUG 1705-1723 1703
    36 GAUAAGGUUACUUGUGUUG 2051-2069 2049
    37 CAGCCAUUAUCACAAUUGA 2157-2175 2155
    38 AGCUCUCAGUAUCAUGCUC 2999-3017 2997
    39 AGAGUGAGGUUGACAAGUU 3021-3038 3019
    40 GAGUGAGGUUGACAAGUUC 3022-3040 3020
    41 UCAACAAGGAGAAUUGUUG 3039-3057 3037
    42 AACAUACCACAGAACAUGG 4138-4156 4136
    43 ACAUGGAUCUAUUAAAGUC 4151-4169 4149
    44 CAUGGAUCUAUUAAAGUCA 4152-4170 4150
    45 CCUAAAUGGUGAAUAUGCA 4291-4309 4289
    46 ACCUCUAGAAGCUUAAACC 4448-4466 4446
    47 CCUUCAAACCAAUGAACAG 4507-4525 4505
    48 AAUGAACAGCAAAGCAUAA 4517-4535 4515
    49 UGAACAGCAAAGCAUAACC 4519-4537 4517
    50 GAACAGCAAAGCAUAACCU 4520-4538 4518
    51 ACAGCAAAGCAUAACCUUG 4522-4540 4520
    52 AAAGCAUAACCUUGAAUCU 4527-4545 4525
    53 AACCAACUCAAUUAUUGAG 4702-4720 4700
    54 UCCUGUGAUCCAUUUUACU 5288-5306 5286
    55 UUUUCUUACUGUCAUAUGA 5422-5440 5420
    56 GGAGAAAAAUGCAGAUCCA 124-142 122
    57 CAGAGACAACCCUUUUGGC 141-159 139
    58 CUCCAAGUAUGAUCGUCUG 241-259 239
    59 AACUGUGGAAGGAAUAGGA 334-352 332
    60 GCAUCGUCAUGAGUAUGUA 432-450 430
    61 CUUCCAAGGAAAUCUGUGC 502-520 500
    62 GGCAUUGAGAUGAAGUUCA 869-887 867
    63 UGAAGUUCAAGAAUAUGCU 879-897 877
    64 AAUAUGCUGUUUCCUAUGA 890-908 888
    65 UGCUCUCCAUAGAGAUCCC 1287-1305 1285
    66 GUAUUUCUCAGCAUUCAAG 1324-1342 1322
    67 CCAAGAUCAAGUCCAUAGA 1923-1941 1921
    68 CAGGGUUUGUUUGUUUCAU 1965-1983 1963
    69 CACCUAUGAAGAACUACCA 2140-2158 2138
    70 GAACUACCAGCCAUUAUCA 2150-2168 2148
    71 GCCAUUAUCACAAUUGAGG 2159-2177 2157
    72 AGCUGAAGAUCGAGAAAGG 2211-2229 2209
    73 GCACCAUUGCUGUUCCAAA 2322-2340 2320
    74 GGAGCUCUUUGUGUCUACA 2359-2377 2357
    75 CUCUUUGUGUCUACACAGA 2363-2381 2361
    76 CUCUCAGAGUAUUAUGGAA 2698-2716 2696
    77 AGAGUAUUAUGGAACGAGC 2703-2721 2701
    78 AGGGUUUGUUUGUUUCAUU 1966-1984 1964
    79 GGGUUUGUUUGUUUCAUUU 1967-1985 1965
    80 GUUUGUUUGUUUCAUUUCC 1969-1987 1967
    81 UCUCCAAGUAUGAUCGUCU 240-258 238
    82 AGGAGAUUGAGAAUGCCUU 486-504 484
    83 AGAAUGCCUUCCAAGGAAA 495-513 493
    84 UGCCUUCCAAGGAAAUCUG 499-517 497
    85 AGAAUAUGCUGUUUCCUAU 888-906 886
    86 UUGGAGGGAACAUCAUCAC 1119-1137 1117
    87 GCUUCUUCUUCAAGUUCUA 1617-1635 1615
    88 GUUGGGCAUAUCAUUGGUG 2066-2084 2064
    89 UCUACACAGAACACCAUGA 2372-2390 2370
    90 CACCCAGGAUCUCUCUCAG 2686-2704 2684
    91 CAAGCUCUCAGUAUCAUGC 2997-3015 2995
    92 GGAAGAGUGAGGUUGACAA 3018-3036 3016
    93 CAAGGAGAAUUGUUGGAAA 3043-3061 3041
    94 AGCUUUGAGACUAACUCAG 3500-3518 3498
    95 UCCGCACAGAUAUUGUCAU 3600-3618 3598
    96 CGCACAGAUAUUGUCAUGG 3602-3620 3600
    97 CUGCUUCUAUCUUCUUUGC 3879-3897 3877
    98 CACACAGGUAAUAACGUGA 3932-3950 3930
    99 UGUAUAACCUCAAGUUCUG 4396-4414 4394
    100 CCAAUGAACAGCAAAGCAU 4515-4533 4513
    101 UAACCUUGAAUCUAUACUC 4533-4551 4531
    102 CAUAAAGCAAGAUUACUCU 4668-4686 4666
    103 CACCUAGAAAUGAUGCUAU 4845-4863 4843
    104 AGCUCUGUCUCUUCUGUCU 5236-5254 5234
    105 AAGGCUUGGUUUUCUUACU 5413-5431 5411
    106 GUGAUGCUCUCCAAGUAUG 233-251 231
    107 CAAGUAUGAUCGUCUGCAG 244-262 242
    108 GCAUGAGAGUUUUAUUCAA 1386-1404 1384
    109 CAAGAUCGUCCACUUUUCU 265-283 263
    110 CAUGUUGCAGUGACAACUG 320-338 318
    111 UGACAACUGUGGAAGGAAU 330-348 328
    112 GGAGGAGAUUGAGAAUGCC 484-502 482
    113 CACGGAGAUUGGCAUUGAG 859-877 857
    114 AGAUGAAGUUCAAGAAUAU 876-894 874
    115 GAGAUACUGCUCUCCAUAG 1280-1298 1278
    116 GGAGUAUUUCUCAGCAUUC 1321-1339 1319
    117 GAGUAUUUCUCAGCAUUCA 1322-1340 1320
    118 GGAGAGAAGAUGACAUUGC 1353-1371 1351
    119 UAACAUAACUGGAAUUUGU 2008-2026 2006
    120 AGCCAUUAUCACAAUUGAG 2158-2176 2156
    121 GCUUUGUUGCAAAAAUGUU 2400-2418 2398
    122 UUUGUUGCAAAAAUGUUGG 2402-2420 2400
    123 GAUUGUGGUUCGAGUGAAG 2437-2455 2435
    124 GAUUGAGAAUGCCUUCCAA 490-508 488
  • In some aspects, an XDH RNAi agent includes an antisense strand wherein position 19 of the antisense strand (5′→3′) is capable of forming a base pair with position 1 of a 19-mer target sequence disclosed in Table 1. In some aspects, an XDH RNAi agent includes an antisense strand wherein position 1 of the antisense strand (5′→3′) is capable of forming a base pair with position 19 of the 19-mer target sequence disclosed in Table 1.
  • In some aspects, an XDH RNAi agent includes an antisense strand wherein position 2 of the antisense strand (5′→3′) is capable of forming a base pair with position 18 of the 19-mer target sequence disclosed in Table 1. In some aspects, an XDH RNAi agent includes an antisense strand wherein positions 2 through 18 of the antisense strand (5′→3′) are capable of forming base pairs with each of the respective complementary bases located at positions 18 through 2 of the 19-mer target sequence disclosed in Table 1.
  • For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) can be perfectly complementary to the XDH gene, or can be non-complementary to the XDH gene. In some aspects, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) is a U. A. or dT. In some aspects, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair with the sense strand.
  • In some aspects, an XDH RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end→3′ end) at positions 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences in Table 2, Table 3, or Table 5C. In some aspects, an XDH RNAi sense strand comprises the sequence of nucleotides (from 5′ end→3′ end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2, Table 4, or Table 5C.
  • In some aspects, an XDH RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end→3′ end) at positions 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences of Table 2, Table 3, or Table 5C. In some aspects, an XDH RNAi sense strand comprises the sequence of nucleotides (from 5′ end→3′ end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences of Table 2, Table 4, or Table 5C.
  • In some aspects, an XDH RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5′ end→3′ end) at positions 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising the sequence of nucleotides (from 5′ end→3′ end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2 or Table 4.
  • In some aspects, the XDH RNAi agents include core 19-mer nucleotide sequences shown in the following Table 2.
  • TABLE 2
    XDH RNAi Agent Antisense Strand and Sense Strand Core Stretch Base 
    Sequences (N = any nucleobase; 1 = hypoxanthine (inosine nucleotide);
    (A2N) = 2-aminoadenine nucleotide)
    Antisense Strand  Sense Strand 
    Base Sequence Base Sequence Corresponding
    (5′ → 3′) (5′ → 3′) Positions of
    SEQ (Shown as an  SEQ (Shown as an  Identified Targeted
    ID Unmodified ID Unmodified Sequence on Gene
    No. Nucleotide Sequence) No. Nucleotide Sequence) SEQ ID NO: 1 Position
    125 AACUUGAAGAAGAAGCUGA 535 UCAGCUUCUUCUUCAAGUU 1614-1632 1612
    126 UACUUGAAGAAGAAGCUGA 536 UCAGCUUCUUCUUCAAGUA 1614-1632 1612
    127 NACUUGAAGAAGAAGCUGA 537 UCAGCUUCUUCUUCAAGUN 1614-1632 1612
    128 NACUUGAAGAAGAAGCUGN 538 NCAGCUUCUUCUUCAAGUN 1614-1632 1612
    129 AGAACUUGAAGAAGAAGCU 539 AGCUUCUUCUUCAAGUUCU 1616-1634 1614
    130 UGAACUUGAAGAAGAAGCU 540 AGCUUCUUCUUCAAGUUCA 1616-1634 1614
    131 NGAACUUGAAGAAGAAGCU 541 AGCUUCUUCUUCAAGUUCN 1616-1634 1614
    132 NGAACUUGAAGAAGAAGCN 542 NGCUUCUUCUUCAAGUUCN 1616-1634 1614
    133 UGUAGAACUUGAAGAAGAA 543 UUCUUCUUCAAGUUCUACA 1619-1637 1617
    134 NGUAGAACUUGAAGAAGAA 544 UUCUUCUUCAAGUUCUACN 1619-1637 1617
    135 NGUAGAACUUGAAGAAGAN 545 NUCUUCUUCAAGUUCUACN 1619-1637 1617
    136 UCAUAGGUGAUUUUCACCC 546 GGGUGAAAAUCACCUAUGA 2130-2148 2128
    137 NCAUAGGUGAUUUUCACCC 547 GGGUGAAAAUCACCUAUGN 2130-2148 2128
    138 NCAUAGGUGAUUUUCACCN 548 NGGUGAAAAUCACCUAUGN 2130-2148 2128
    139 UUUCAUAGGUGAUUUUCAC 549 GUGAAAAUCACCUAUGAAA 2132-2150 2130
    140 NUUCAUAGGUGAUUUUCAC 550 GUGAAAAUCACCUAUGAAN 2132-2150 2130
    141 NUUCAUAGGUGAUUUUCAN 551 NUGAAAAUCACCUAUGAAN 2132-2150 2130
    142 UCUUCAUAGGUGAUUUUCA 552 UGAAAAUCACCUAUGAAGA 2133-2151 2131
    143 NCUUCAUAGGUGAUUUUCA 553 UGAAAAUCACCUAUGAAGN 2133-2151 2131
    144 NCUUCAUAGGUGAUUUUCN 554 NGAAAAUCACCUAUGAAGN 2133-2151 2131
    145 UUCUUCAUAGGUGAUUUUC 555 GAAAAUCACCUAUGAAGAA 2134-2152 2132
    146 NUCUUCAUAGGUGAUUUUC 556 GAAAAUCACCUAUGAAGAN 2134-2152 2132
    147 NUCUUCAUAGGUGAUUUUN 557 NAAAAUCACCUAUGAAGAN 2134-2152 2132
    148 AAUUGUGAUAAUGGCUGGU 558 ACCAGCCAUUAUCACAAUU 2155-2173 2153
    149 UAUUGUGAUAAUGGCUGGU 559 ACCAGCCAUUAUCACAAUA 2155-2173 2153
    150 NAUUGUGAUAAUGGCUGGU 560 ACCAGCCAUUAUCACAAUN 2155-2173 2153
    151 NAUUGUGAUAAUGGCUGGN 561 NCCAGCCAUUAUCACAAUN 2155-2173 2153
    152 UCAUAAAAGGAGUUGUUCU 562 AGAACAACUCCUUUUAUGA 2187-2205 2185
    153 NCAUAAAAGGAGUUGUUCU 563 AGAACAACUCCUUUUAUGN 2187-2205 2185
    154 NCAUAAAAGGAGUUGUUCN 564 NGAACAACUCCUUUUAUGN 2187-2205 2185
    155 UCCAUAAAAGGAGUUGUUC 565 GAACAACUCCUUUUAUGGA 2188-2206 2186
    156 NCCAUAAAAGGAGUUGUUC 566 GAACAACUCCUUUUAUGGN 2188-2206 2186
    157 NCCAUAAAAGGAGUUGUUN 567 NAACAACUCCUUUUAUGGN 2188-2206 2186
    158 UACAGUGUUAGUGCUUGUC 568 GACAAGCACUAACACUGUA 3274-3292 3272
    159 NACAGUGUUAGUGCUUGUC 569 GACAAGCACUAACACUGUN 3274-3292 3272
    160 NACAGUGUUAGUGCUUGUN 570 NACAAGCACUAACACUGUN 3274-3292 3272
    161 UUGUGUACAUACUCAUGAC 571 GUCAUGAGUAUGUACACAA 437-455 435
    162 NUGUGUACAUACUCAUGAC 572 GUCAUGAGUAUGUACACAN 437-455 435
    163 NUGUGUACAUACUCAUGAN 573 NUCAUGAGUAUGUACACAN 437-455 435
    164 UACCAGUUAUCAGCAUGUC 574 GACAUGCUGAUAACUGIUA 2573-2591 2571
    165 NACCAGUUAUCAGCAUGUC 575 GACAUGCUGAUAACUGIUN 2573-2591 2571
    166 NACCAGUUAUCAGCAUGUN 576 NACAUGCUGAUAACUGIUN 2573-2591 2571
    167 UACCAGUUAUCAGCAUGUC 577 GACAUGCUGAUAACUGGUA 2573-2591 2571
    168 NACCAGUUAUCAGCAUGUC 578 GACAUGCUGAUAACUGGUA 2573-2591 2571
    169 NACCAGUUAUCAGCAUGUN 579 GACAUGCUGAUAACUGGUA 2573-2591 2571
    170 UAUGAAGCCAACCUUGUAU 580 AUACAAGGUUGGCUUCAUA 2614-2632 2612
    171 NAUGAAGCCAACCUUGUAU 581 AUACAAGGUUGGCUUCAUN 2614-2632 2612
    172 NAUGAAGCCAACCUUGUAN 582 NUACAAGGUUGGCUUCAUN 2614-2632 2612
    173 UCUUCAUGAAGCCAACCUU 583 AAGGUUGGCUUCAUGAAGA 2618-2636 2616
    174 NCUUCAUGAAGCCAACCUU 584 AAGGUUGGCUUCAUGAAGN 2618-2636 2616
    175 NCUUCAUGAAGCCAACCUN 585 NAGGUUGGCUUCAUGAAGN 2618-2636 2616
    176 UUCUUCAUGAAGCCAACCU 586 AGGUUGGCUUCAUGAAGAA 2619-2637 2617
    177 NUCUUCAUGAAGCCAACCU 587 AGGUUGGCUUCAUGAAGAN 2619-2637 2617
    178 NUCUUCAUGAAGCCAACCN 588 NGGUUGGCUUCAUGAAGAN 2619-2637 2617
    179 UAGUCUUCAUGAAGCCAAC 589 GUUGGCUUCAUGAAGACUA 2621-2639 2619
    180 NAGUCUUCAUGAAGCCAAC 590 GUUGGCUUCAUGAAGACUN 2621-2639 2619
    181 NAGUCUUCAUGAAGCCAAN 591 NUUGGCUUCAUGAAGACUN 2621-2639 2619
    182 UCUUUUUCCAACAAUUCUC 592 GAGAAUUGUUGGAAAAAGA 3047-3065 3045
    183 NCUUUUUCCAACAAUUCUC 593 GAGAAUUGUUGGAAAAAGN 3047-3065 3045
    184 NCUUUUUCCAACAAUUCUN 594 NAGAAUUGUUGGAAAAAGN 3047-3065 3045
    185 UUCUACUUCAGAGCAAGCC 595 GGCUUGCUCUGAAGUAGAA 3550-3568 3548
    186 NUCUACUUCAGAGCAAGCC 596 GGCUUGCUCUGAAGUAGAN 3550-3568 3548
    187 NUCUACUUCAGAGCAAGCN 597 NGCUUGCUCUGAAGUAGAN 3550-3568 3548
    188 UAUUUCUACUUCAGAGCAA 598 UUGCUCUGAAGUAGAAAUA 3553-3571 3551
    189 NAUUUCUACUUCAGAGCAA 599 UUGCUCUGAAGUAGAAAUN 3553-3571 3551
    190 NAUUUCUACUUCAGAGCAN 600 NUGCUCUGAAGUAGAAAUN 3553-3571 3551
    191 UGUCCAAUAUCAAUGGCAG 601 CUGCCAUUGAUAUUIGACA 3642-3660 3640
    192 NGUCCAAUAUCAAUGGCAG 602 CUGCCAUUGAUAUUIGACN 3642-3660 3640
    193 NGUCCAAUAUCAAUGGCAN 603 NUGCCAUUGAUAUUIGACN 3642-3660 3640
    194 UCAGAAAAGUGGACGAUCU 604 AGAUCGUCCACUUUUCUGA 267-285 265
    195 NCAGAAAAGUGGACGAUCU 605 AGAUCGUCCACUUUUCUGN 267-285 265
    196 NCAGAAAAGUGGACGAUCN 606 NGAUCGUCCACUUUUCUGN 267-285 265
    197 ACAACAUUAUCUGCUUCGG 607 CCGAAGCAGAUAAUGUUGU 2250-2268 2248
    198 UCAACAUUAUCUGCUUCGG 608 CCGAAGCAGAUAAUGUUGU 2250-2268 2248
    199 NCAACAUUAUCUGCUUCGG 609 CCGAAGCAGAUAAUGUUGN 2250-2268 2248
    200 NCAACAUUAUCUGCUUCGN 610 NCGAAGCAGAUAAUGUUGN 2250-2268 2248
    201 UCAUAAUACUCUGAGAGAG 611 CUCUCUCAGAGUAUUAUGA 2696-2714 2694
    202 NCAUAAUACUCUGAGAGAG 612 CUCUCUCAGAGUAUUAUGN 2696-2714 2694
    203 NCAUAAUACUCUGAGAGAN 613 NUCUCUCAGAGUAUUAUGN 2696-2714 2694
    204 UCUUAUUCCAAACUUGGUG 614 CACCAAGUUUGGAAUAAGA 3085-3103 3083
    205 NCUUAUUCCAAACUUGGUG 615 CACCAAGUUUGGAAUAAGN 3085-3103 3083
    206 NCUUAUUCCAAACUUGGUN 616 NACCAAGUUUGGAAUAAGN 3085-3103 3083
    207 UAGUAAUCUUGCUUUAUGC 617 GCAUAAAGCAAGAUUACUA 4667-4685 4665
    208 NAGUAAUCUUGCUUUAUGC 618 GCAUAAAGCAAGAUUACUN 4667-4685 4665
    209 NAGUAAUCUUGCUUUAUGN 619 NCAUAAAGCAAGAUUACUN 4667-4685 4665
    210 AAAGAAAUCUAGAACAUUG 620 CAAUGUUCUAGAUUUCUUU 4727-4745 4725
    211 UAAGAAAUCUAGAACAUUG 621 CAAUGUUCUAGAUUUCUUA 4727-4745 4725
    212 NAAGAAAUCUAGAACAUUG 622 CAAUGUUCUAGAUUUCUUN 4727-4745 4725
    213 NAAGAAAUCUAGAACAUUN 623 NAAUGUUCUAGAUUUCUUN 4727-4745 4725
    214 UAACUUCACUCAUCCAGCA 624 UGCUGGAUGAGUGAAGUUA 2852-2870 2850
    215 NAACUUCACUCAUCCAGCA 625 UGCUGGAUGAGUGAAGUUN 2852-2870 2850
    216 NAACUUCACUCAUCCAGCN 626 NGCUGGAUGAGUGAAGUUN 2852-2870 2850
    217 UCAACUUCACUCAUCCAGC 627 GCUIGAUGAGUGAAGUUGA 2853-2871 2851
    218 NCAACUUCACUCAUCCAGC 628 GCUIGAUGAGUGAAGUUGN 2853-2871 2851
    219 NCAACUUCACUCAUCCAGN 629 NCUIGAUGAGUGAAGUUGN 2853-2871 2851
    220 UGCAACUUCACUCAUCCAG 630 CUGGAUGAGUGAAGUUICA 2854-2872 2852
    221 NGCAACUUCACUCAUCCAG 631 CUGGAUGAGUGAAGUUICN 2854-2872 2852
    222 NGCAACUUCACUCAUCCAN 632 NUGGAUGAGUGAAGUUICN 2854-2872 2852
    223 UGAUCAUACUUGGAGAGCA 633 UGCUCUCCAAGUAUGAUCA 237-255 235
    224 NGAUCAUACUUGGAGAGCA 634 UGCUCUCCAAGUAUGAUCN 237-255 235
    225 NGAUCAUACUUGGAGAGCN 635 NGCUCUCCAAGUAUGAUCN 237-255 235
    226 UCUUGUUCUGCAGACGAUC 636 GAUCGUCUGCAGAACAAGA 251-269 249
    227 NCUUGUUCUGCAGACGAUC 637 GAUCGUCUGCAGAACAAGN 251-269 249
    228 NCUUGUUCUGCAGACGAUC 638 GAUCGUCUGCAGAACAAGN 251-269 249
    229 UGAUCUUGUUCUGCAGACG 639 CGUCUGCAGAACAAGAUCA 254-272 252
    230 NGAUCUUGUUCUGCAGACG 640 CGUCUGCAGAACAAGAUCN 254-272 252
    231 NGAUCUUGUUCUGCAGACN 641 NGUCUGCAGAACAAGAUCN 254-272 252
    232 UAGUAAAGUUGCACUGGCG 642 CGCCAGUGCAACUUUACUA 1705-1723 1703
    233 NAGUAAAGUUGCACUGGCG 643 CGCCAGUGCAACUUUACUN 1705-1723 1703
    234 NAGUAAAGUUGCACUGGCN 644 NGCCAGUGCAACUUUACUN 1705-1723 1703
    235 UAACACAAGUAACCUUAUC 645 GAUAAGGUUACUUGUGUUA 2051-2069 2049
    236 NAACACAAGUAACCUUAUC 646 GAUAAGGUUACUUGUGUUN 2051-2069 2049
    237 NAACACAAGUAACCUUAUN 647 NAUAAGGUUACUUGUGUUN 2051-2069 2049
    238 UCAAUUGUGAUAAUGGCUG 648 CAGCCAUUAUCACAAUUGA 2157-2175 2155
    239 NCAAUUGUGAUAAUGGCUG 649 CAGCCAUUAUCACAAUUGN 2157-2175 2155
    240 NCAAUUGUGAUAAUGGCUN 650 NAGCCAUUAUCACAAUUGN 2157-2175 2155
    241 UAGCAUGAUACUGAGAGCU 651 AGCUCUCAGUAUCAUGCUA 2999-3017 2997
    242 NAGCAUGAUACUGAGAGCU 652 AGCUCUCAGUAUCAUGCUN 2999-3017 2997
    243 NAGCAUGAUACUGAGAGCN 653 NGCUCUCAGUAUCAUGCUN 2999-3017 2997
    244 AACUUGUCAACCUCACUCU 654 AGAGUGAGGUUGACAAGUU 3021-3039 3019
    245 UACUUGUCAACCUCACUCU 655 AGAGUGAGGUUGACAAGUA 3021-3039 3019
    246 NACUUGUCAACCUCACUCU 656 AGAGUGAGGUUGACAAGUN 3021-3039 3019
    247 NACUUGUCAACCUCACUCN 657 NGAGUGAGGUUGACAAGUN 3021-3039 3019
    248 UAACUUGUCAACCUCACUC 658 GAGUGAGGUUGACAAGUUA 3022-3040 3020
    249 NAACUUGUCAACCUCACUC 659 GAGUGAGGUUGACAAGUUN 3022-3040 3020
    250 NAACUUGUCAACCUCACUN 660 NAGUGAGGUUGACAAGUUN 3022-3040 3020
    251 UAACAAUUCUCCUUGUUGA 661 UCAACAAGGAGAAUUGUUA 3039-3057 3037
    252 NAACAAUUCUCCUUGUUGA 662 UCAACAAGGAGAAUUGUUN 3039-3057 3037
    253 NAACAAUUCUCCUUGUUGN 663 NCAACAAGGAGAAUUGUUN 3039-3057 3037
    254 UCAUGUUCUGUGGUAUGUU 664 AACAUACCACAGAACAUGA 4138-4156 4136
    255 NCAUGUUCUGUGGUAUGUU 665 AACAUACCACAGAACAUGN 4138-4156 4136
    256 NCAUGUUCUGUGGUAUGUN 666 NACAUACCACAGAACAUGN 4138-4156 4136
    257 UACUUUAAUAGAUCCAUGU 667 ACAUGGAUCUAUUAAAGUA 4151-4169 4149
    258 NACUUUAAUAGAUCCAUGU 668 ACAUGGAUCUAUUAAAGUN 4151-4169 4149
    259 NACUUUAAUAGAUCCAUGN 669 NCAUGGAUCUAUUAAAGUN 4151-4169 4149
    260 UGACUUUAAUAGAUCCAUG 670 CAUGGAUCUAUUAAAGUCA 4152-4170 4150
    261 NGACUUUAAUAGAUCCAUG 671 CAUGGAUCUAUUAAAGUCN 4152-4170 4150
    262 NGACUUUAAUAGAUCCAUN 672 NAUGGAUCUAUUAAAGUCN 4152-4170 4150
    263 UGCAUAUUCACCAUUUAGG 673 CCUAAAUGGUGAAUAUGCA 4291-4309 4289
    264 NGCAUAUUCACCAUUUAGG 674 CCUAAAUGGUGAAUAUGCN 4291-4309 4289
    265 NGCAUAUUCACCAUUUAGN 675 NCUAAAUGGUGAAUAUGCN 4291-4309 4289
    266 UGUUUAAGCUUCUAGAGGU 676 ACCUCUAGAAGCUUAAACA 4448-4466 4446
    267 NGUUUAAGCUUCUAGAGGU 677 ACCUCUAGAAGCUUAAACN 4448-4466 4446
    268 NGUUUAAGCUUCUAGAGGN 678 NCCUCUAGAAGCUUAAACN 4448-4466 4446
    269 UUGUUCAUUGGUUUGAAGG 679 CCUUCAAACCAAUGAACAA 4507-4525 4505
    270 NUGUUCAUUGGUUUGAAGG 680 CCUUCAAACCAAUGAACAN 4507-4525 4505
    271 NUGUUCAUUGGUUUGAAGN 681 NCUUCAAACCAAUGAACAN 4507-4525 4505
    272 UUAUGCUUUGCUGUUCAUU 682 AAUGAACAGCAAAGCAUAA 4517-4535 4515
    273 NUAUGCUUUGCUGUUCAUU 683 AAUGAACAGCAAAGCAUAN 4517-4535 4515
    274 NUAUGCUUUGCUGUUCAUN 684 NAUGAACAGCAAAGCAUAN 4517-4535 4515
    275 UGUUAUGCUUUGCUGUUCA 685 UGAACAGCAAAGCAUAACA 4519-4537 4517
    276 NGUUAUGCUUUGCUGUUCA 686 UGAACAGCAAAGCAUAACN 4519-4537 4517
    277 NGUUAUGCUUUGCUGUUCN 687 NGAACAGCAAAGCAUAACN 4519-4537 4517
    278 AGGUUAUGCUUUGCUGUUC 688 GAACAGCAAAGCAUAACCU 4520-4538 4518
    279 UGGUUAUGCUUUGCUGUUC 689 GAACAGCAAAGCAUAACCA 4520-4538 4518
    280 NGGUUAUGCUUUGCUGUUC 690 GAACAGCAAAGCAUAACCN 4520-4538 4518
    281 NGGUUAUGCUUUGCUGUUN 691 NAACAGCAAAGCAUAACCN 4520-4538 4518
    282 UAAGGUUAUGCUUUGCUGU 692 ACAGCAAAGCAUAACCUUA 4522-4540 4520
    283 NAAGGUUAUGCUUUGCUGU 693 ACAGCAAAGCAUAACCUUN 4522-4540 4520
    284 NAAGGUUAUGCUUUGCUGN 694 NCAGCAAAGCAUAACCUUN 4522-4540 4520
    285 AGAUUCAAGGUUAUGCUUU 695 AAAGCAUAACCUUGAAUCU 4527-4545 4525
    286 UGAUUCAAGGUUAUGCUUU 696 AAAGCAUAACCUUGAAUCA 4527-4545 4525
    287 NGAUUCAAGGUUAUGCUUU 697 AAAGCAUAACCUUGAAUCN 4527-4545 4525
    288 NGAUUCAAGGUUAUGCUUN 698 NAAGCAUAACCUUGAAUCN 4527-4545 4525
    289 UUCAAUAAUUGAGUUGGUU 699 AACCAACUCAAUUAUUGAA 4702-4720 4700
    290 NUCAAUAAUUGAGUUGGUU 700 AACCAACUCAAUUAUUGAN 4702-4720 4700
    291 NUCAAUAAUUGAGUUGGUN 701 NACCAACUCAAUUAUUGAN 4702-4720 4700
    292 AGUAAAAUGGAUCACAGGA 702 UCCUGUGAUCCAUUUUACU 5288-5306 5286
    293 UGUAAAAUGGAUCACAGGA 703 UCCUGUGAUCCAUUUUACA 5288-5306 5286
    294 NGUAAAAUGGAUCACAGGA 704 UCCUGUGAUCCAUUUUACN 5288-5306 5286
    295 NGUAAAAUGGAUCACAGGN 705 NCCUGUGAUCCAUUUUACN 5288-5306 5286
    296 UCAUAUGACAGUAAGAAAA 706 UUUUCUUACUGUCAUAUGA 5422-5440 5420
    297 NCAUAUGACAGUAAGAAAA 707 UUUUCUUACUGUCAUAUGN 5422-5440 5420
    298 NCAUAUGACAGUAAGAAAN 708 NUUUCUUACUGUCAUAUGN 5422-5440 5420
    299 UGGAUCUGCAUUUUUCUCC 709 GGAGAAAAAUGCAIAUCCA 124-142 122
    300 NGGAUCUGCAUUUUUCUCC 710 GGAGAAAAAUGCAIAUCCN 124-142 122
    301 NGGAUCUGCAUUUUUCUCN 711 NGAGAAAAAUGCAIAUCCN 124-142 122
    302 UCCAAAAGGGUUGUCUCUG 712 CAGAGACAACUCUUUUGGA 141-159 139
    303 NCCAAAAGGGUUGUCUCUG 713 CAGAGACAACUCUUUUGGN 141-159 139
    304 NCCAAAAGGGUUGUCUCUN 714 NAGAGACAACUCUUUUGGN 141-159 139
    305 UAGACGAUCAUACUUGGAG 715 CUCCAAGUAUGAUCIUCUA 241-259 239
    306 NAGACGAUCAUACUUGGAG 716 CUCCAAGUAUGAUCIUCUN 241-259 239
    307 NAGACGAUCAUACUUGGAN 717 NUCCAAGUAUGAUCIUCUN 241-259 239
    308 UCCUAUUCCUUCCACAGUU 718 AACUGUGGAAGGAAUAGGA 334-352 332
    309 NCCUAUUCCUUCCACAGUU 719 AACUGUGGAAGGAAUAGGN 334-352 332
    310 NCCUAUUCCUUCCACAGUN 720 NACUGUGGAAGGAAUAGGN 334-352 332
    311 UACAUACUCAUGACGAUGC 721 GCAUCGUCAUGAGUAUGUA 432-450 430
    312 NACAUACUCAUGACGAUGC 722 GCAUCGUCAUGAGUAUGUN 432-450 430
    313 NACAUACUCAUGACGAUGN 723 NCAUCGUCAUGAGUAUGUN 432-450 430
    314 UCACAGAUUUCCUUGGAAG 724 CUUCCAAGGAAAUCUGUIA 502-520 500
    315 NCACAGAUUUCCUUGGAAG 725 CUUCCAAGGAAAUCUGUIN 502-520 500
    316 NCACAGAUUUCCUUGGAAN 726 NUUCCAAGGAAAUCUGUIN 502-520 500
    317 UGAACUUCAUCUCAAUGCC 727 GGCAUUGAGAUGAAGUUCA 869-887 867
    318 NGAACUUCAUCUCAAUGCC 728 GGCAUUGAGAUGAAGUUCN 869-887 867
    319 NGAACUUCAUCUCAAUGCN 729 NGCAUUGAGAUGAAGUUCN 869-887 867
    320 AGCAUAUUCUUGAACUUCA 730 UGAAGUUCAAGAAUAUGCU 879-897 877
    321 UGCAUAUUCUUGAACUUCA 731 UGAAGUUCAAGAAUAUGCA 879-897 877
    322 NGCAUAUUCUUGAACUUCA 732 UGAAGUUCAAGAAUAUGCN 879-897 877
    323 NGCAUAUUCUUGAACUUCN 733 NGAAGUUCAAGAAUAUGCN 879-897 877
    324 UCAUAGGAAACAGCAUAUU 734 AAUAUGCUGUUUCCUAUGA 890-908 888
    325 NCAUAGGAAACAGCAUAUU 735 AAUAUGCUGUUUCCUAUGN 890-908 888
    326 NCAUAGGAAACAGCAUAUN 736 NAUAUGCUGUUUCCUAUGN 890-908 888
    327 UGGAUCUCUAUGGAGAGCA 737 UGCUCUCCAUAGAIAUCCA 1287-1305 1285
    328 NGGAUCUCUAUGGAGAGCA 738 UGCUCUCCAUAGAIAUCCN 1287-1305 1285
    329 NGGAUCUCUAUGGAGAGCN 739 NGCUCUCCAUAGAIAUCCN 1287-1305 1285
    330 UUUGAAUGCUGAGAAAUAC 740 GUAUUUCUCAGCAUUCAAA 1324-1342 1322
    331 NUUGAAUGCUGAGAAAUAC 741 GUAUUUCUCAGCAUUCAAN 1324-1342 1322
    332 NUUGAAUGCUGAGAAAUAN 742 NUAUUUCUCAGCAUUCAAN 1324-1342 1322
    333 UCUAUGGACUUGAUCUUGG 743 CCAAGAUCAAGUCCAUAGA 1923-1941 1921
    334 NCUAUGGACUUGAUCUUGG 744 CCAAGAUCAAGUCCAUAGN 1923-1941 1921
    335 NCUAUGGACUUGAUCUUGN 745 NCAAGAUCAAGUCCAUAGN 1923-1941 1921
    336 AUGAAACAAACAAACCCUG 746 CAGGGUUUGUUUGUUUCAU 1965-1983 1963
    337 UUGAAACAAACAAACCCUG 747 CAGGGUUUGUUUGUUUCAA 1965-1983 1963
    338 NUGAAACAAACAAACCCUG 748 CAGGGUUUGUUUGUUUCAN 1965-1983 1963
    339 NUGAAACAAACAAACCCUN 749 NAGGGUUUGUUUGUUUCAN 1965-1983 1963
    340 UGGUAGUUCUUCAUAGGUG 750 CACCUAUGAAGAACUACCA 2140-2158 2138
    341 NGGUAGUUCUUCAUAGGUG 751 CACCUAUGAAGAACUACCN 2140-2158 2138
    342 NGGUAGUUCUUCAUAGGUN 752 NACCUAUGAAGAACUACCN 2140-2158 2138
    343 UGAUAAUGGCUGGUAGUUC 753 GAACUACCAGCCAUUAUCA 2150-2168 2148
    344 NGAUAAUGGCUGGUAGUUC 754 GAACUACCAGCCAUUAUCN 2150-2168 2148
    345 NGAUAAUGGCUGGUAGUUN 755 NAACUACCAGCCAUUAUCN 2150-2168 2148
    346 UCUCAAUUGUGAUAAUGGC 756 GCCAUUAUCACAAUUGAGA 2159-2177 2157
    347 NCUCAAUUGUGAUAAUGGC 757 GCCAUUAUCACAAUUGAGN 2159-2177 2157
    348 NCUCAAUUGUGAUAAUGGN 758 NCCAUUAUCACAAUUGAGN 2159-2177 2157
    349 UCUUUCUCGAUCUUCAGCU 759 AGCUGAAGAUCGAGAAAGA 2211-2229 2209
    350 NCUUUCUCGAUCUUCAGCU 760 AGCUGAAGAUCGAGAAAGN 2211-2229 2209
    351 NCUUUCUCGAUCUUCAGCN 761 NGCUGAAGAUCGAGAAAGN 2211-2229 2209
    352 UUUGGAACAGCAAUGGUGC 762 GCACCAUUGCUGUUCCAAA 2322-2340 2320
    353 NUUGGAACAGCAAUGGUGC 763 GCACCAUUGCUGUUCCAAN 2322-2340 2320
    354 NUUGGAACAGCAAUGGUGN 764 NCACCAUUGCUGUUCCAAN 2322-2340 2320
    355 UGUAGACACAAAGAGCUCC 765 GGAGCUCUUUGUGUUUACA 2359-2377 2357
    356 NGUAGACACAAAGAGCUCC 766 GGAGCUCUUUGUGUUUACN 2359-2377 2357
    357 NGUAGACACAAAGAGCUCN 767 NGAGCUCUUUGUGUUUACN 2359-2377 2357
    358 UCUGUGUAGACACAAAGAG 768 CUCUUUGUGUCUACACAIA 2363-2381 2361
    359 NCUGUGUAGACACAAAGAG 769 CUCUUUGUGUCUACACAIN 2363-2381 2361
    360 NCUGUGUAGACACAAAGAN 770 NUCUUUGUGUCUACACAIN 2363-2381 2361
    361 UUCCAUAAUACUCUGAGAG 771 CUCUCAGAGUAUUAUGGAA 2698-2716 2696
    362 NUCCAUAAUACUCUGAGAG 772 CUCUCAGAGUAUUAUGGAN 2698-2716 2696
    363 NUCCAUAAUACUCUGAGAN 773 NUCUCAGAGUAUUAUGGAN 2698-2716 2696
    364 UCUCGUUCCAUAAUACUCU 774 AGAGUAUUAUGGAACGAIA 2703-2721 2701
    365 NCUCGUUCCAUAAUACUCU 775 AGAGUAUUAUGGAACGAIN 2703-2721 2701
    366 NCUCGUUCCAUAAUACUCN 776 NGAGUAUUAUGGAACGAIN 2703-2721 2701
    367 AAUGAAACAAACAAACCCU 777 AGGGUUUGUUUGUUUCAUU 1966-1984 1964
    368 UAUGAAACAAACAAACCCU 778 AGGGUUUGUUUGUUUCAUA 1966-1984 1964
    369 NAUGAAACAAACAAACCCU 779 AGGGUUUGUUUGUUUCAUN 1966-1984 1964
    370 NAUGAAACAAACAAACCCN 780 NGGGUUUGUUUGUUUCAUN 1966-1984 1964
    371 AAAUGAAACAAACAAACCC 781 GGGUUUGUUUGUUUCAUUU 1967-1985 1965
    372 UAAUGAAACAAACAAACCC 782 GGGUUUGUUUGUUUCAUUA 1967-1985 1965
    373 NAAUGAAACAAACAAACCC 783 GGGUUUGUUUGUUUCAUUN 1967-1985 1965
    374 NAAUGAAACAAACAAACCN 784 NGGUUUGUUUGUUUCAUUN 1967-1985 1965
    375 UGAAAUGAAACAAACAAAC 785 GUUUGUUUGUUUCAUUUCA 1969-1987 1967
    376 NGAAAUGAAACAAACAAAC 786 GUUUGUUUGUUUCAUUUCN 1969-1987 1967
    377 NGAAAUGAAACAAACAAAN 787 NUUUGUUUGUUUCAUUUCN 1969-1987 1967
    378 AGACGAUCAUACUUGGAGA 788 UCUCCAAGUAUGAUCIUCU 240-258 238
    379 UGACGAUCAUACUUGGAGA 789 UCUCCAAGUAUGAUCIUCA 240-258 238
    380 NGACGAUCAUACUUGGAGA 790 UCUCCAAGUAUGAUCIUCN 240-258 238
    381 NGACGAUCAUACUUGGAGN 791 NCUCCAAGUAUGAUCIUCN 240-258 238
    382 AAGGCAUUCUCAAUCUCCU 792 AGGAGAUUGAGAAUICCUU 486-504 484
    383 UAGGCAUUCUCAAUCUCCU 793 AGGAGAUUGAGAAUICCUA 486-504 484
    384 NAGGCAUUCUCAAUCUCCU 794 AGGAGAUUGAGAAUICCUN 486-504 484
    385 NAGGCAUUCUCAAUCUCCN 795 NGGAGAUUGAGAAUICCUN 486-504 484
    386 UUUCCUUGGAAGGCAUUCU 796 AGAAUGCCUUCCAAGGAAA 495-513 493
    387 NUUCCUUGGAAGGCAUUCU 797 AGAAUGCCUUCCAAGGAAN 495-513 493
    388 NUUCCUUGGAAGGCAUUCN 798 NGAAUGCCUUCCAAGGAAN 495-513 493
    389 UAGAUUUCCUUGGAAGGCA 799 UGCCUUCCAAGGAAAUCUA 499-517 497
    390 NAGAUUUCCUUGGAAGGCA 800 UGCCUUCCAAGGAAAUCUN 499-517 497
    391 NAGAUUUCCUUGGAAGGCN 801 NGCCUUCCAAGGAAAUCUN 499-517 497
    392 AUAGGAAACAGCAUAUUCU 802 AGAAUAUGCUGUUUCCUAU 888-906 886
    393 UUAGGAAACAGCAUAUUCU 803 AGAAUAUGCUGUUUCCUAA 888-906 886
    394 NUAGGAAACAGCAUAUUCU 804 AGAAUAUGCUGUUUCCUAN 888-906 886
    395 NUAGGAAACAGCAUAUUCN 805 NGAAUAUGCUGUUUCCUAN 888-906 886
    396 UUGAUGAUGUUCCCUCCAA 806 UUGGAGGGAACAUCAUCAA 1119-1137 1117
    397 NUGAUGAUGUUCCCUCCAA 807 UUGGAGGGAACAUCAUCAN 1119-1137 1117
    398 NUGAUGAUGUUCCCUCCAN 808 NUGGAGGGAACAUCAUCAN 1119-1137 1117
    399 UAGAACUUGAAGAAGAAGC 809 GCUUCUUCUUCAAGUUCUA 1617-1635 1615
    400 NAGAACUUGAAGAAGAAGC 810 GCUUCUUCUUCAAGUUCUN 1617-1635 1615
    401 NAGAACUUGAAGAAGAAGN 811 NCUUCUUCUUCAAGUUCUN 1617-1635 1615
    402 UACCAAUGAUAUGCCCAAC 812 GUUGGGCAUAUCAUUGGUA 2066-2084 2064
    403 NACCAAUGAUAUGCCCAAC 813 GUUGGGCAUAUCAUUGGUN 2066-2084 2064
    404 NACCAAUGAUAUGCCCAAN 814 NUUGGGCAUAUCAUUGGUN 2066-2084 2064
    405 UCAUGGUGUUCUGUGUAGA 815 UCUACACAGAACACCAUGA 2372-2390 2370
    406 NCAUGGUGUUCUGUGUAGA 816 UCUACACAGAACACCAUGN 2372-2390 2370
    407 NCAUGGUGUUCUGUGUAGN 817 NCUACACAGAACACCAUGN 2372-2390 2370
    408 UUGAGAGAGAUCCUGGGUG 818 CACCCAGGAUCUCUUUCAA 2686-2704 2684
    409 NUGAGAGAGAUCCUGGGUG 819 CACCCAGGAUCUCUUUCAN 2686-2704 2684
    410 NUGAGAGAGAUCCUGGGUN 820 NACCCAGGAUCUCUUUCAN 2686-2704 2684
    411 UCAUGAUACUGAGAGCUUG 821 CAAGCUCUCAGUAUCAUGA 2997-3015 2995
    412 NCAUGAUACUGAGAGCUUG 822 CAAGCUCUCAGUAUCAUGN 2997-3015 2995
    413 NCAUGAUACUGAGAGCUUN 823 NAAGCUCUCAGUAUCAUGN 2997-3015 2995
    414 UUGUCAACCUCACUCUUCC 824 GGAAGAGUGAGGUUGACAA 3018-3036 3016
    415 NUGUCAACCUCACUCUUCC 825 GGAAGAGUGAGGUUGACAN 3018-3036 3016
    416 NUGUCAACCUCACUCUUCN 826 NGAAGAGUGAGGUUGACAN 3018-3036 3016
    417 UUUCCAACAAUUCUCCUUG 827 CAAGGAGAAUUGUUGGAAA 3043-3061 3041
    418 NUUCCAACAAUUCUCCUUG 828 CAAGGAGAAUUGUUGGAAN 3043-3061 3041
    419 NUUCCAACAAUUCUCCUUN 829 NAAGGAGAAUUGUUGGAAN 3043-3061 3041
    420 UUGAGUUAGUCUCAAAGCU 830 AGCUUUGAGACUAACUCAA 3500-3518 3498
    421 NUGAGUUAGUCUCAAAGCU 831 AGCUUUGAGACUAACUCAN 3500-3518 3498
    422 NUGAGUUAGUCUCAAAGCN 832 NGCUUUGAGACUAACUCAN 3500-3518 3498
    423 AUGACAAUAUCUGUGCGGA 833 UCCGCACAGAUAUUGUCAU 3600-3618 3598
    424 UUGACAAUAUCUGUGCGGA 834 UCCGCACAGAUAUUGUCAA 3600-3618 3598
    425 NUGACAAUAUCUGUGCGGA 835 UCCGCACAGAUAUUGUCAN 3600-3618 3598
    426 NUGACAAUAUCUGUGCGGN 836 NCCGCACAGAUAUUGUCAN 3600-3618 3598
    427 UCAUGACAAUAUCUGUGCG 837 CGCACAGAUAUUGUCAUGA 3602-3620 3600
    428 NCAUGACAAUAUCUGUGCG 838 CGCACAGAUAUUGUCAUGN 3602-3620 3600
    429 NCAUGACAAUAUCUGUGCN 839 NGCACAGAUAUUGUCAUGN 3602-3620 3600
    430 UCAAAGAAGAUAGAAGCAG 840 CUGCUUCUAUCUUCUUUGA 3879-3897 3877
    431 NCAAAGAAGAUAGAAGCAG 841 CUGCUUCUAUCUUCUUUGN 3879-3897 3877
    432 NCAAAGAAGAUAGAAGCAN 842 NUGCUUCUAUCUUCUUUGN 3879-3897 3877
    433 UCACGUUAUUACCUGUGUG 843 CACACAGGUAAUAACGUIA 3932-3950 3930
    434 NCACGUUAUUACCUGUGUG 844 CACACAGGUAAUAACGUIN 3932-3950 3930
    435 NCACGUUAUUACCUGUGUN 845 NACACAGGUAAUAACGUIN 3932-3950 3930
    436 UAGAACUUGAGGUUAUACA 846 UGUAUAACCUCAAGUUCUA 4396-4414 4394
    437 NAGAACUUGAGGUUAUACA 847 UGUAUAACCUCAAGUUCUN 4396-4414 4394
    438 NAGAACUUGAGGUUAUACN 848 NGUAUAACCUCAAGUUCUN 4396-4414 4394
    439 AUGCUUUGCUGUUCAUUGG 849 CCAAUGAACAGCAAAGCAU 4515-4533 4513
    440 UUGCUUUGCUGUUCAUUGG 850 CCAAUGAACAGCAAAGCAA 4515-4533 4513
    441 NUGCUUUGCUGUUCAUUGG 851 CCAAUGAACAGCAAAGCAN 4515-4533 4513
    442 NUGCUUUGCUGUUCAUUGN 852 NCAAUGAACAGCAAAGCAN 4515-4533 4513
    443 UAGUAUAGAUUCAAGGUUA 853 UAACCUUGAAUCUAUACUA 4533-4551 4531
    444 NAGUAUAGAUUCAAGGUUA 854 UAACCUUGAAUCUAUACUN 4533-4551 4531
    445 NAGUAUAGAUUCAAGGUUN 855 NAACCUUGAAUCUAUACUN 4533-4551 4531
    446 AGAGUAAUCUUGCUUUAUG 856 CAUAAAGCAAGAUUACUCU 4668-4686 4666
    447 UGAGUAAUCUUGCUUUAUG 857 CAUAAAGCAAGAUUACUCA 4668-4686 4666
    448 NGAGUAAUCUUGCUUUAUG 858 CAUAAAGCAAGAUUACUCN 4668-4686 4666
    449 NGAGUAAUCUUGCUUUAUN 859 NAUAAAGCAAGAUUACUCN 4668-4686 4666
    450 AUAGCAUCAUUUCUAGGUG 860 CACCUAGAAAUGAUGCUAU 4845-4863 4843
    451 UUAGCAUCAUUUCUAGGUG 861 CACCUAGAAAUGAUGCUAA 4845-4863 4843
    452 NUAGCAUCAUUUCUAGGUG 862 CACCUAGAAAUGAUGCUAN 4845-4863 4843
    453 NUAGCAUCAUUUCUAGGUN 863 NACCUAGAAAUGAUGCUAN 4845-4863 4843
    454 AGACAGAAGAGACAGAGCU 864 AGCUCUGUCUCUUCUIUCU 5236-5254 5234
    455 UGACAGAAGAGACAGAGCU 865 AGCUCUGUCUCUUCUIUCA 5236-5254 5234
    456 NGACAGAAGAGACAGAGCU 866 AGCUCUGUCUCUUCUIUCN 5236-5254 5234
    457 NGACAGAAGAGACAGAGCN 867 NGCUCUGUCUCUUCUIUCN 5236-5254 5234
    458 AGUAAGAAAACCAAGCCUU 868 (A2N)AGGCUUGGUUUUCUUACU 5413-5431 5411
    459 UGUAAGAAAACCAAGCCUU 869 (A2N)AGGCUUGGUUUUCUUACA 5413-5431 5411
    460 NGUAAGAAAACCAAGCCUU 870 (A2N)AGGCUUGGUUUUCUUACN 5413-5431 5411
    461 NGUAAGAAAACCAAGCCUN 871 NAGGCUUGGUUUUCUUACN 5413-5431 5411
    462 AGUAAGAAAACCAAGCCUU 872 AAGGCUUGGUUUUCUUACU 5413-5431 5411
    463 UGUAAGAAAACCAAGCCUU 873 AAGGCUUGGUUUUCUUACA 5413-5431 5411
    464 NGUAAGAAAACCAAGCCUU 874 AAGGCUUGGUUUUCUUACN 5413-5431 5411
    465 NGUAAGAAAACCAAGCCUN 875 NAGGCUUGGUUUUCUUACN 5413-5431 5411
    466 UAUACUUGGAGAGCAUCAC 876 GUGAUGCUCUCCAAGUAUA 233-251 231
    467 NAUACUUGGAGAGCAUCAC 877 GUGAUGCUCUCCAAGUAUN 233-251 231
    468 NAUACUUGGAGAGCAUCAN 878 NUGAUGCUCUCCAAGUAUN 233-251 231
    469 UUGCAGACGAUCAUACUUG 879 CAAGUAUGAUCGUCUICAA 244-262 242
    470 NUGCAGACGAUCAUACUUG 880 CAAGUAUGAUCGUCUICAN 244-262 242
    471 NUGCAGACGAUCAUACUUN 881 NAAGUAUGAUCGUCUICAN 244-262 242
    472 UUGAAUAAAACUCUCAUGC 882 GCAUGAGAGUUUUAUUCAA 1386-1404 1384
    473 NUGAAUAAAACUCUCAUGC 883 GCAUGAGAGUUUUAUUCAN 1386-1404 1384
    474 NUGAAUAAAACUCUCAUGN 884 NCAUGAGAGUUUUAUUCAN 1386-1404 1384
    475 UUGAAUAAAACUCUCAUGC 885 GCAUGAGAGUUUU(A2N)UUCAA 1386-1404 1384
    476 NUGAAUAAAACUCUCAUGC 886 GCAUGAGAGUUUU(A2N)UUCAN 1386-1404 1384
    477 NUGAAUAAAACUCUCAUGN 887 NCAUGAGAGUUUU(A2N)UUCAN 1386-1404 1384
    478 AGAAAAGUGGACGAUCUUG 888 CAAGAUCGUCCACUUUUCU 265-283 263
    479 UGAAAAGUGGACGAUCUUG 889 CAAGAUCGUCCACUUUUCU 265-283 263
    480 NGAAAAGUGGACGAUCUUG 890 CAAGAUCGUCCACUUUUCN 265-283 263
    481 NGAAAAGUGGACGAUCUUN 891 NAAGAUCGUCCACUUUUCN 265-283 263
    482 UAGUUGUCACUGCAACAUG 892 CAUGUUGCAGUGACAACUA 320-338 318
    483 NAGUUGUCACUGCAACAUG 893 CAUGUUGCAGUGACAACUN 320-338 318
    484 NAGUUGUCACUGCAACAUN 894 NAUGUUGCAGUGACAACUN 320-338 318
    485 AUUCCUUCCACAGUUGUCA 895 UGACAACUGUGGAAGGAAU 330-348 328
    486 UUUCCUUCCACAGUUGUCA 896 UGACAACUGUGGAAGGAAA 330-348 328
    487 NUUCCUUCCACAGUUGUCA 897 UGACAACUGUGGAAGGAAN 330-348 328
    488 NUUCCUUCCACAGUUGUCN 898 NGACAACUGUGGAAGGAAN 330-348 328
    489 UGCAUUCUCAAUCUCCUCC 899 GGAGGAGAUUGAGAAUGCA 484-502 482
    490 NGCAUUCUCAAUCUCCUCC 900 GGAGGAGAUUGAGAAUGCN 484-502 482
    491 NGCAUUCUCAAUCUCCUCN 901 NGAGGAGAUUGAGAAUGCN 484-502 482
    492 UUCAAUGCCAAUCUCCGUG 902 CACGGAGAUUGGCAUUGAA 859-877 857
    493 NUCAAUGCCAAUCUCCGUG 903 CACGGAGAUUGGCAUUGAN 859-877 857
    494 NUCAAUGCCAAUCUCCGUN 904 NACGGAGAUUGGCAUUGAN 859-877 857
    495 AUAUUCUUGAACUUCAUCU 905 AGAUGAAGUUCAAGAAU(A2N)U 876-894 874
    496 UUAUUCUUGAACUUCAUCU 906 AGAUGAAGUUCAAGAAU(A2N)A 876-894 874
    497 NUAUUCUUGAACUUCAUCU 907 AGAUGAAGUUCAAGAAU(A2N)N 876-894 874
    498 NUAUUCUUGAACUUCAUCN 908 NGAUGAAGUUCAAGAAU(A2N)N 876-894 874
    499 AUAUUCUUGAACUUCAUCU 909 AGAUGAAGUUCAAGAAUAU 876-894 874
    500 UUAUUCUUGAACUUCAUCU 910 AGAUGAAGUUCAAGAAUAA 876-894 874
    501 NUAUUCUUGAACUUCAUCU 911 AGAUGAAGUUCAAGAAUAN 876-894 874
    502 NUAUUCUUGAACUUCAUCN 912 NGAUGAAGUUCAAGAAUAN 876-894 874
    503 UUAUGGAGAGCAGUAUCUC 913 GAGAUACUGCUCUCCAUAA 1280-1298 1278
    504 NUAUGGAGAGCAGUAUCUC 914 GAGAUACUGCUCUCCAUAN 1280-1298 1278
    505 NUAUGGAGAGCAGUAUCUN 915 NAGAUACUGCUCUCCAUAN 1280-1298 1278
    506 UAAUGCUGAGAAAUACUCC 916 GGAGUAUUUCUCAGCAUUA 1321-1339 1319
    507 NAAUGCUGAGAAAUACUCC 917 GGAGUAUUUCUCAGCAUUN 1321-1339 1319
    508 NAAUGCUGAGAAAUACUCN 918 NGAGUAUUUCUCAGCAUUN 1321-1339 1319
    509 UGAAUGCUGAGAAAUACUC 919 GAGUAUUUCUCAGCAUUCA 1322-1340 1320
    510 NGAAUGCUGAGAAAUACUC 920 GAGUAUUUCUCAGCAUUCN 1322-1340 1320
    511 NGAAUGCUGAGAAAUACUN 921 NAGUAUUUCUCAGCAUUCN 1322-1340 1320
    512 UCAAUGUCAUCUUCUCUCC 922 GGAGAGAAGAUGACAUUGA 1353-1371 1351
    513 NCAAUGUCAUCUUCUCUCC 923 GGAGAGAAGAUGACAUUGN 1353-1371 1351
    514 NCAAUGUCAUCUUCUCUCN 924 NGAGAGAAGAUGACAUUGN 1353-1371 1351
    515 ACAAAUUCCAGUUAUGUUA 925 UAACAUAACUGGAAUUUGU 2008-2026 2006
    516 UCAAAUUCCAGUUAUGUUA 926 UAACAUAACUGGAAUUUGA 2008-2026 2006
    517 NCAAAUUCCAGUUAUGUUA 927 UAACAUAACUGGAAUUUGN 2008-2026 2006
    518 NCAAAUUCCAGUUAUGUUN 928 NAACAUAACUGGAAUUUGN 2008-2026 2006
    519 UUCAAUUGUGAUAAUGGCU 929 AGCCAUUAUCACAAUUGAA 2158-2176 2156
    520 NUCAAUUGUGAUAAUGGCU 930 AGCCAUUAUCACAAUUGAN 2158-2176 2156
    521 NUCAAUUGUGAUAAUGGCN 931 NGCCAUUAUCACAAUUGAN 2158-2176 2156
    522 AACAUUUUUGCAACAAAGC 932 GCUUUGUUGCAAAAAUGUU 2400-2418 2398
    523 UACAUUUUUGCAACAAAGC 933 GCUUUGUUGCAAAAAUGUA 2400-2418 2398
    524 NACAUUUUUGCAACAAAGC 934 GCUUUGUUGCAAAAAUGUN 2400-2418 2398
    525 NACAUUUUUGCAACAAAGN 935 NCUUUGUUGCAAAAAUGUN 2400-2418 2398
    526 UCAACAUUUUUGCAACAAA 936 UUUGUUGCAAAAAUGUUGA 2402-2420 2400
    527 NCAACAUUUUUGCAACAAA 937 UUUGUUGCAAAAAUGUUGN 2402-2420 2400
    528 NCAACAUUUUUGCAACAAN 938 NUUGUUGCAAAAAUGUUGN 2402-2420 2400
    529 UUUCACUCGAACCACAAUC 939 GAUUGUGGUUCGAGUGAAA 2437-2455 2435
    530 NUUCACUCGAACCACAAUC 940 GAUUGUGGUUCGAGUGAAN 2437-2455 2435
    531 NUUCACUCGAACCACAAUN 941 NAUUGUGGUUCGAGUGAAN 2437-2455 2435
    532 UUGGAAGGCAUUCUCAAUC 942 GAUUGAGAAUGCCUUCCAA 490-508 488
    533 NUGGAAGGCAUUCUCAAUC 943 GAUUGAGAAUGCCUUCCAN 490-508 488
    534 NUGGAAGGCAUUCUCAAUN 944 NAUUGAGAAUGCCUUCCAN 490-508 488
  • The XDH RNAi agent sense strands and antisense strands that comprise or consist of the sequences in Table 2 can be modified nucleotides or unmodified nucleotides. In some aspects, the XDH RNAi agents having the sense and antisense strand sequences that comprise or consist of the sequences in Table 2 are all or substantially all modified nucleotides.
  • In some aspects, the antisense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2. In some aspects, the sense strand of an XDH RINAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2.
  • As used herein, each N listed in a sequence disclosed in Table 2 may be independently selected from any and all nucleobases (including those found on both modified and unmodified nucleotides). In some aspects, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand. In some aspects, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand. In some aspects, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is the same as the N nucleotide at the corresponding position on the other strand. In some aspects, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is different from the N nucleotide at the corresponding position on the other strand.
  • Certain modified XDH RNAi agent antisense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 3. Certain modified XDH RNAi agent sense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 4. In forming XDH RNAi agents, each of the nucleotides in each of the underlying base sequences listed in Tables 3 and 4, as well as in Table 2, above, can be a modified nucleotide.
  • The XDH RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2 or Table 4, can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.
  • In some aspects, an XDH RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3.
  • In some aspects, an XDH RNAi agent comprises or consists of a duplex having the nucleobase sequences of the sense strand and the antisense strand of any of the sequences in Table 2, Table 3, or Table 4.
  • Examples of antisense strands containing modified nucleotides are provided in Table 3 and Table 5C. Examples of sense strands containing modified nucleotides are provided in Table 4 and Table 5C.
  • As used in Tables 3, 4, and 5C the following notations are used to indicate modified nucleotides and linking groups:
      • A=adenosine-3′-phosphate;
      • C=cytidine-3′-phosphate;
      • G=guanosine-3′-phosphate;
      • U=uridine-3′-phosphate
      • I=inosine-3′-phosphate
      • a=2′-O-methyladenosine-3′-phosphate
      • as=2′-O-methyladenosine-3′-phosphorothioate
      • c=2′-O-methylcytidine-3′-phosphate
      • cs=2′-O-methylcytidine-3′-phosphorothioate
      • g=2′-O-methylguanosine-3′-phosphate
      • gs=2′-O-methylguanosine-3′-phosphorothioate
      • t=2′-O-methyl-5-methyluridine-3′-phosphate
      • ts=2′-O-methyl-5-methyluridine-3′-phosphorothioate
      • u=2′-O-methyluridine-3′-phosphate
      • us=2′-O-methyluridine-3′-phosphorothioate
      • i=2′-O-methylinosine-3′-phosphate
      • is=2′-O-methylinosine-3′-phosphorothioate
      • Af=2′-fluoroadenosine-3′-phosphate
      • Afs=2′-fluoroadenosine-3′-phosporothioate
      • Cf=2′-fluorocytidine-3′-phosphate
      • Cfs=2′-fluorocytidine-3′-phosphorothioate
      • Gf=2′-fluoroguanosine-3′-phosphate
      • Gfs=2′-fluoroguanosine-3′-phosphorothioate
      • Tf=2′-fluoro-5′-methyluridine-3′-phosphate
      • Tfs=2′-fluoro-5′-methyluridine-3′-phosphorothioate
      • Uf=2′-fluorouridine-3′-phosphate
      • Ufs=2′-fluorouridine-3′-phosphorothioate
      • AUNA=2′,3′-seco-adenosine-3′-phosphate, see Table 6
      • AUNAS=2′,3′-seco-adenosine-3′-phosphorothioate, see Table 6
      • CUNA=2′,3′-seco-cytidine-3′-phosphate, see Table 6
      • CUNAS=2′,3′-seco-cytidine-3′-phosphorothioate, see Table 6
      • GUNA=2′,3′-seco-guanosine-3′-phosphate, see Table 6
      • GUNAS=2′,3′-seco-guanosine-3′-phosphorothioate, see Table 6
      • UUNA=2′,3′-seco-uridine-3′-phosphate, see Table 6
      • UUNAS=2′,3′-seco-uridine-3′-phosphorothioate, see Table 6
      • a_2N=2′-O-methyl-2-aminoadenosine-3′-phosphate, see Table 6
      • a_2Ns=2′-O-methyl-2-aminoadenosine-3′-phosphorothioate, see Table 6
      • (invAb)=inverted abasic deoxyribonucleotide, see Table 6
      • (invAb)s=inverted abasic deoxyribonucleotide-5′-phosphorothioate, see Table 6
      • cPrpa=5′-cyclopropyl phosphonate-2′-O-methyladenosine-3′-phosphate (see Table 6)
      • cPrpas=5′-cyclopropyl phosphonate-2′-O-methyladenosine-3′-phosphorothioate (see Table 6)
      • cPrpu=5′-cyclopropyl phosphonate-2′-O-methyluridne-3′-phosphate (see Table 6)
      • cPrpus=5′-cyclopropyl phosphonate-2′-O-methylundine-3′-phosphorothioate (see Table 6)
  • As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence (such as, for example, by a phosphorothioate linkage “s”), when present in an oligonucleotide, the nucleotide monomers are mutually linked by 5-3′-phosphodiester bonds. As the person of ordinary skill in the art would clearly understand, the inclusion of a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides. Further, the person of ordinary skill in the art would readily understand that the terminal nucleotide at the 3′ end of a given oligonucleotide sequence would typically have a hydroxyl (—OH) group at the respective 3′ position of the given monomer instead of a phosphate moiety ex vivo. Additionally, for the various aspects disclosed herein, when viewing the respective strand 5′→3′, the inverted abasic residues are inserted such that the 3′ position of the deoxyribose is linked at the 3′ end of the preceding monomer on the respective strand (see, e.g., Table 6). Moreover, as the person of ordinary skill would readily understand and appreciate, while the phosphorothioate chemical structures depicted herein typically show the anion on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers and resonance structures (e.g., where the sulfur atom has a double-bond and the anion is on an oxygen atom). Unless expressly indicated otherwise herein, such understandings of the person of ordinary skill in the art are used when describing the XDH RNAi agents and compositions of XDH RNAi agents disclosed herein.
  • Certain examples of targeting ligands, targeting groups, and linking groups used with the XDH RNAi agents disclosed herein are provided below in Table 6. More specifically, targeting groups and linking groups (which together can form a targeting ligand) include (NAG37) and (NAG37)s, for which their chemical structures are provided below in Table 6. Each sense strand and/or antisense strand can have any targeting ligands, targeting groups, or linking groups listed herein, as well as other groups, conjugated to the 5′ and/or 3′ end of the sequence.
  • TABLE 3
    XDH RNAi Agent Strand Sequences
    Underlying Base Sequence
    (5′ → 3′)
    Antisense Modified Antisense Strand SEQ ID (Shown as an Unmodified SEQ ID
    Strand ID: (5′ → 3′) NO. Nucleotide Sequence) NO.
    AM13029-AS usUfsgsFgaAfgGfcAfuUgcUfcAfaUgcUfsc 945 UUGGAAGGCAUUCUCAAUCUC 1352
    AM13031-AS usUfsggaAfgGfCfauucUfcAfaucusc 946 UUGGAAGGCAUUCUCAAUCUC 1352
    AM13033-AS asAfscsUfuGfaAfgAfaGfaAfgCfuGfaGfsg 947 AACUUGAAGAAGAAGCUGAGG 1353
    AM13035-AS asGfsasAfcUfuGfaAfgAfaGfaAfgCfuGgsc 948 AGAACUUGAAGAAGAAGCUGC 1354
    AM13037-AS usGfsusAfgAfaCfuUfgAfaGfaAgfAfaGfsc 949 UGUAGAACUUGAAGAAGAAGC 1355
    AM13039-AS usCfsasUfaGfgUfgAfuUfuUfcAfcCfcCfsu 950 UCAUAGGUGAUUUUCACCCCU 1356
    AM13041-AS usUfsusCfaUfaGfgUfgAfuUfuUfcAfcCfsc 951 UUUCAUAGGUGAUUUUCACCC 1357
    AM13043-AS usCfsusUfcAfuAfgGfuGfaUfuUfuCfaCfsc 952 UCUUCAUAGGUGAUUUUCACC 1358
    AM13045-AS usUfscsUfuCfaUfaGfgUfgAfuUfuUfcAfsc 953 UUCUUCAUAGGUGAUUUUCAC 1359
    AM13047-AS asAfsusUfgUfgAfuAfaUfgGfcUfgGfuAfsg 954 AAUUGUGAUAAUGGCUGGUAG 1360
    AM13049-AS usCfsasUfaAfaAfgGfaGfuUfgUfuCfuUfsc 955 UCAUAAAAGGAGUUGUUCUUC 1361
    AM13051-AS usCfscsAfuAfaAfaGfgAgfUfuGfuUfcUfsc 956 UCCAUAAAAGGAGUUGUUCUC 1362
    AM13053-AS usAfscsAfgUfgUfuAgfUgfCfuUfgUfcUfsc 957 UACAGUGUUAGUGCUUGUCUC 1363
    AM13055-AS usUfsgsUfgUfaCfaUfaCfuCfaUfgAgcGfsa 958 UUGUGUACAUACUCAUGACGA 1364
    AM13057-AS usAfscsCfaGfuUfaUfcAfgCfaUfgUfcCfsu 959 UACCAGUUAUCAGCAUGUCCU 1365
    AM13059-AS usAfsusGfaAfgCfcAfaCfcUfuGfuAfuCfsc 960 UAUGAAGCCAACCUUGUAUCC 1366
    AM13061-AS usCfsusUfcAfuGfaAfgCgcAfaCfcUfuGfsc 961 UCUUCAUGAAGCCAACCUUGC 1367
    AM13063-AS usUfscsUfuCfaUfgAfaGfcCfaAfcCfuUfsg 962 UUCUUCAUGAAGCCAACCUUG 1368
    AM13065-AS usAfsgsUfcUfuCfaUfgAfaGfcCfaAfcCfsu 963 UAGUCUUCAUGAAGCCAACCU 1369
    AM13067-AS usCfsusUfuUfuCfcAfaCfaAfuUfcUfcCfsu 964 UCUUUUUCCAACAAUUCUCCU 1370
    AM13069-AS usUfscsUfaCfuUfcAfgAfgCfaAfgCfcAfsc 965 UUCUACUUCAGAGCAAGCCAC 1371
    AM13071-AS usAfsusUfuCfuAfcUfuCfaGfaGfcAfaGfsc 966 UAUUUCUACUUCAGAGCAAGC 1372
    AM13073-AS usGfsusCfcAfaUfaUfcAfaUfgGfcAfgGfsg 967 UGUCCAAUAUCAAUGGCAGGG 1373
    AM13164-AS usCfsasGfaAfaAfgUfgGfaCfgAfuCfuUfsg 968 UCAGAAAAGUGGACGAUCUUG 1374
    AM13166-AS asCfsasAfcAfuUfaUfcUfgCfuUfcGfgAfsc 696 ACAACAUUAUCUGCUUCGGAC 1375
    AM13168-AS usCfsasUfaAfuAfcUfcUfgAgfAgfAgfAfsc 970 UCAUAAUACUCUGAGAGAGAC 1376
    AM13170-AS usCfsusUfaUfuCfcAfaAfcUfuGfgUfgGfsg 971 UCUUAUUCCAAACUUGGUGGG 1377
    AM13172-AS usAfsgsUfaAfuCfuUfgCfuUfuAfuGfcAfsg 972 UAGUAAUCUUGCUUUAUGCAG 1378
    AM13174-AS asAfsasGfaAfaUfcUfaGfaAfcAfuUfgUfsc 973 AAAGAAAUCUAGAACAUUGUC 1379
    AM13176-AS usCfsasgaaaagugGfaCfgAfuCfuUfsg 974 UCAGAAAAGUGGACGAUCUUG 1374
    AM13177-AS asCfsasacauUfaUfcUfgCfuUfcggasc 975 ACAACAUUAUCUGCUUCGGAC 1375
    AM13179-AS usCfsasUfaAfuacucUfgAfgAfgagasc 976 UCAUAAUACUCUGAGAGAGAC 1376
    AM13181-AS asAfsasGfaAfaUfcUfaGfaAfcAfuUfuUfsc 977 AAAGAAAUCUAGAACAUUUUC 1380
    AM13204-AS usCfsasGfaAfaagugGfaCgfAfuCfuUfsg 978 UCAGAAAAGUGGACGAUCUUG 1374
    AM13205-AS usCfsasUfaAfuacucUfgAgfAfgAfgAfsc 979 UCAUAAUACUCUGAGAGAGAC 1376
    AM13206-AS usCfsusUfaUfuccaaAfcUfuGfgUfggsg 980 UCUUAUUCCAAACUUGGUGGG 1377
    AM13207-AS usAfsgsUfaAfucuugCfuUfuAfuGfcAgsg 981 UAGUAAUCUUGCUUUAUGCAG 1378
    AM13600-AS usAfsasCfuUfcacucAfuCfcAfgCfacsu 982 UAACUUCACUCAUCCAGCACU 1381
    AM13602-AS usCfsasAfcuucacuCfaUfcCfagcasc 983 UCAACUUCACUCAUCCAGCAC 1382
    AM13604-AS usGfscsAfacuucacUfcAfuCfcagcsa 984 UGCAACUUCACUCAUCCAGCA 1383
    AM13648-AS usGfsasucauacuuGfgAfgAfgcausc 985 UGAUCAUACUUGGAGAGCAUC 1384
    AM13650-AS usCfsusuguucugcAfgAfcGfaucasc 986 UCUUGUUCUGCAGACGAUCAC 1385
    AM13652-AS usGfsasucuuguucUfgCfaGfacgasc 987 UGAUCUUGUUCUGCAGACGAC 1386
    AM13654-AS usAfsgsuaaaguugCfaCfuGfgcgasc 988 UAGUAAAGUUGCACUGGCGAC 1387
    AM13656-AS usAfsasCfacaaguaAfcCfuUfauccsu 989 UAACACAAGUAACCUUAUCCU 1388
    AM13658-AS usCfsasAfuugugauAfaUfgGfcuggsu 990 UCAAUUGUGAUAAUGGCUGGU 1389
    AM13660-AS usAfsgscaugauacUfgAfgAfgcuusg 991 UAGCAUGAUACUGAGAGCUUG 1390
    AM13662-AS asAfscsUfugucaacCfuCfaCfucuusc 992 AACUUGUCAACCUCACUCUUC 1391
    AM13664-AS usAfsasCfuugucaaCfcUfcAfcucusc 993 UAACUUGUCAACCUCACUCUC 1392
    AM13666-AS usAfsasCfaauucucCfuUfgUfugaasc 994 UAACAAUUCUCCUUGUUGAAC 1393
    AM13668-AS usCfsasuguucuguGfgUfaUfguucsc 995 UCAUGUUCUGUGGUAUGUUCC 1394
    AM13670-AS usAfscsUfuUfaauagAfuCfcAfuguusc 996 UACUUUAAUAGAUCCAUGUUC 1395
    AM13672-AS usGfsascuuuAfaUfaGfaUfcCfaugusc 997 UGACUUUAAUAGAUCCAUGUC 1396
    AM13674-AS usGfscsauauucacCfaUfuUfaggcsa 998 UGCAUAUUCACCAUUUAGGCA 1397
    AM13676-AS usGfsusuUfuaagcuuCfuAfgAfgguusc 999 UGUUUAAGCUUCUAGAGGUUC 1398
    AM13678-AS usUfsgsuucauuggUfuUfgAfaggcsc 1000 UUGUUCAUUGGUUUGAAGGCC 1399
    AM13680-AS usUfsasUfgCfuuugcUfgUfuCfauugsg 1001 UUAUGCUUUGCUGUUCAUUGG 1400
    AM13682-AS usGfsusUfaugcuuuGfcUfgUfuCfausc 1002 UGUUAUGCUUUGCUGUUCAUC 1401
    AM13684-AS asGfsgsUfuaugcuuUfgCfuGfuucasc 1003 AGGUUAUGCUUUGCUGUUCAC 1402
    AM13686-AS usAfsasgguuaugcUfuUfgCfuguusc 1004 UAAGGUUAUGCUUUGCUGUUC 1403
    AM13688-AS asGfsasUfucaagguUfaUfgCfuuugsc 1005 AGAUUCAAGGUUAUGCUUUGC 1404
    AM13690-AS usUfscsAfauaauugAfgUfuGfguugsg 1006 UUCAAUAAUUGAGUUGGUUGG 1405
    AM13692-AS asGfsusAfaaauggaUfcAfcAfggaasg 1007 AGUAAAAUGGAUCACAGGAAG 1406
    AM13694-AS usCfsasUfaugaagUfaAfgAfaaacsc 1008 UCAUAUGACAGUAAGAAAACC 1407
    AM13696-AS usUfsgsgaaggcauUfcUfcGfaucusc 1009 UUGGAAGGCAUUCUCGAUCUC 1408
    AM13698-AS usCfsasUfcauugaaAfaUfgCfcagusc 1010 UCAUCAUUGAAAAUGCCAGUC 1409
    AM13700-AS asAfsasGfacaguuuCfaUfcAfuugasc 1011 AAAGACAGUUUCAUCAUUGAC 1410
    AM13702-AS asAfscsacaaguaaCfcUfcAfuccusc 1012 AACACAAGUAACCUCAUCCUC 1411
    AM13704-AS asGfsascaacauugUfcAfgCfuucasg 1013 AGACAACAUUGUCAGCUUCAG 1412
    AM13706-AS usCfsasacaucuuuGfcAfaUfaaagsc 1014 UCAACAUCUUUGCAAUAAAGC 1413
    AM13708-AS asGfsasUfuagucuuAfcAfaAfuccusc 1015 AGAUUAGUCUUACAAAUCCUC 1414
    AM13710-AS usCfsusUfauuccaaAfcUfuAfgucgsg 1016 UCUUAUUCCAAACUUAGUCGG 1415
    AM13712-AS usCfsasGfaaaagaaAfgUfgUfgaagsc 1017 UCAGAAAAGAAAGUGUGAAGC 1416
    AM13714-AS usAfsgsAfguuugucUfcAfaAfgcugsc 1018 UAGAGUUUGUCUCAAAGCUGC 1417
    AM13716-AS usUfsgsUfuaagcagUfcAfaUfuUfcusc 1019 UUGUUAAGCAGUCAAUUUCUC 1418
    AM13718-AS usUfsgsGfaaaucugGfaUfaCfuacgsg 1020 UUGGAAAUCUGGAUACUACGG 1419
    AM13720-AS usCfsusUfgaaaaugCfcAfuCfcugcsu 1021 UCUUGAAAAUGCCAUCCUGCU 1420
    AM13722-AS asUfsgsAfuuuggauCfaCfaAfuugusc 1022 AUGAUUUGGAUCACAAUUGUC 1421
    AM13724-AS usAfsgsAfauuacucAfaAfaCfugccsa 1023 UAGAAUUACUCAAAACUGCCA 1422
    AM13726-AS usGfsasucaaAfAfauGfgAfcUfcagasc 1024 UGAUCAAAAAUGGACUCAGAC 1423
    AM13728-AS usAfsasGfaaagcauGfcAfgAfucuasg 1025 UAAGAAAGCAUGCAGAUCUAG 1424
    AM13730-AS usCfsasgauauaagCfuCfuCfugaasg 1026 UCAGAUAUAAGCUCUCUGAAG 1425
    AM13747-AS usAfsusGfaagccaaCfcUfuGfuAfucsc 1027 UAUGAAGCCAACCUUGUAUCC 1366
    AM13748-AS usAfsusGfaagccaaCfcUfuGfuaucsc 1028 UAUGAAGCCAACCUUGUAUCC 1366
    AM13749-AS usAfsusGfaagCUNAcaaCfcUfuGfuaucsc 1029 UAUGAAGCCAACCUUGUAUCC 1366
    AM13753-AS usAfsusGfaagucaaCfcUfuGfuaucsc 1030 UAUGAAGUCAACCUUGUAUCC 1426
    AM13754-AS usAfsusGfaagcuaaCfcUfuGfuaucsc 1031 UAUGAAGCUAACCUUGUAUCC 1427
    AM13755-AS cPrpusAfsusGfaagccaaCfcUfuGfuaucsc 1032 UAUGAAGCCAACCUUGUAUCC 1366
    AM13758-AS usCfsusUfcaugaagCfcAfaCfcuugsc 1033 UCUUCAUGAAGCCAACCUUGC 1367
    AM13759-AS cPrpusCfsusUfcaugaagCfcAfaCfcuugsc 1034 UCUUCAUGAAGCCAACCUUGC 1367
    AM13761-AS usCfsusUfcaUUNAgaagCfcAfaCfcuugsc 1035 UCUUCAUGAAGCCAACCUUGC 1367
    AM13858-AS usGfsgsAfuCfugcauUfuUfuCfuCfcasc 1036 UGGAUCUGCAUUUUUCUCCAC 1428
    AM13860-AS usCfscsAfaAfaggguUfgUfcUfcUfggsa 1037 UCCAAAAGGGUUGUCUCUGGA 1429
    AM13862-AS usAfsgsAfcGfaucauAfcUfuGfgAfgasg 1038 UAGACGAUCAUACUUGGAGAG 1430
    AM13864-AS usCfscsUfaUfuccuuCfcAfcAfgUfugsc 1039 UCCUAUUCCUUCCACAGUUGC 1431
    AM13866-AS usAfscsAfuAfcucauGfaCfgAfuGfccsa 1040 UACAUACUCAUGACGAUGCCA 1432
    AM13868-AS usCfsasCfaGfauuucCfuUfgGfaAfggsc 1041 UCACAGAUUUCCUUGGAAGGC 1433
    AM13870-AS usGfsasAfcUfucaucUfcAfaUfgCfcasc 1042 UGAACUUCAUCUCAAUGCCAC 1434
    AM13872-AS asGfscsAfuAfuucuuGfaAfcUfuCfausc 1043 AGCAUAUUCUUGAACUUCAUC 1435
    AM13874-AS usCfsasUfaGfgaaacAfgCfaUfaUfucsc 1044 UCAUAGGAAACAGCAUAUUCC 1436
    AM13876-AS usGfsgsAfuCfucuauGfgAfgAfgCfagsc 1045 UGGAUCUCUAUGGAGAGCAGC 1437
    AM13878-AS usUfsusGfaAfugcugAgfAgaAguAfcusc 1046 UUUGAAUGCUGAGAAAUACUC 1438
    AM13880-AS usCfsusAfuGfgacuuGfaUfcUfuGfgcsg 1047 UCUAUGGACUUGAUCUUGGCG 1439
    AM13882-AS asUfsgsAfaAfcaaacAfaAfcCfcUfggsa 1048 AUGAAACAAACAAACCCUGGA 1440
    AM13884-AS usGfsgsUfaGfuucuuCfaUfaGfgUfgasc 1049 UGGUAGUUCUUCAUAGGUGAC 1441
    AM13886-AS usGfsasUfaAfuggcuGfgUfaGfuUfcusc 1050 UGAUAAUGGCUGGUAGUUCUC 1442
    AM13888-AS usCfsusCfaAfuugugAfuAfaUfgGfcusg 1051 UCUCAAUUGUGAUAAUGGCUG 1443
    AM13890-AS usCfsusUfuCfucgauCfuUfcAfgCfucsa 1052 UCUUUCUCGAUCUUCAGCUCA 1444
    AM13892-AS usUfsusGfgAfacagcAfaUfgGfuGfcasg 1053 UUUGGAACAGCAAUGGUGCAG 1445
    AM13894-AS usGfsusAfgAfcacaaAfgAfgCfuCfcasc 1054 UGUAGACACAAAGAGCUCCAC 1446
    AM13896-AS usCfsusGfuGfuagacAfcAfaAfgAfgcsu 1055 UCUGUGUAGACACAAAGAGCU 1447
    AM13898-AS usUfscsCfaUfaauacUfcUfgAfgAfgasg 1056 UUCCAUAAUACUCUGAGAGAG 1448
    AM13900-AS usCfsusCfgUfuccauAfaUfaCfuCfugsc 1057 UCUCGUUCCAUAAUACUCUGC 1449
    AM14175-AS cPrpusUfsgsAfaAfcaaacAfaAfcCfcufggsa 1058 UUGAAACAAACAAACCCUGGA 1450
    AM14176-AS cPrpusUfscsCfaUfaauacUfcUfgAgfAfgasg 1059 UUCCAUAAUACUCUGAGAGAG 1448
    AM14204-AS asAfsusGfaaacaaaCfaAfaCfccugsg 1060 AAUGAAACAAACAAACCCUGG 1451
    AM14206-AS asAfsasUfgaaacaaAfcAfaAfcccusg 1061 AAAUGAAACAAACAAACCCUG 1452
    AM14208-AS usGfsasAfaugaaacAfaAfcAfaaccsc 1062 UGAAAUGAAACAAACAAACCC 1453
    AM14209-AS usUfsgsAfaAfcaaacAfaAfcCfcUfggsa 1063 UUGAAACAAACAAACCCUGGA 1450
    AM14210-AS cPrpusUfsgsAfaacaaacAfaAfcCfcuggsa 1064 UUGAAACAAACAAACCCUGGA 1450
    AM14211-AS cPrpuUfgAfaacaaacAfaAfcCfcuggsa 1065 UUGAAACAAACAAACCCUGGA 1450
    AM14212-AS cPrpuUfgAfaacaaacAfaAfcCfcugsgsa 1066 UUGAAACAAACAAACCCUGGA 1450
    AM14216-AS asGfsasCfgaucauaCfuUfgGfagagsc 1067 AGACGAUCAUACUUGGAGAGC 1454
    AM14218-AS asAfsgsGfcauucucAfaUfcUfccucsc 1068 AAGGCAUUCUCAAUCUCCUCC 1455
    AM14220-AS usUfsusCfcuuggaaGfgCfaUfucucsg 1069 UUUCCUUGGAAGGCAUUCUCG 1456
    AM14222-AS usAfsgsAfuuuccuuGfgAfaGfgcausc 1070 UAGAUUUCCUUGGAAGGCAUC 1457
    AM14224-AS asUfsasGfgaaacagCfaUfaUfucuusg 1071 AUAGGAAACAGCAUAUUCUUG 1458
    AM14226-AS usUfsgsAfugauguuCfcCfuCfcaacsg 1072 UUGAUGAUGUUCCCUCCAACG 1459
    AM14228-AS usAfsgsAfacuugaaGfaAfgAgagcusg 1073 UAGAACUUGAAGAAGAAGCUG 1460
    AM14230-AS usAfscsCfaaugauaUfgCfcCfaacasc 1074 UACCAAUGAUAUGCCCAACAC 1461
    AM14232-AS usCfsasUfggUUNAguucUfgUfgUfagacsg 1075 UCAUGGUGUUCUGUGUAGACG 1462
    AM14234-AS usUfsgsAfgagagauCfcUfgGfgugusc 1076 UUGAGAGAGAUCCUGGGUGUC 1463
    AM14236-AS usCfsasUfgauacugAfgAgfCfuugcsu 1077 UCAUGAUACUGAGAGCUUGCU 1464
    AM14238-AS usUfsgsUfcaaccucAfcUfcUfuccgsa 1078 UUGUCAACCUCACUCUUCCGA 1465
    AM14240-AS usUfsusCfcaacaauUfcUfcCfuugusc 1079 UUUCCAACAAUUCUCCUUGUC 1466
    AM14242-AS usUfsgsAfguuagucUfcAfaAfgcugsc 1080 UUGAGUUAGUCUCAAAGCUGC 1467
    AM14244-AS asUfsgsAfcaauaucUfgUfgCfggagsg 1081 AUGACAAUAUCUGUGCGGAGG 1468
    AM14246-AS usCfsasUfgacaauaUfcUfgUfgcggsa 1082 UCAUGACAAUAUCUGUGCGGA 1469
    AM14248-AS usCfsasAfagaagauAfgAgaGfcagcsc 1083 UCAAAGAAGAUAGAAGCAGCC 1470
    AM14250-AS usCfsasCfguuauuaCfcUfgUfgugcsu 1084 UCACGUUAUUACCUGUGUGCU 1471
    AM14252-AS usAfsgsAfacuugagGfuUfaUfacagsg 1085 UAGAACUUGAGGUUAUACAGG 1472
    AM14254-AS asUfsgsCfuuugcugUfuCfaUfuggusc 1086 AUGCUUUGCUGUUCAUUGGUC 1473
    AM14256-AS usAfsgsUfauagauuCfaAfgGfuuausg 1087 UAGUAUAGAUUCAAGGUUAUG 1474
    AM14258-AS asGfsasGfuaaucuuGfcUfuUfaugcsc 1088 AGAGUAAUCUUGCUUUAUGCC 1475
    AM14260-AS asUfsasGfcaucauuUfcUfaGfguggsa 1089 AUAGCAUCAUUUCUAGGUGGA 1476
    AM14262-AS asGfsasCfagaagagAfcAfgAgfcuasg 1090 AGACAGAAGAGACAGAGCUAG 1477
    AM14264-AS asGfsusAfagaaaacCfaAfgCfcuuasg 1091 AGUAAGAAAACCAAGCCUUAG 1478
    AM14280-AS usUfscsCfauaauacUfcUfgAfgagasg 1092 UUCCAUAAUACUCUGAGAGAG 1448
    AM14281-AS cPrpusUfscsCfauaauacUfcUfgAfgagasg 1093 UUCCAUAAUACUCUGAGAGAG 1448
    AM14282-AS cPrpuUfcCfauaauacUfcUfgAfgagasg 1094 UUCCAUAAUACUCUGAGAGAG 1448
    AM14283-AS cPrpuUfcCfauaauacUfcUfgAfgagsasg 1095 UUCCAUAAUACUCUGAGAGAG 1448
    AM14285-AS cPrpuUfccauaaUfacUfcUfgAfgagasg 1096 UUCCAUAAUACUCUGAGAGAG 1448
    AM14288-AS usAfsusAfcuuggagAgfCfaUfcacusg 1097 UAUACUUGGAGAGCAUCACUG 1479
    AM14290-AS usUfsgsCfagacgauCfUfaCfuuggsc 1098 UUGCAGACGAUCAUACUUGGC 1480
    AM14292-AS usUfsgsAfaUfaaaacUfcUfcAfugccsa 1099 UUGAAUAAAACUCUCAUGCCA 1481
    AM14293-AS cPrpusUfsgsAfaUfaaaacUfcUfcAfugccsa 1100 UUGAAUAAAACUCUCAUGCCA 1482
    AM14296-AS usAfscsUfuGfaAfgAfaGfaAfgCfuGfaGfsg 1101 UACUUGAAGAAGAAGCUGAGG 1482
    AM14297-AS usAfscsUfugaagaaGfaAfgCfugagsg 1102 UACUUGAAGAAGAAGCUGAGG 1482
    AM14298-AS cPrpusAfscsUfugaagaaGfaAfgCfugagsg 1103 UACUUGAAGAAGAAGCUGAGG 1482
    AM14299-AS cPrpuAfcUfugaagaaGfaAfgCfugagsg 1104 UACUUGAAGAAGAAGCUGAGG 1482
    AM14301-AS cPrpuAfcUfugaagaaGfaAgfCfugasgsg 1105 UACUUGAAGAAGAAGCUGAGG 1482
    AM14304-AS cPrpuAfcuugAgagaaGfaAgfCfugagsg 1106 UACUUGAAGAAGAAGCUGAGG 1482
    AM14305-AS cPrpuAfcuugaaGfaaGfaAgfCfugagsg 1107 UACUUGAAGAAGAAGCUGAGG 1482
    AM14383-AS cPrpusUfsusGfaAfucugAgfAgaAguAgcusc 1108 UUUGAAUGCUGAGAAAUACUC 1438
    AM14384-AS cPrpusUfsusGfaaugcugAgfAgaAguacusc 1109 UUUGAAUGCUGAGAAAUACUC 1438
    AM14385-AS cPrpusUfsusgaaUfgcugAfgAgaAguacusc 1110 UUUGAAUGCUGAGAAAUACUC 1438
    AM14387-AS cPrpuUfuGgaaugcugAfgAfaAfuacusc 1111 UUUGAAUGCUGAGAAAUACUC 1438
    AM14388-AS cPrpuUfuGfaaugcugAgfAgaAguacsusc 1112 UUUGAAUGCUGAGAAAUACUC 1438
    AM14391-AS asGfsasAfaAfguggaCfgAfuCfuUfgusc 1113 AGAAAAGUGGACGAUCUUGUC 1483
    AM14393-AS usAfsgsUfuGfucacuGfcAfaCfaUfggsu 1114 UAGUUGUCACUGCAACAUGGU 1484
    AM14395-AS asUfsusCfcUfuccacAfgUfuGfuCfacsc 1115 AUUCCUUCCACAGUUGUCACC 1485
    AM14397-AS usGfscsAfuUfcucaaUfcUfcCfuCfcasc 1116 UGCAUUCUCAAUCUCCUCCAC 1486
    AM14399-AS usUfscsAfaUfgccaaUfcUfcCfgUfgusc 1117 UUCAAUGCCAAUCUCCGUGUC 1487
    AM14401-AS asUfsasUfuCfuugaaCfuUfcAfuCfucsg 1118 AUAUUCUUGAACUUCAUCUCG 1488
    AM14403-AS usUfsasUfgGfagagcAfgUfaUfcUfccsu 1119 UUAUGGAGAGCAGUAUCUCCU 1489
    AM14405-AS usAfsasUfgCfugagaAgaUgaCfuCfccsc 1120 UAAUGCUGAGAAAUACUCCCC 1490
    AM14407-AS usGfsasAfuGfcugagAgaAguAgcUfccsc 1121 UGAAUGCUGAGAAAUACUCCC 1491
    AM14409-AS usCfsasAfuGfucaucUfuCfuCfuCfcgsg 1122 UCAAUGUCAUCUUCUCUCCGG 1492
    AM14411-AS asCfsasAfaUfuccagUfuAfuGfuUfacsc 1123 ACAAAUUCCAGUUAUGUUACC 1493
    AM14413-AS usUfscsAfaUfugugaUfaAfuGfgCfugsg 1124 UUCAAUUGUGAUAAUGGCUGG 1494
    AM14415-AS asAfscsAfuUfuuugcAfaCfaAfaGfcusc 1125 AACAUUUUUGCAACAAAGCUC 1495
    AM14417-AS usCfsasAfcAfuuuuuGfcAfaCfaAfagsc 1126 UCAACAUUUUUGCAACAAAGC 1496
    AM14419-AS usUfsusCfaCfucgaaCgcAfcAfaUfccsg 1127 UCUUAUUCCAAACUUGGUGGG 1497
    AM14522-AS cPrpusCfsusUfaUfuccaaAfcUfuGfgUfggsg 1128 UCUUAUUCCAAACUUGGUGGG 1377
    AM14523-AS cPrpuCfuUfaUfuccaaAfcUfuGfgUfggsg 1229 UCUUAUUCCAAACUUGGUGGG 1377
    AM14524-AS cPrpuCfuuauucCfaaAfcUfuGfguggsg 1130 UCUUAUUCCAAACUUGGUGGG 1377
    AM14527-AS cPrpuGfcauauucacCfaUfuUfaggcsa 1131 UGCAUAUUCACCAUUUAGGCA 1397
    AM14529-AS cPrpuGfcauaUfucacCfaUfuUfaggcsa 1132 UGCAUAUUCACCAUUUAGGCA 1397
    AM14530-AS cPrpuGfcauauuCfacCfaUfuUfaggcsa 1133 UGCAUAUUCACCAUUUAGGCA 1397
    AM14543-AS usGfscauauucacCfaUfuUfaggcsa 1134 UGCAUAUUCACCAUUUAGGCA 1397
    AM14544-AS usGfscauaUfucacCfaUfuUfaggcsa 1135 UGCAUAUUCACCAUUUAGGCA 1397
    AM14545-AS usGfscauauuCfacCfaUfuUfaggcsa 1136 UGCAUAUUCACCAUUUAGGCA 1397
    AM14642-AS cPrpasUfsgsAfaacaaacAfaAfcCfcuggsa 1137 AUGAAACAAACAAACCCUGGA 1440
    AM14643-AS cPrpasUfsgsAfaacaaacAfaAfcCfcugsgsa 1138 AUGAAACAAACAAACCCUGGA 1440
    AM14644-AS cPrpasUfsgAfaacaaacAfaAfcCfcugsgsa 1139 AUGAAACAAACAAACCCUGGA 1440
    AM14645-AS cPrpaUfgAfaacaaacAfaAfcCfcugsgsa 1140 AUGAAACAAACAAACCCUGGA 1440
    AM14647-AS cPrpasUfsgaaacaAfacAfaAfcCfcugsgsa 1141 AUGAAACAAACAAACCCUGGA 1440
    AM14648-AS cPrpasUfsgaaaCfaaacAfaAfcCfcugsgsa 1142 AUGAAACAAACAAACCCUGGA 1440
    AM14649-AS cPrpasUfsgaAfacaaacAfaAfcCfcugsgsa 1143 AUGAAACAAACAAACCCUGGA 1440
    AM14650-AS cPrpasUfsgAfaaCfaAfacAfaAfcCfcugsgsa 1144 AUGAAACAAACAAACCCUGGA 1440
    AM15134-AS cPrpusUfscCfauaauacUfcUfgAfgagasg 1145 UUCCAUAAUACUCUGAGAGAG 1448
    AM15135-AS cPrpusUfscCfauaauacUfcUfgAfgagsasg 1146 UUCCAUAAUACUCUGAGAGAG 1448
    AM15137-AS cPrpuUfcCfauaauacUfcUfgAfgagasc 1147 UUCCAUAAUACUCUGAGAGAG 1498
    AM15139-AS cPrpuUfcCfauaauacUfcUfgAfgaggsg 1148 UUCCAUAAUACUCUGAGAGGG 1499
    AM15141-AS cPrpuUfcCfauaauacUfcUfgAfgaggsc 1149 UUCCAUAAUACUCUGAGAGGC 1500
    AM15143-AS cPrpuUfcCfauaauacUfcUfgAgfaggsu 1150 UUCCAUAAUACUCUGAGAGGA 1501
    AM15145-AS cPrpuUfcCfauaauacUfcUfgAfgaggsa 1151 UUCCAUAAUACUCUGAGAGGA 1502
    AM15146-AS cPrpuUfccauAfauacUfcUfgAfgagasg 1152 UUCCAUAAUACUCUGAGAGAG 1448
    AM15147-AS cPrpusGfscsauauuCfacCfaUfuUfaggcsa 1153 UGCAUAUUCACCAUUUAGGCA 1397
    AM15148-AS cPrpusGfscauauuCfacCfaUfuUfaggcsa 1154 UGCAUAUUCACCAUUUAGGCA 1397
    AM15149-AS cPrpusGfscauauuCfacCfaUfuUfaggscsa 1155 UGCAUAUUCACCAUUUAGGCA 1397
    AM15150-AS cPrpuGfcauauuCfacCfaUfuUfaggscsa 1156 UGCAUAUUCACCAUUUAGGCA 1397
    AM15151-AS cPrpusGfscsauaUfucacCfaufuUfaggcsa 1157 UGCAUAUUCACCAUUUAGGCA 1397
    AM15152-AS cPrpusGfscauaUfucacCfaUfuUfaggcsa 1158 UGCAUAUUCACCAUUUAGGCA 1397
    AM15153-AS cPrpusGfscauaUfucacCfaUfuUfaggscsa 1159 UGCAUAUUCACCAUUUAGGCA 1397
    AM15154-AS cPrpuGfcauaUfucacCfaUfuUfaggscsa 1160 UGCAUAUUCACCAUUUAGGCA 1397
    AM15285-AS asUfsgsacaAfuaucUfgUfgCfggagsg 1161 AUGACAAUAUCUGUGCGGAGG 1468
    AM15286-AS asUfsgsacaauAfucUfgUfgCfggagsg 1162 AUGACAAUAUCUGUGCGGAGG 1468
    AM15287-AS cPrpasUfsgsacaauAfucUfgUfgCfggagsg 1163 AUGACAAUAUCUGUGCGGAGG 1468
    AM15289-AS cPrpusUfsgsacaauAfucUfgUfgCfggagsg 1164 AUGACAAUAUCUGUGCGGAGG 1503
    AM15290-AS cPrpaUfgacaauAfucUfgUfgCfggagsg 1165 AUGACAAUAUCUGUGCGGAGG 1468
    AM15291-AS cPrpaUfgacaauAfucUfgUfgCfggasgsg 1166 AUGACAAUAUCUGUGCGGAGG 1468
    AM15292-AS cPrpasUfsgacaauAfucUfgUfgCfggasgsg 1167 AUGACAAUAUCUGUGCGGAGG 1468
    AM15294-AS cPrpasUfsgsacaauAfucUfgUfgCfggasg 1168 AUGACAAUAUCUGUGCGGAG 1504
    AM15296-AS cPrpasUfsgsacaauAfucUfgUfgCfggsa 1169 AUGACAAUAUCUGUGCGGA 1505
    AM15606-AS cPrpusUfsccauaaUfacUfcUfgAfgagsasg 1170 UUCCAUAAUACUCUGAGAGAG 1448
    AM15607-AS cPrpusUfscCfauaauacUfcUfgAfgagsasc 1171 UUCCAUAAUACUCUGAGAGAC 1498
    AM15608-AS cPrpusUfsgaaaCfaaacAfaAfcCfcugsgsa 1172 UUGAAACAAACAAACCCUGGA 1450
    AM15626-AS asUfsgAfaAfcaaacAfaAfcCfcUfgsgsa 1173 AUGAAACAAACAAACCCUGGA 1440
    AM15627-AS asUfsgAfaacaaacAfaAfcCfcugsgsa 1174 AUGAAACAAACAAACCCUGGA 1440
    AM17243-AS asCfsucgUfuccauaaUfaCfucugasgsa 1672 ACUCGUUCCAUAAUACUCUGAGA 1674
    AM17245-AS asUfsccaUfaauacucUfgAfgagagsasu 1673 AUCCAUAAUACUCUGAGAGAGAU 1675
  • TABLE 4
    XDH RNAi Agent Strand Sequences
    Underlying Base Sequence
    Sense SEQ (5′ → 3′) SEQ
    Strand Modified Sense Strand ID (Shown as an Unmodified ID
    ID: (5′ → 3′) NO. Nucleotide Sequence) NO.
    AM13028-SS (NAG37)s(invAb)sgagauugaGfAfAfugccuuccaas(invAb) 1175 GAGAUUGAGAAUGCCUUCCAA 1506
    AM13030-SS (NAG37)s(invAb)sgagauuGfaGfAfAfugccuuccaas(invAb) 1176 GAGAUUGAGAAUGCCUUCCAA 1506
    AM13032-SS (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguus(invAb) 1177 CCUCAGCUUCUUCUUCAAGUU 1507
    AM13034-SS (NAG37)s(invAb)sgcagcuucUfUfCfuucaaguucus(invAb) 1178 GCAGCUUCUUCUUCAAGUUCU 1508
    AM13036-SS (NAG37)s(invAb)sgcuucuucUfUfCfaaguucuacas(invAb) 1179 GCUUCUUCUUCAAGUUCUACA 1509
    AM13038-SS (NAG37)s(invAb)saggggugaAfAfAfucaccuaugas(invAb) 1180 AGGGGUGAAAAUCACCUAUGA 1510
    AM13040-SS (NAG37)s(invAb)sgggugaaaAfUfCfaccuaugaaas(invAb) 1181 GGGUGAAAAUCACCUAUGAAA 1511
    AM13042-SS (NAG37)s(invAb)sggugaaaaUfCfAfccuaugaagas(invAb) 1182 GGUGAAAAUCACCUAUGAAGA 1512
    AM13044-SS (NAG37)s(invAb)sgugaaaauCfAfCfcuaugaagaas(invAb) 1183 GUGAAAAUCACCUAUGAAGAA 1513
    AM13046-SS (NAG37)s(invAb)scuaccagcCfAfUfuaucacaauus(invAb) 1184 CUACCAGCCAUUAUCACAAUU 1514
    AM13048-SS (NAG37)s(invAb)sgaagaacaAfCfUfccuuuuaugas(invAb) 1185 GAAGAACAACUCCUUUUAUGA 1515
    AM13050-SS (NAG37)s(invAb)sgagaacaaCfUfCfcuuuuauggas(invAb) 1186 GAGAACAACUCCUUUUAUGGA 1516
    AM13052-SS (NAG37)s(invAb)sgagacaagCfAfCfuaacacuguas(invAb) 1187 GAGACAAGCACUAACACUGUA 1517
    AM13054-SS (NAG37)s(invAb)sucgucaugAfGfUfauguacacaas(invAb) 1188 UCGUCAUGAGUAUGUACACAA 1518
    AM13056-SS (NAG37)s(invAb)saggacaugCfUfGfauaacugiuas(invAb) 1189 AGGACAUGCUGAUAACUGIUA 1519
    AM13058-SS (NAG37)s(invAb)sggauacaaGfGfufuggcuucauas(invAb) 1190 GGAUACAAGGUUGGCUUCAUA 1520
    AM13060-SS (NAG37)s(invAb)sgcaagguuGfGfCfuucaugaagas(invAb) 1191 GCAAGGUUGGCUUCAUGAAGA 1521
    AM13062-SS (NAG37)s(invAb)scagguugGfCfUfucaugaagasas(invAb) 1992 CAAGGUUGGCUUCAUGAAGAA 1522
    AM13064-SS (NAG37)s(invAb)sagguuggcUfUfCfaugaagacuas(invAb) 1193 AGGUUGGCUUCAUGAAGACUA 1523
    AM13066-SS (NAG37)s(invAb)saggagaauUfGfUfuggaaaaagas(invAb) 1194 AGGAGAAUUGUUGGAAAAAGA 1524
    AM13068-SS (NAG37)s(invAb)sguggcuugCfUfCfugaaguagaas(invAb) 1195 GUGGCUUGCUCUGAAGUAGAA 1525
    AM13070-SS (NAG37)s(invAb)sgcuugcucUfGfAfaguagaaauas(invAb) 1196 GCUUGCUCUGAAGUAGAAAUA 1526
    AM13072-SS (NAG37)s(invAb)scccugccaUfUfGfauauuigacas(invAb) 1197 CCCUGCCAUUGAUAUUIGACA 1527
    AM13163-SS (NAG37)s(invAb)scaagaucgUfCfCfacuuuucugas(invAb) 1198 CAAGAUCGUCCACUUUUCUGA 1528
    AM13165-SS (NAG37)s(invAb)sguccgaagCfAfGfauaauguugus(invAb) 1199 GUCCGAAGCAGAUAAUGUUGU 1529
    AM13167-SS (NAG37)s(invAb)sgucucucuCfAfGfaguauuaugas(invAb) 1200 GUCUCUCUCAGAGUAUUAUGA 1530
    AM13169-SS (NAG37)s(invAb)scccaccaaGfUfUfuggaauaagas(invAb) 1201 CCCACCAAGUUUGGAAUAAGA 1531
    AM13171-SS (NAG37)s(invAb)scugcauaaAfGfCfaagauuacuas(invAb) 1202 CUGCAUAAAGCAAGAUUACUA 1532
    AM13173-SS (NAG37)s(invAb)sgacaauguUfCfUfagauuucuuus(invAb) 1203 GACAAUGUUCUAGAUUUCUUU 1533
    AM13175-SS (NAG37)s(invAb)scaagaucgUfcCfaCfuuuucugas(invAb) 1204 CAAGAUCGUCCACUUUUCUGA 1528
    AM13178-SS (NAG37)s(invAb)sgucucucuCfaGfaGfuauuaugas(invAb) 1205 GUCUCUCUCAGAGUAUUAUGA 1530
    AM13180-SS (NAG37)s(invAb)sgaaaauguUfCfUfagauuucuuus(invAb) 1206 GAAAAUGUUCUAGAUUUCUUU 1534
    AM13599-SS (NAG37)s(invAb)sagugcuggAfUfGfagugaaguuas(invAb) 1207 AGUGCUGGAUGAGUGAAGUUA 1535
    AM13601-SS (NAG37)s(invAb)sgugcuigaUfGfAfgugaaguugas(invAb) 1208 GUGCUIGAUGAGUGAAGUUGA 1536
    AM13603-SS (NAG37)s(invAb)sugcuggauGfAfGfugaaguuicas(invAb) 1209 UGCUGGAUGAGUGAAGUUICA 1537
    AM13647-SS (NAG37)s(invAb)sgaugcucuCfcAfaGfuaugaucas(invAb) 1210 GAUGCUCUCCAAGUAUGAUCA 1538
    AM13649-SS (NAG37)s(invAb)sgugaucguCfuGfcAfgaacaagas(invAb) 1211 GUGAUCGUCUGCAGAACAAGA 1539
    AM13651-SS (NAG37)s(invAb)sgucgucugCfaGfaafcaagaucas(invAb) 1212 GUCGUCUGCAGAACAAGAUCA 1540
    AM13653-SS (NAG37)s(invAb)sgucgccagUfgCfaAfcuuuacuas(invAb) 1213 GUCGCCAGUGCAACUUUACUA 1541
    AM13655-SS (NAG37)s(invAb)saggauaAfgGfuUfacuuguguuas(invAb) 1214 AGGAUAAGGUUACUUGUGUUA 1542
    AM13657-SS (NAG37)s(invAb)saccagccaUfuAfuCfacaauugas(invAb) 1215 ACCAGCCAUUAUCACAAUUGA 1543
    AM13659-SS (NAG37)s(invAb)scaagcucuCfaGfuAfucaugcuas(invAb) 1216 CAAGCUCUCAGUAUCAUGCUA 1544
    AM13661-SS (NAG37)s(invAb)sgaagagugAfgGfuUfgacaaguus(invAb) 1217 GAAGAGUGAGGUUGACAAGUU 1545
    AM13663-SS (NAG37)s(invAb)sgagagugaGfGfUfugacaaguuas(invAb) 1218 GAGAGUGAGGUUGACAAGUUA 1546
    AM13665-SS (NAG37)s(invAb)sguucaacaAfGfGfagaauuguuas(invAb) 1219 GUUCAACAAGGAGAAUUGUUA 1547
    AM13667-SS (NAG37)s(invAb)sggaacaUfaCfcAfcagaacaugas(invAb) 1220 GGAACAUACCACAGAACAUGA 1548
    AM13669-SS (NAG37)s(invAb)sgaacauggAfuCfuAfuuaaaguas(invAb) 1221 GAACAUGGAUCUAUUAAAGUA 1549
    AM13671-SS (NAG37)s(invAb)sgacauggaUfcUfaUfuaaagucas(invAb) 1222 GACAUGGAUCUAUUAAAGUCA 1550
    AM13673-SS (NAG37)s(invAb)sugccuaAfaUfgGfugaauaugcas(invAb) 1223 UGCCUAAAUGGUGAAUAUGCA 1551
    AM13675-SS (NAG37)s(invAb)sgaaccucuAfGfAfagcuuaaacas(invAb) 1224 GAACCUCUAGAAGCUUAAACA 1552
    AM13677-SS (NAG37)s(invAb)sggccuucaAfaCfcAfaugaacaas(invAb) 1225 GGCCUUCAAACCAAUGAACAA 1553
    AM13679-SS (NAG37)s(invAb)sccaaugAfaCfaGfcaaagcauaas(invAb) 1226 CCAAUGAACAGCAAAGCAUAA 1554
    AM13681-SS (NAG37)s(invAb)sgaugaacaGfcAfAfagcauaacas(invAb) 1227 GAUGAACAGCAAAGCAUAACA 1555
    AM13683-SS (NAG37)s(invAb)sgugaacagCfAfAfagcauaaccus(invAb) 1228 GUGAACAGCAAAGCAUAACCU 1556
    AM13685-SS (NAG37)s(invAb)sgaacagcaAfaGfcAfuaaccuuas(invAb) 1229 GAACAGCAAAGCAUAACCUUA 1557
    AM13687-SS (NAG37)s(invAb)sgcaaagcaUfAfAfccuugaaucus(invAb) 1230 GCAAAGCAUAACCUUGAAUCU 1558
    AM13689-SS (NAG37)s(invAb)sccaaccaaCfuCfaAfuuauugaas(invAb) 1231 CCAACCAACUCAAUUAUUGAA 1559
    AM13694-SS (NAG37)s(invAb)scuuccuguGfAfUfccauuuuacus(invAb) 1232 CUUCCUGUGAUCCAUUUUACU 1560
    AM13693-SS (NAG37)s(invAb)sgguuuucuUfAfCfugucauaugas(invAb) 1233 GGUUUUCUUACUGUCAUAUGA 1561
    AM13695-SS (NAG37)s(invAb)sgagaucgaGfAfAfugccuuccaas(invAb) 1234 GAGAUCGAGAAUGCCUUCCAA 1562
    AM13697-SS (NAG37)s(invAb)sgacuggcaUfUfUfucaaugaugas(invAb) 1235 GACUGGCAUUUUCAAUGAUGA 1563
    AM13699-SS (NAG37)s(invAb)sgucaaugaUfGfAfaacugucuuus(invAb) 1236 GUCAAUGAUGAAACUGUCUUU 1564
    AM13701-SS (NAG37)s(invAb)sgaggaugaGfGfUfuacuuguguus(invAb) 1237 GAGGAUGAGGUUACUUGUGUU 1565
    AM13703-SS (NAG37)s(invAb)scugaagcuGfAfCfaauguugucus(invAb) 1238 CUGAAGCUGACAAUGUUGUCU 1566
    AM13705-SS (NAG37)s(invAb)sgcuuuauuGfCfAfaagauguugas(invAb) 1239 GCUUUAUUGCAAAGAUGUUGA 1567
    AM13707-SS (NAG37)s(invAb)sgaggauuuGfUfAfagacuaaucus(invAb) 1240 GAGGAUUUGUAAGACUAAUCU 1568
    AM13709-SS (NAG37)s(invAb)sccgacuaaGfUfUfuggaauaagas(invAb) 1241 CCGACUAAGUUUGGAAUAAGA 1569
    AM13711-SS (NAG37)s(invAb)sgcuucacaCfUfUfucuuuucugas(invAb) 1242 GCUUCACACUUUCUUUUCUGA 1570
    AM13713-SS (NAG37)s(invAb)sgcagcuuuGfAfGfacaaacucuas(invAb) 1243 GCAGCUUUGAGACAAACUCUA 1571
    AM13715-SS (NAG37)s(invAb)sgagaaauuGfAfCfugcuuaacaas(invAb) 1244 GAGAAAUUGACUGCUUAACAA 1572
    AM13717-SS (NAG37)s(invAb)sccguaguaUfCfCfagauuuccaas(invAb) 1245 CCGUAGUAUCCAGAUUUCCAA 1573
    AM13719-SS (NAG37)s(invAb)sagcaggauGfGfCfauuuucaagas(invAb) 1246 AGCAGGAUGGCAUUUUCAAGA 1574
    AM13721-SS (NAG37)s(invAb)sgacaauugUfGfAfuccaaaucaus(invAb) 1247 GACAAUUGUGAUCCAAAUCAU 1575
    AM13723-SS (NAG37)s(invAb)suggcaguuUfUfGfaguaauucuas(invAb) 1248 UGGCAGUUUUGAGUAAUUCUA 1576
    AM13725-SS (NAG37)s(invAb)sgucugaguCfCfAfuuuuugaucas(invAb) 1249 GUCUGAGUCCAUUUUUGAUCA 1577
    AM13727-SS (NAG37)s(invAb)scuagaucuGfCfAfugcuuucuuas(invAb) 1250 CUAGAUCUGCAUGCUUUCUUA 1578
    AM13729-SS (NAG37)s(invAb)scuucagagAfGfCfuuauaucugas(invAb) 1251 CUUCAGAGAGCUUAUAUCUGA 1579
    AM13746-SS (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252 GGAUACAAGGUUGGCUUCAUA 1520
    AM13750-SS (NAG37)s(invAb)sggauacAfaGfgUfugicuucauas(invAb) 1253 GGAUACAAGGUUGICUUCAUA 1580
    AM13751-SS (NAG37)s(invAb)sggauacAfaGfgUfuigcuucauas(invAb) 1254 GGAUACAAGGUUIGCUUGAUA 1581
    AM13752-SS (NAG37)s(invAb)sggauacAfaGfgUfugguuucauas(invAb) 1255 GGAUACAAGGUUGGUUUCAUA 1582
    AM13756-SS (NAG37)s(invAb)sggauacaaGfgUfUfggcuucauas(invAb) 1256 GGAUACAAGGUUGGCUUCAUA 1520
    AM13757-SS (NAG37)s(invAb)sggauacaaGfgUfuFgfcuucauas(invAb) 1257 GGAUACAAGGUUGGCUUCAUA 1520
    AM13760-SS (NAG37)s(invAb)sgcaagguuGfgCfuUfcaugaagas(invAb) 1258 GCAAGGUUGGCUUCAUGAAGA 1521
    AM13857-SS (NAG37)s(invAb)sguggagaaAfAfAfugcaiauccas(invAb) 1259 GUGGAGAAAAAUGCAIAUCCA 1583
    AM13859-SS (NAG37)s(invAb)succagagCfAfAfcucuuuuuggas(invAb) 1260 UCCAGAGACAACUCUUUUGGA 1584
    AM13861-SS (NAG37)s(invAb)scucuccaaGfUfAfugauciucuas(invAb) 1261 CUCUCCAAGUAUGAUCIUCUA 1585
    AM13863-SS (NAG37)s(invAb)sgcaacuguGfGfAfaggaauaggas(invAb) 1262 GCAACUGUGGAAGGAAUAGGA 1586
    AM13865-SS (NAG37)s(invAb)suggcaucgUfCfAfugaguauguas(invAb) 1263 UGGCAUCGUCAUGAGUAUGUA 1587
    AM13867-SS (NAG37)s(invAb)sgccuuccaAfGfGfaaaucuguias(invAb) 1264 GCCUUCCAAGGAAAUCUGUIA 1588
    AM13869-SS (NAG37)s(invAb)sguggcauuGfAfGfaugaaguucas(invAb) 1265 GUGGCAUUGAGAUGAAGUUCA 1589
    AM13871-SS (NAG37)s(invAb)sga_2NugaaguUfCfAfagaauaugcus(invAb) 1266 G(A2N)UGAAGUUCAAGAAUAUGCU 1590
    AM13873-SS (NAG37)s(invAb)sggaauaugCfUfGfuuuccuaugas(invAb) 1267 GGAAUAUGCUGUUUCCUAUGA 1591
    AM13875-SS (NAG37)s(invAb)sgcugcucuCfCfAfuagaiauccas(invAb) 1268 GCUGCUCUCCAUAGAIAUCCA 1592
    AM13877-SS (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269 GAGUAUUUCUCAGCAUUCAAA 1593
    AM13879-SS (NAG37)s(invAb)scgccaagaUfCfAfaguccauagas(invAb) 1270 CGCCAAGAUCAAGUCCAUAGA 1594
    AM13881-SS (NAG37)s(invAb)succaggguUfUfGfuuuguuucaus(invAb) 1271 UCCAGGGUUUGUUUGUUUCAU 1595
    AM13883-SS (NAG37)s(invAb)sgucaccuaUfGfAfagaacuaccas(invAb) 1272 GUCACCUAUGAAGAACUACCA 1596
    AM13885-SS (NAG37)s(invAb)sgagaacuaCfCfAfgccauuaucas(invAb) 1273 GAGAACUACCAGCCAUUAUCA 1597
    AM13887-SS (NAG37)s(invAb)scagccauuAfUfCfacaauugagas(invAb) 1274 CAGCCAUUAUCACAAUUGAGA 1598
    AM13889-SS (NAG37)s(invAb)sugagcugaAfGfAfucgagaaagas(invAb) 1275 UGAGCUGAAGAUCGAGAAAGA 1599
    AM13891-SS (NAG37)s(invAb)scugcaccaUfUfGfcuguuccaaas(invAb) 1276 CUGCACCAUUGCUGUUCCAAA 1600
    AM13893-SS (NAG37)s(invAb)sguggagcuCfUfUfuguguuuacas(invAb) 1277 GUGGAGCUCUUUGUGUUUACA 1601
    AM13895-SS (NAG37)s(invAb)sagcucuuuGfUfGfucuacacaias(invAb) 1278 AGCUCUUUGUGUCUACACAIA 1602
    AM13897-SS (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279 CUCUCUCAGAGUAUUAUGGAA 1603
    AM13899-SS (NAG37)s(invAb)sgcagaguaUfUfAfuggaacgaias(invAb) 1280 GCAGAGUAUUAUGGAACGAIA 1604
    AM14174-SS (NAG37)s(invAb)succaggguUfUfGfuuuguuucaas(invAb) 1281 UCCAGGGUUUGUUUGUUUCAA 1605
    AM14203-SS (NAG37)s(invAb)sccaggguuUfGfUfuuguuucauus(invAb) 1282 CCAGGGUUUGUUUGUUUCAUU 1606
    AM14205-SS (NAG37)s(invAb)scaggguuuGfUfUfuguuucauuus(invAb) 1283 CAGGGUUUGUUUGUUUCAUUU 1607
    AM14207-SS (NAG37)s(invAb)sggguuuguUfUfGfuuucauuucas(invAb) 1284 GGGUUUGUUUGUUUCAUUUCA 1608
    AM14213-SS (NAG37)s(invAb)succaggguUfuGfuUfuguuucaas(invAb) 1285 UCCAGGGUUUGUUUGUUUCAA 1605
    AM14214-SS (NAG37)s(invAb)succaggguUfuGfUfuuguuucaas(invAb) 1286 UCCAGGGUUUGUUUGUUUCAA 1605
    AM14215-SS (NAG37)s(invAb)sgcucuccaAfGfUfaugauciucus(invAb) 1287 GCUCUCCAAGUAUGAUCIUCU 1609
    AM14217-SS (NAG37)s(invAb)sggaggagaUfUfGfagaauiccuus(invAb) 1288 GGAGGAGAUUGAGAAUICCUU 1610
    AM14219-SS (NAG37)s(invAb)scgagaaugCfCfUfuccaaggaaas(invAb) 1289 CGAGAAUGCCUUCCAAGGAAA 1611
    AM14221-SS (NAG37)s(invAb)sgaugccuuCfCfAfaggaaaucuas(invAb) 1290 GAUGCCUUCCAAGGAAAUCUA 1612
    AM14223-SS (NAG37)s(invAb)sca_2NagaauaUfGfCfuguuuccuaus(invAb) 1291 C(A2N)AGAAUAUGCUGUUUCCUAU 1613
    AM14225-SS (NAG37)s(invAb)scguuggagGfGfAfacaucaucaas(invAb) 1292 CGUUGGAGGGAACAUCAUCAA 1614
    AM14227-SS (NAG37)s(invAb)scagcuucuUfCfUfucaaguucuas(invAb) 1293 CAGCUUCUUCUUCAAGUUCUA 1615
    AM14229-SS (NAG37)s(invAb)sguguugggCfAfUfaucauugguas(invAb) 1294 GUGUUGGGCAUAUCAUUGGUA 1616
    AM14231-SS (NAG37)s(invAb)scgucuacaCfAfGfaacaccaugas(invAb) 1295 CGUCUACACAGAACACCAUGA 1617
    AM14233-SS (NAG37)s(invAb)sgacacccaGfGfAfucucuuucaas(invAb) 1296 GACACCCAGGAUCUCUUUCAA 1618
    AM14235-SS (NAG37)s(invAb)sagcaagcuCfUfCfaguaucaugas(invAb) 1297 AGCAAGCUCUCAGUAUCAUGA 1619
    AM14237-SS (NAG37)s(invAb)sucggaagaGfUfGfagguugacaas(invAb) 1298 UCGGAAGAGUGAGGUUGACAA 1620
    AM14239-SS  (NAG37)s(invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) 1299 GACAAGGAGAAUUGUUGGAAA 1621
    AM14241-SS (NAG37)s(invAb)sgcagcuuuGfAfGfacuaacucaas(invAb) 1300 GCAGCUUUGAGACUAACUCAA 1622
    AM14243-SS (NAG37)s(invAb)sccuccgcaCfAfGfauauugucaus(invAb) 1301 CCUCCGCACAGAUAUUGUCAU 1623
    AM14245-SS (NAG37)s(invAb)succgcacaGfAfUfauugucaugas(invAb) 1302 UCCGCACAGAUAUUGUCAUGA 1624
    AM14247-SS (NAG37)s(invAb)sggcugcuuCfUfAfucuucuuugas(invAb) 1303 GGCUGCUUCUAUCUUCUUUGA 1625
    AM14249-SS (NAG37)s(invAb)sagcacacaGfGfUfaauaacguias(invAb) 1304 AGCACACAGGUAAUAACGUIA 1626
    AM14251-SS (NAG37)s(invAb)sccuguauaAfCfCfucaaguucuas(invAb) 1305 CCUGUAUAACCUCAAGUUCUA 1627
    AM14253-SS (NAG37)s(invAb)sgaccaaugAgAgCfagcaaagcaus(invAb) 1306 GACCAAUGAACAGCAAAGCAU 1628
    AM14255-SS (NAG37)s(invAb)sca_2NuaaccuUfGfAfaucuauacuas(invAb) 1307 C(A2n)UAACCUUGAAUCUAUACUA 1629
    AM14257-SS (NAG37)s(invAb)sggcauaaaGfCfAfagauuacucus(invAb) 1308 GGCAUAAAGCAAGAUUACUCU 1630
    AM14259-SS (NAG37)s(invAb)succaccuaGfAfAfaugaugcuaus(invAb) 1309 UCCACCUAGAAAUGAUGCUAU 1631
    AM14261-SS (NAG37)s(invAb)scuagcucuGfUfCfucuucuiucus(invAb) 1310 CUAGCUCUGUCUCUUCUIUCU 1632
    AM14263-SS (NAG37)s(invAb)scua_2NaggcuUfGfGfuuuucuuacus(invAb) 1311 CU(A2N)AGGCUUGGUUUUCUUACU 1633
    AM14284-SS (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312 CUCUCUCAGAGUAUUAUGGAA 1603
    AM14286-SS (NAG37)s(invAb)scucucucaGfaGfUfauuauggaas(invAb) 1313 CUCUCUCAGAGUAUUAUGGAA 1603
    AM14287-SS (NAG37)s(invAb)scagugaugCfUfCfuccaaguauas(invAb) 1314 CAGUGAUGCUCUCCAAGUAUA 1634
    AM14289-SS (NAG37)s(invAb)sgccaaguaUfGfAfucgucuicaas(invAb) 1315 GCCAAGUAUGAUCGUCUICAA 1635
    AM14291-SS (NAG37)s(invAb)suggcaugaGfAgGfuuuuauucaas(invAb) 1316 UGGCAUGAGAGUUUUAUUCAA 1636
    AM14294-SS (NAG37)s(invAb)suggcaugaGfAfGfuuuua_2Nuucaas(invAb) 1317 UGGCAUGAGAGUUUU(A2N)UUCAA 1367
    AM14295-SS (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguas(invAb) 1318 CCUCAGCUUCUUCUUCAAGUA 1638
    AM14300-SS (NAG37)s(invAb)sccucagcuUfCfUfucuuuaaguas(invAb) 1319 CCUCAGCUUCUUCUUUAAGUA 1639
    AM14302-SS (NAG37)s(invAb)sccucagcuUfcUfUfcuucaaguas(invAb) 1320 CCUCAGCUUCUUCUUCAAGUA 1638
    AM14303-SS (NAG37)s(invAb)sccucagcuUfcUfuCfuucaaguas(invAb) 1321 CCUCAGCUUCUUCUUCAAGUA 1638
    AM14386-SS (NAG37)s(invAb)sgaguauuuCfuCfAfgcauucaaas(invAb) 1322 GAGUAUUUCUCAGCAUUCAAA 1593
    AM14390-SS (NAG37)s(invAb)sgacaagauCfGfUfccacuuuucus(invAb) 1323 GACAAGAUCGUCCACUUUUCU 1640
    AM14392-SS (NAG37)s(invAb)saccauguuGfCfAfgugacaacuas(invAb) 1324 ACCAUGUUGCAGUGACAACUA 1641
    AM14394-SS (NAG37)s(invAb)sggugacaaCfUfGfuggaaggaaus(invAb) 1325 GGUGACAACUGUGGAAGGAAU 1642
    AM14396-SS (NAG37)s(invAb)sguggaggaGfAfUfugagaaugcas(invAb) 1326 GUGGAGGAGAUUGAGAAUGCA 1643
    AM14398-SS (NAG37)s(invAb)sgacacggaGfAfUfuggcauugaas(invAb) 1327 GACACGGAGAUUGGCAUUGAA 1644
    AM14400-SS (NAG37)s(invAb)scgagaugaAgGfUfucaagaaua_2Nus(invAb) 1328 CGAGAUGAAGUUCAAGAAU(A2N)U 1645
    AM14402-SS (NAG37)s(invAb)saggagauaCfUfGfcucuccauaas(invAb) 1329 AGGAGAUACUGCUCUCCAUAA 1646
    AM14404-SS (NAG37)s(invAb)sggggaguaUfUfUfcucagcauuas(invAb) 1330 GGGGAGUAUUUCUCAGCAUUA 1647
    AM14406-SS (NAG37)s(invAb)sgggaguauUfUfCfucagcauucas(invAb) 1331 GGGAGUAUUUCUCAGCAUUCA 1648
    AM14408-SS (NAG37)s(invAb)sccggagagAgAgGfaugacauugas(invAb) 1332 CCGGAGAGAAGAUGACAUUGA 1649
    AM14410-SS (NAG37)s(invAb)sgguaacauAgAgCguggaauuugus(invAb) 1333 GGUAACAUAACUGGAAUUUGU 1650
    AM14412-SS (NAG37)s(invAb)sccagccauUfAfUfcacaauugaas(invAb) 1334 CCAGCCAUUAUCACAAUUGAA 1651
    AM14414-SS (NAG37)s(invAb)sgagcuuugUfUfGfcaaaaauguus(invAb) 1335 GAGCUUUGUUGCAAAAAUGUU 1652
    AM14416-SS (NAG37)s(invAb)sgcuuuguuGfCfAfaaaauguugas(invAb) 1336 GCUUUGUUGCAAAAAUGUUGA 1653
    AM14418-SS (NAG37)s(invAb)scggauuguGfGfUfucgagugaaas(invAb) 1337 CGGAUUGUGGUUCGAGUGAAA 1654
    AM14525-SS (NAG37)s(invAb)scccaccaaGfuUfuGfgaauaagas(invAb) 1338 CCCACCAAGUUUGGAAUAAGA 1531
    AM14526-SS (NAG37)s(invAb)scccaccaaGfuUfUfggaauaagas(invAb) 1339 CCCACCAAGUUUGGAAUAAGA 1531
    AM14528-SS (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340 UGCCUAAAUGGUGAAUAUGCA 1551
    AM14531-SS (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341 UGCCUAAAUGGUGAAUAUGCA 1551
    AM14646-SS (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342 UCCAGGGUUUGUUUGUUUCAU 1595
    AM15136-SS (NAG37)s(invAb)sgucucucaGfaGfuAfuuauggaas(invAb) 1343 GUCUCUCAGAGUAUUAUGGAA 1655
    AM15138-SS (NAG37)s(invAb)scccucucaGfaGfuAfuuauggaas(invAb) 1344 CCCUCUCAGAGUAUUAUGGAA 1656
    AM15140-SS (NAG37)s(invAb)sgccucucaGfaGfuAfuuauggaas(invAb) 1345 GCCUCUCAGAGUAUUAUGGAA 1657
    AM15142-SS (NAG37)s(invAb)saccucucaGfaGfuAfuuauggaas(invAb) 1346 ACCUCUCAGAGUAUUAUGGAA 1658
    AM15144-SS (NAG37)s(invAb)succucucaGfaGfuAfuuauggaas(invAb) 1347 UCCUCUCAGAGUAUUAUGGAA 1659
    AM15284-SS (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348 CCUCCGCACAGAUAUUGUCAU 1623
    AM15288-SS (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaas(invAb) 1349 CCUCCGCACAGAUAUUGUCAA 1660
    AM15293-SS (NAG37)s(invAb)scuccgcaCfaGfaUfauugucaus(invAb) 1350 CUCCGCACAGAUAUUGUCAU 1661
    AM15295-SS (NAG37)s(invAb)succgcaCfaGfaUfauugucaus(invAb) 1351 UCCGCACAGAUAUUGUCAU 1662
    AM17242-SS (NAG37)suscagagUfaUfUfAfuggaacgagus(invAb) 1676 UCAGAGUAUUAUGGAACGAGU 1678
    AM17244-SS (NAG37)scsucucuCfaGfAfGfuauuauggaus(invAb) 1677 CUCUCUCAGAGUAUUAUGGAU 1679
    (A2N) = 2-aminoadenine nucleotide;
    I = hypoxanthine(inosine)nucleotide
  • The XDH RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, or Table 5C can be hybridized to any antisense strand containing a sequence listed in Table 2, Table 3, or Table 5C provided the two sequences have a region of at least 85% complementarity over a contiguous 15, 16, 17, 18, 19, 20, or 21 nucleotide sequence.
  • In some aspects, the antisense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3 or Table 5C. In some aspects, the sense strand of an XDH RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4 or Table 5C.
  • In some aspects, an XDH RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2, Table 3, or Table 5C. In some aspects, an XDH RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end→3′ end) at positions 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, of any of the sequences in Table 2, Table 3, or Table 5C. In certain aspects, an XDH RNAi agent antisense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 3 or Table 5C.
  • In some aspects, an XDH RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2, Table 4, or Table 5C. In some aspects, an XDH RNAi agent sense strand comprises the sequence of nucleotides (from 5′ end→3′ end) at positions 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, or 4-21, of any of the sequences in Table 2, Table 4, or Table 5C. In certain aspects, an XDH RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4 or Table 5C.
  • For the XDH RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) can be perfectly complementary to an XDH gene, or can be non-complementary to an XDH gene. In some aspects, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) is a U, A, or dT (or a modified version thereof). In some aspects, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair with the sense strand.
  • A sense strand containing a sequence listed in Table 2, Table 4, or Table 5C can be hybridized to any antisense strand containing a sequence listed in Table 2. Table 3, or Table 5C, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some aspects, the XDH RNAi agent has a sense strand consisting of the modified sequence of any of the modified sequences in Table 4 or Table 5C, and an antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 5C. Certain representative sequence pairings are exemplified by the Duplex ID Nos, shown in Tables 5A, 5B, and 5C.
  • In some aspects, an XDH RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the Duplex ID Nos. presented herein. In some aspects, an XDH RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the duplexes represented by any of the Duplex ID NOs. presented herein. In some aspects, an XDH RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the duplexes represented by any of the Duplex ID NOs. presented herein and a targeting group and/or linking group wherein the targeting group and/or linking group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some aspects, an XDH RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID NOs. presented herein. In some aspects, an XDH RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID NOs. presented herein and a targeting group and/or linking group, wherein the targeting group and/or linking group is covalently linked to the sense strand or the antisense strand.
  • In some aspects, an XDH RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Table 2 or Tables 5A, 5B, and 5C, and further comprises a targeting group or targeting ligand. In some aspects, an XDH RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Table 2 or Tables 5A, 5B, and 5C, and further comprises an asialoglycoprotein receptor ligand targeting group.
  • A targeting group, with or without a linker, can be linked to the 5′ or 3′ end of any of the sense and/or antisense strands disclosed in Tables 2, 3, 4, or 5C. A linker, with or without a targeting group, can be attached to the 5′ or 3′ end of any of the sense and/or antisense strands disclosed in Tables 2, 3, 4, and 5C.
  • In some aspects, an XDH RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Table 2 or Tables 5A, 5B and 5C, and further comprises a targeting ligand selected from the group consisting of: (NAG37) and (NAG37)s, each as defined in Table 6.
  • In some aspects, an XDH RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequence of any of the antisense strand and/or sense strand nucleotide sequences in Table 3 or Table 4.
  • In some aspects, an XDH RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand and/or sense strand nucleotide sequences of any of the duplexes Tables 5A, 5B, and 5C, and further comprises an asialoglycoprotein receptor ligand targeting group.
  • In some aspects, an XDH RNAi agent comprises, consists of, or consists essentially ofany of the duplexes of Tables 5A, 5B, and 5C.
  • TABLE 5A
    XDH RNAi Agents Duplexes with Corresponding Sense and Antisense Strand ID
    Numbers and Sequence ID numbers for the modified and unmodified nucleotide sequences.
    AS AS SS SS
    modified unmodified modified unmodified
    SEQ ID SEQ ID SEQ ID SEQ ID
    Duplex AS ID NO: NO: SS ID NO: NO:
    AD09217 AM13029-AS 945 1352 AM13028-SS 1175 1506
    AD09218 AM13031-AS 946 1352 AM13030-SS 1176 1506
    AD09219 AM13033-AS 947 1353 AM13032-SS 1177 1507
    AD09220 AM13035-AS 948 1354 AM13034-SS 1178 1508
    AD09221 AM13037-AS 949 1355 AM13036-SS 1179 1509
    AD09222 AM13039-AS 950 1356 AM13038-SS 1180 1510
    AD09223 AM13041-AS 951 1357 AM13040-SS 1181 1511
    AD09224 AM13043-AS 952 1358 AM13042-SS 1182 1512
    AD09225 AM13045-AS 953 1359 AM13044-SS 1183 1513
    AD09226 AM13047-AS 954 1360 AM13046-SS 1184 1514
    AD09227 AM13049-AS 955 1361 AM13048-SS 1185 1515
    AD09228 AM13051-AS 956 1362 AM13050-SS 1186 1516
    AD09229 AM13053-AS 957 1363 AM13052-SS 1187 1517
    AD09230 AM13055-AS 958 1364 AM13054-SS 1188 1518
    AD09231 AM13057-AS 959 1365 AM13056-SS 1189 1519
    AD09232 AM13059-AS 960 1366 AM13058-SS 1190 1520
    AD09233 AM13061-AS 961 1367 AM13060-SS 1191 1521
    AD09234 AM13063-AS 962 1368 AM13062-SS 1192 1522
    AD09235 AM13065-AS 963 1369 AM13064-SS 1193 1523
    AD09236 AM13067-AS 964 1370 AM13066-SS 1194 1524
    AD09237 AM13069-AS 965 1371 AM13068-SS 1195 1525
    AD09238 AM13071-AS 966 1372 AM13070-SS 1196 1526
    AD09239 AM13073-AS 967 1373 AM13072-SS 1197 1527
    AD09302 AM13164-AS 968 1374 AM13163-SS 1198 1528
    AD09303 AM13166-AS 969 1375 AM13165-SS 1199 1529
    AD09304 AM13168-AS 970 1376 AM13167-SS 1200 1530
    AD09305 AM13170-AS 971 1377 AM13169-SS 1201 1531
    AD09306 AM13172-AS 972 1378 AM13171-SS 1202 1532
    AD09307 AM13174-AS 973 1379 AM13173-SS 1203 1533
    AD09308 AM13176-AS 974 1374 AM13175-SS 1204 1528
    AD09309 AM13177-AS 975 1375 AM13165-SS 1199 1529
    AD09310 AM13179-AS 976 1376 AM13178-SS 1205 1530
    AD09311 AM13181-AS 977 1380 AM13180-SS 1206 1534
    AD09323 AM13204-AS 978 1374 AM13163-SS 1198 1528
    AD09324 AM13205-AS 979 1376 AM13167-SS 1200 1530
    AD09325 AM13206-AS 980 1377 AM13169-SS 1201 1531
    AD09326 AM13207-AS 981 1378 AM13171-SS 1202 1532
    AD09571 AM13600-AS 982 1381 AM13599-SS 1207 1535
    AD09572 AM13602-AS 983 1382 AM13601-SS 1208 1536
    AD09573 AM13604-AS 984 1383 AM13603-SS 1209 1537
    AD09598 AM13648-AS 985 1384 AM13647-SS 1210 1538
    AD09599 AM13650-AS 986 1385 AM13649-SS 1211 1539
    AD09600 AM13652-AS 987 1386 AM13651-SS 1212 1540
    AD09601 AM13654-AS 988 1387 AM13653-SS 1213 1541
    AD09602 AM13656-AS 989 1388 AM13655-SS 1214 1542
    AD09603 AM13658-AS 990 1389 AM13657-SS 1215 1543
    AD09604 AM13660-AS 991 1390 AM13659-SS 1216 1544
    AD09605 AM13662-AS 992 1391 AM13661-SS 1217 1545
    AD09606 AM13664-AS 993 1392 AM13663-SS 1218 1546
    AD09607 AM13666-AS 994 1393 AM13665-SS 1219 1547
    AD09608 AM13668-AS 995 1394 AM13667-SS 1220 1548
    AD09609 AM13670-AS 996 1395 AM13669-SS 1221 1549
    AD09610 AM13672-AS 997 1396 AM13671-SS 1222 1550
    AD09611 AM13674-AS 998 1397 AM13673-SS 1223 1551
    AD09612 AM13676-AS 999 1398 AM13675-SS 1224 1552
    AD09613 AM13678-AS 1000 1399 AM13677-SS 1225 1553
    AD09614 AM13680-AS 1001 1400 AM13679-SS 1226 1554
    AD09615 AM13682-AS 1002 1401 AM13681-SS 1227 1555
    AD09616 AM13684-AS 1003 1402 AM13683-SS 1228 1556
    AD09617 AM13686-AS 1004 1403 AM13685-SS 1229 1557
    AD09618 AM13688-AS 1005 1404 AM13687-SS 1230 1558
    AD09619 AM13690-AS 1006 1405 AM13689-SS 1231 1559
    AD09620 AM13692-AS 1007 1406 AM13691-SS 1232 1560
    AD09621 AM13694-AS 1008 1407 AM13693-SS 1233 1561
    AD09623 AM13696-AS 1009 1408 AM13695-SS 1234 1262
    AD09624 AM13698-AS 1010 1409 AM13697-SS 1235 1563
    AD09625 AM13700-AS 1011 1410 AM13699-SS 1236 1564
    AD09626 AM13702-AS 1012 1411 AM13701-SS 1237 1565
    AD09627 AM13704-AS 1013 1412 AM13703-SS 1238 1566
    AD09628 AM13706-AS 1014 1413 AM13705-SS 1239 1567
    AD09629 AM13708-AS 1015 1414 AM13707-SS 1240 1568
    AD09630 AM13710-AS 1016 1415 AM13709-SS 1241 1569
    AD09631 AM13712-AS 1017 1416 AM13711-SS 1242 1570
    AD09632 AM13714-AS 1018 1417 AM13713-SS 1243 1571
    AD09633 AM13716-AS 1019 1418 AM13715-SS 1244 1572
    AD09634 AM13718-AS 1020 1419 AM13717-SS 1245 1573
    AD09635 AM13720-AS 1021 1420 AM13719-SS 1246 1574
    AD09636 AM13722-AS 1022 1421 AM13721-SS 1247 1575
    AD09637 AM13724-AS 1023 1422 AM13723-SS 1248 1576
    AD09638 AM13726-AS 1024 1423 AM13725-SS 1249 1577
    AD09639 AM13728-AS 1025 1424 AM13727-SS 1250 1578
    AD09640 AM13730-AS 1026 1425 AM13729-SS 1251 1579
    AD09650 AM13747-AS 1027 1366 AM13746-SS 1252 1520
    AD09651 AM13748-AS 1028 1366 AM13746-SS 1252 1520
    AD09652 AM13749-AS 1029 1366 AM13746-SS 1252 1520
    AD09653 AM13748-AS 1028 1366 AM13750-SS 1253 1580
    AD09654 AM13748-AS 1028 1366 AM13751-SS 1254 1581
    AD09655 AM13748-AS 1028 1366 AM13752-SS 1255 1582
    AD09656 AM13753-AS 1030 1426 AM13746-SS 1252 1520
    AD09657 AM13754-AS 1031 1427 AM13746-SS 1252 1520
    AD09658 AM13755-AS 1032 1366 AM13746-SS 1252 1520
    AD09659 AM13748-AS 1028 1366 AM13058-SS 1190 1520
    AD09660 AM13748-AS 1028 1366 AM13756-SS 1256 1520
    AD09661 AM13748-AS 1028 1366 AM13757-SS 1257 1520
    AD09662 AM13758-AS 1028 1366 AM13060-SS 1191 1521
    AD09663 AM13759-AS 1034 1367 AM13060-SS 1191 1521
    AD09664 AM13758-AS 1033 1367 AM13760-SS 1258 1521
    AD09665 AM13761-AS 1035 1367 AM13760-SS 1258 1521
    AD09724 AM13858-AS 1036 1428 AM13857-SS 1259 1583
    AD09725 AM13860-AS 1037 1429 AM13859-SS 1260 1584
    AD09726 AM13862-AS 1038 1430 AM13861-SS 1261 1585
    AD09727 AM13864-AS 1039 1431 AM13863-SS 1262 1586
    AD09728 AM13866-AS 1040 1432 AM13865-SS 1263 1587
    AD09729 AM13868-AS 1041 1433 AM13867-SS 1264 1588
    AD09730 AM13870-AS 1042 1434 AM13869-SS 1265 1589
    AD09731 AM13872-AS 1043 1435 AM13871-SS 1266 1590
    AD09732 AM13874-AS 1044 1436 AM13873-SS 1267 1591
    AD09733 AM13876-AS 1045 1437 AM13875-SS 1268 1592
    AD09734 AM13878-AS 1046 1438 AM13877-SS 1269 1593
    AD09735 AM13880-AS 1047 1439 AM13879-SS 1270 1594
    AD09736 AM13882-AS 1048 1440 AM13881-SS 1271 1595
    AD09737 AM13884-AS 1049 1441 AM13883-SS 1272 1596
    AD09738 AM13886-AS 1050 1442 AM13885-SS 1273 1597
    AD09739 AM13888-AS 1051 1443 AM13887-SS 1274 1598
    AD09740 AM13890-AS 1052 1444 AM13889-SS 1275 1599
    AD09741 AM13892-AS 1053 1445 AM13891-SS 1276 1600
    AD09742 AM13894-AS 1054 1446 AM13893-SS 1277 1601
    AD09743 AM13896-AS 1055 1447 AM13895-SS 1278 1602
    AD09744 AM13898-AS 1056 1448 AM13897-SS 1279 1603
    AD09745 AM13900-AS 1057 1449 AM13899-SS 1280 1604
    AD09937 AM14175-AS 1058 1450 AM14174-SS 1281 1605
    AD09938 AM14176-AS 1059 1448 AM13897-SS 1279 1603
    AD09962 AM14204-AS 1060 1451 AM14203-SS 1282 1606
    AD09963 AM14206-AS 1061 1452 AM14205-SS 1283 1607
    AD09964 AM14208-AS 1062 1453 AM14207-SS 1284 1608
    AD09965 AM14209-AS 1063 1450 AM14174-SS 1281 1605
    AD09966 AM14210-AS 1064 1450 AM14174-SS 1281 1605
    AD09967 AM14211-AS 1065 1450 AM14174-SS 1281 1605
    AD09968 AM14212-AS 1066 1450 AM14174-SS 1281 1605
    AD09969 AM14211-AS 1065 1450 AM14213-SS 1285 1605
    AD09970 AM14211-AS 1065 1450 AM14214-SS 1286 1605
    AD09971 AM14216-AS 1067 1454 AM14215-SS 1287 1609
    AD09972 AM14218-AS 1068 1455 AM14217-SS 1288 1610
    AD09973 AM14220-AS 1069 1456 AM14219-SS 1289 1611
    AD09974 AM14222-AS 1070 1457 AM14221-SS 1290 1612
    AD09975 AM14224-AS 1071 1458 AM14223-SS 1291 1613
    AD09976 AM14226-AS 1072 1459 AM14225-SS 1292 1614
    AD09977 AM14228-AS 1073 1460 AM14227-SS 1293 1615
    AD09978 AM14230-AS 1074 1461 AM14229-SS 1294 1616
    AD09979 AM14232-AS 1075 1462 AM14231-SS 1295 1617
    AD09980 AM14234-AS 1076 1463 AM14233-SS 1296 1618
    AD09981 AM14236-AS 1077 1464 AM14235-SS 1297 1619
    AD09982 AM14238-AS 1078 1465 AM14237-SS 1298 1620
    AD09983 AM14240-AS 1079 1466 AM14239-SS 1299 1621
    AD09984 AM14242-AS 1080 1467 AM14241-SS 1300 1622
    AD09985 AM14244-AS 1081 1468 AM14243-SS 1301 1623
    AD09986 AM14246-AS 1082 1469 AM14245-SS 1302 1624
    AD09987 AM14248-AS 1083 1470 AM14247-SS 1303 1625
    AD09988 AM14250-AS 1084 1471 AM14249-SS 1304 1626
    AD09989 AM14252-AS 1085 1472 AM14251-SS 1305 1627
    AD09990 AM14254-AS 1086 1473 AM14253-SS 1306 1628
    AD09991 AM14256-AS 1087 1474 AM14255-SS 1307 1629
    AD09992 AM14258-AS 1088 1475 AM14257-SS 1308 1630
    AD09993 AM14260-AS 1089 1476 AM14259-SS 1309 1631
    AD09994 AM14262-AS 1090 1477 AM14261-SS 1310 1632
    AD09995 AM14264-AS 1091 1478 AM14263-SS 1311 1633
    AD10008 AM14280-AS 1092 1448 AM13897-SS 1279 1603
    AD10009 AM14281-AS 1093 1448 AM13897-SS 1279 1603
    AD10010 AM14282-AS 1094 1448 AM13897-SS 1279 1603
    AD10011 AM14283-AS 1095 1448 AM13897-SS 1279 1603
    AD10012 AM14282-AS 1094 1448 AM14284-SS 1312 1603
    AD10013 AM14285-AS 1096 1448 AM14284-SS 1312 1603
    AD10014 AM14282-AS 1094 1448 AM14286-SS 1313 1603
    AD10015 AM14285-AS 1096 1448 AM14286-SS 1313 1603
    AD10016 AM14288-AS 1097 1479 AM14287-SS 1314 1634
    AD10017 AM14290-AS 1098 1480 AM14289-SS 1315 1635
    AD10018 AM14292-AS 1099 1481 AM14291-SS 1316 1636
    AD10019 AM14293-AS 1100 1482 AM14291-SS 1316 1636
    AD10020 AM14292-AS 1099 1481 AM14294-SS 1317 1637
    AD10021 AM14296-AS 1101 1482 AM14295-SS 1318 1638
    AD10022 AM14297-AS 1102 1482 AM14295-SS 1318 1638
    AD10023 AM14298-AS 1103 1482 AM14295-SS 1318 1638
    AD10024 AM14299-AS 1104 1482 AM14295-SS 1318 1638
    AD10025 AM14299-AS 1104 1482 AM14300-SS 1319 1639
    AD10026 AM14301-AS 1105 1482 AM14295-SS 1318 1638
    AD10027 AM14299-AS 1104 1482 AM14302-SS 1320 1638
    AD10028 AM14299-AS 1104 1482 AM14303-SS 1321 1638
    AD10029 AM14304-AS 1106 1482 AM14303-SS 1321 1638
    AD10030 AM14305-AS 1107 1482 AM14302-SS 1320 1638
    AD10091 AM14383-AS 1108 1438 AM13877-SS 1269 1593
    AD10092 AM14384-AS 1109 1438 AM13877-SS 1269 1593
    AD10093 AM14385-AS 1110 1438 AM13877-SS 1269 1593
    AD10094 AM14384-AS 1109 1438 AM14386-SS 1322 1593
    AD10095 AM14385-AS 1110 1438 AM14386-SS 1322 1593
    AD10096 AM14387-AS 1111 1438 AM13877-SS 1269 1593
    AD10097 AM14388-AS 1112 1438 AM13877-SS 1269 1593
    AD10099 AM14391-AS 1113 1483 AM14390-SS 1323 1640
    AD10100 AM14393-AS 1114 1484 AM14392-SS 1324 1641
    AD10101 AM14395-AS 1115 1485 AM14394-SS 1325 1642
    AD10102 AM14397-AS 1116 1486 AM14396-SS 1326 1643
    AD10103 AM14399-AS 1117 1487 AM14398-SS 1327 1644
    AD10104 AM14401-AS 1118 1488 AM14400-SS 1328 1645
    AD10105 AM14403-AS 1119 1489 AM14402-SS 1329 1646
    AD10106 AM14405-AS 1120 1490 AM14404-SS 1330 1647
    AD10107 AM14407-AS 1121 1491 AM14406-SS 1331 1648
    AD10108 AM14409-AS 1122 1492 AM14408-SS 1332 1649
    AD10109 AM14411-AS 1123 1493 AM14410-SS 1333 1650
    AD10110 AM14413-AS 1124 1494 AM14412-SS 1334 1651
    AD10111 AM14415-AS 1125 1495 AM14414-SS 1335 1652
    AD10112 AM14417-AS 1126 1496 AM14416-SS 1336 1653
    AD10113 AM14419-AS 1127 1497 AM14418-SS 1337 1654
    AD10176 AM14522-AS 1128 1377 AM13169-SS 1201 1531
    AD10177 AM14523-AS 1129 1377 AM13169-SS 1201 1531
    AD10178 AM14524-AS 1130 1377 AM13169-SS 1201 1531
    AD10179 AM14524-AS 1130 1377 AM14525-SS 1338 1531
    AD10180 AM14524-AS 1130 1377 AM14526-SS 1339 1531
    AD10181 AM14527-AS 1131 1397 AM13673-SS 1223 1551
    AD10182 AM14529-AS 1132 1397 AM14528-SS 1340 1551
    AD10183 AM14530-AS 1133 1397 AM14528-SS 1340 1551
    AD10184 AM14529-AS 1132 1397 AM14531-SS 1341 1551
    AD10200 AM14543-AS 1134 1397 AM13673-SS 1223 1551
    AD10201 AM14544-AS 1135 1397 AM13673-SS 1223 1551
    AD10202 AM14545-AS 1136 1397 AM13673-SS 1223 1551
    AD10203 AM14544-AS 1135 1397 AM14528-SS 1340 1551
    AD10204 AM14545-AS 1136 1397 AM14528-SS 1340 1551
    AD10205 AM14544-AS 1135 1397 AM14531-SS 1341 1551
    AD10275 AM14642-AS 1137 1440 AM13881-SS 1271 1595
    AD10276 AM14643-AS 1138 1440 AM13881-SS 1271 1595
    AD10277 AM14644-AS 1139 1440 AM13881-SS 1271 1595
    AD10278 AM14645-AS 1140 1440 AM13881-SS 1271 1595
    AD10279 AM14644-AS 1139 1440 AM14646-SS 1342 1595
    AD10280 AM14647-AS 1141 1440 AM14646-SS 1342 1595
    AD10281 AM14648-AS 1142 1440 AM14646-SS 1342 1595
    AD10282 AM14649-AS 1143 1440 AM14646-SS 1342 1595
    AD10283 AM14650-AS 1144 1440 AM14646-SS 1342 1595
    AD10619 AM14281-AS 1093 1448 AM14284-SS 1312 1603
    AD10620 AM15134-AS 1145 1448 AM14284-SS 1312 1603
    AD10621 AM15135-AS 1146 1448 AM14284-SS 1312 1603
    AD10622 AM14283-AS 1095 1448 AM14284-SS 1312 1603
    AD10623 AM15137-AS 1147 1498 AM15136-SS 1343 1655
    AD10624 AM15139-AS 1148 1499 AM15138-SS 1344 1656
    AD10625 AM15141-AS 1149 1500 AM15140-SS 1345 1657
    AD10626 AM15143-AS 1150 1501 AM15142-SS 1346 1658
    AD10627 AM15145-AS 1151 1502 AM15144-SS 1347 1659
    AD10628 AM15146-AS 1152 1448 AM14284-SS 1312 1603
    AD10629 AM15147-AS 1153 1397 AM14528-SS 1340 1551
    AD10630 AM15148-AS 1154 1397 AM14528-SS 1340 1551
    AD10631 AM15149-AS 1155 1397 AM14528-SS 1340 1551
    AD10632 AM15150-AS 1156 1397 AM14528-SS 1340 1551
    AD10633 AM15151-AS 1157 1397 AM14531-SS 1341 1551
    AD10634 AM15152-AS 1158 1397 AM14531-SS 1341 1551
    AD10635 AM15153-AS 1159 1397 AM14531-SS 1341 1551
    AD10636 AM15154-AS 1160 1397 AM14531-SS 1341 1551
    AD10728 AM14244-AS 1081 1468 AM15284-SS 1348 1623
    AD10729 AM15285-AS 1161 1468 AM15284-SS 1348 1623
    AD10730 AM15286-AS 1162 1468 AM15284-SS 1348 1623
    AD10731 AM15287-AS 1163 1468 AM15284-SS 1348 1623
    AD10732 AM15289-AS 1164 1503 AM15288-SS 1349 1660
    AD10733 AM15290-AS 1165 1468 AM15284-SS 1348 1623
    AD10734 AM15291-AS 1166 1468 AM15284-SS 1348 1623
    AD10735 AM15292-AS 1167 1468 AM15284-SS 1348 1623
    AD10736 AM15294-AS 1168 1504 AM15293-SS 1350 1661
    AD10737 AM15296-AS 1169 1505 AM15295-SS 1351 1662
    AD10952 AM15606-AS 1170 1448 AM14284-SS 1312 1603
    AD10953 AM15607-AS 1171 1498 AM15136-SS 1343 1655
    AD10954 AM15608-AS 1172 1450 AM14213-SS 1285 1605
    AD10967 AM13882-AS 1048 1440 AM14646-SS 1342 1595
    AD10968 AM15626-AS 1173 1440 AM14646-SS 1342 1595
    AD10969 AM15627-AS 1174 1440 AM14646-SS 1342 1595
    AD12167 AM17243-AS 1672 1674 AM17242-SS 1676 1678
    AD12168 AM17245-AS 1673 1675 AM17244-SS 1677 1679
  • TABLE 5B
    XDH RNAi Agents Duplexes with Corresponding
    Sense and Antisense Strand ID Numbers Referencing
    Position Targeted on XDH Gene (SEQ ID NO: 1)
    Antisense Targeted XDH
    Duplex Strand Sense Gene Position
    ID ID Strand ID (Of SEQ ID NO:1)
    AD09217 AM13029-AS AM13028-SS 488
    AD09218 AM13031-AS AM13030-SS 488
    AD09219 AM13033-AS AM13032-SS 1612
    AD09220 AM13035-AS AM13034-SS 1614
    AD09221 AM13037-AS AM13036-SS 1617
    AD09222 AM13039-AS AM13038-SS 2128
    AD09223 AM13041-AS AM13040-SS 2130
    AD09224 AM13043-AS AM13042-SS 2131
    AD09225 AM13045-AS AM13044-SS 2132
    AD09226 AM13047-AS AM13046-SS 2153
    AD09227 AM13049-AS AM13048-SS 2185
    AD09228 AM13051-AS AM13050-SS 2186
    AD09229 AM13053-AS AM13052-SS 3272
    AD09230 AM13055-AS AM13054-SS 435
    AD09231 AM13057-AS AM13056-SS 2571
    AD09232 AM13059-AS AM13058-SS 2612
    AD09233 AM13061-AS AM13060-SS 2616
    AD09234 AM13063-AS AM13062-SS 2617
    AD09235 AM13065-AS AM13064-SS 2619
    AD09236 AM13067-AS AM13066-SS 3045
    AD09237 AM13069-AS AM13068-SS 3548
    AD09238 AM13071-AS AM13070-SS 3551
    AD09239 AM13073-AS AM13072-SS 3640
    AD09302 AM13164-AS AM13163-SS 265
    AD09303 AM13166-AS AM13165-SS 2248
    AD09304 AM13168-AS AM13167-SS 2694
    AD09305 AM13170-AS AM13169-SS 3083
    AD09306 AM13172-AS AM13171-SS 4665
    AD09307 AM13174-AS AM13173-SS 4725
    AD09308 AM13176-AS AM13175-SS 265
    AD09309 AM13177-AS AM13165-SS 2248
    AD09310 AM13179-AS AM13178-SS 2694
    AD09311 AM13181-AS AM13180-SS 4725
    AD09323 AM13204-AS AM13163-SS 265
    AD09324 AM13205-AS AM13167-SS 2694
    AD09325 AM13206-AS AM13169-SS 3083
    AD09326 AM13207-AS AM13171-SS 4665
    AD09571 AM13600-AS AM13599-SS 2850
    AD09572 AM13602-AS AM13601-SS 2851
    AD09573 AM13604-AS AM13603-SS 2852
    AD09598 AM13648-AS AM13647-SS 235
    AD09599 AM13650-AS AM13649-SS 249
    AD09600 AM13652-AS AM13651-SS 252
    AD09601 AM13654-AS AM13653-SS 1703
    AD09602 AM13656-AS AM13655-SS 2049
    AD09603 AM13658-AS AM13657-SS 2155
    AD09604 AM13660-AS AM13659-SS 2997
    AD09605 AM13662-AS AM13661-SS 3019
    AD09606 AM13664-AS AM13663-SS 3020
    AD09607 AM13666-AS AM13665-SS 3037
    AD09608 AM13668-AS AM13667-SS 4136
    AD09609 AM13670-AS AM13669-SS 4149
    AD09610 AM13672-AS AM13671-SS 4150
    AD09611 AM13674-AS AM13673-SS 4289
    AD09612 AM13676-AS AM13675-SS 4446
    AD09613 AM13678-AS AM13677-SS 4505
    AD09614 AM13680-AS AM13679-SS 4515
    AD09615 AM13682-AS AM13681-SS 4517
    AD09616 AM13684-AS AM13683-SS 4518
    AD09617 AM13686-AS AM13685-SS 4520
    AD09618 AM13688-AS AM13687-SS 4525
    AD09619 AM13690-AS AM13689-SS 4700
    AD09620 AM13692-AS AM13691-SS 5286
    AD09621 AM13694-AS AM13693-SS 5420
    AD09623 AM13696-AS AM13695-SS N/A (mouse-
    specific RNAi agent)
    AD09624 AM13698-AS AM13697-SS N/A (mouse-
    specific RNAi agent)
    AD09625 AM13700-AS AM13699-SS N/A (mouse-
    specific RNAi agent)
    AD09626 AM13702-AS AM13701-SS N/A (mouse-
    specific RNAi agent)
    AD09627 AM13704-AS AM13703-SS N/A (mouse-
    specific RNAi agent)
    AD09628 AM13706-AS AM13705-SS N/A (mouse-
    specific RNAi agent)
    AD09629 AM13708-AS AM13707-SS N/A (mouse-
    specific RNAi agent)
    AD09630 AM13710-AS AM13709-SS N/A (mouse-
    specific RNAi agent)
    AD09631 AM13712-AS AM13711-SS N/A (mouse-
    specific RNAi agent)
    AD09632 AM13714-AS AM13713-SS N/A (mouse-
    specific RNAi agent)
    AD09633 AM13716-AS AM13715-SS N/A (mouse-
    specific RNAi agent)
    AD09634 AM13718-AS AM13717-SS N/A (mouse-
    specific RNAi agent)
    AD09635 AM13720-AS AM13719-SS N/A (mouse-
    specific RNAi agent)
    AD09636 AM13722-AS AM13721-SS N/A (mouse-
    specific RNAi agent)
    AD09637 AM13724-AS AM13723-SS N/A (mouse-
    specific RNAi agent)
    AD09638 AM13726-AS AM13725-SS N/A (mouse-
    specific RNAi agent)
    AD09639 AM13728-AS AM13727-SS N/A (mouse-
    specific RNAi agent)
    AD09640 AM13730-AS AM13729-SS N/A (mouse-
    specific RNAi agent)
    AD09650 AM13747-AS AM13746-SS 2612
    AD09651 AM13748-AS AM13746-SS 2612
    AD09652 AM13749-AS AM13746-SS 2612
    AD09653 AM13748-AS AM13750-SS 2612
    AD09654 AM13748-AS AM13751-SS 2612
    AD09655 AM13748-AS AM13752-SS 2612
    AD09656 AM13753-AS AM13746-SS 2612
    AD09657 AM13754-AS AM13746-SS 2612
    AD09658 AM13755-AS AM13746-SS 2612
    AD09659 AM13748-AS AM13058-SS 2612
    AD09660 AM13748-AS AM13756-SS 2612
    AD09661 AM13748-AS AM13757-SS 2612
    AD09662 AM13758-AS AM13060-SS 2616
    AD09663 AM13759-AS AM13060-SS 2616
    AD09664 AM13758-AS AM13760-SS 2616
    AD09665 AM13761-AS AM13760-SS 2616
    AD09724 AM13858-AS AM13857-SS 122
    AD09725 AM13860-AS AM13859-SS 139
    AD09726 AM13862-AS AM13861-SS 239
    AD09727 AM13864-AS AM13863-SS 332
    AD09728 AM13866-AS AM13865-SS 430
    AD09729 AM13868-AS AM13867-SS 500
    AD09730 AM13870-AS AM13869-SS 867
    AD09731 AM13872-AS AM13871-SS 877
    AD09732 AM13874-AS AM13873-SS 888
    AD09733 AM13876-AS AM13875-SS 1285
    AD09734 AM13878-AS AM13877-SS 1322
    AD09735 AM13880-AS AM13879-SS 1921
    AD09736 AM13882-AS AM13881-SS 1963
    AD09737 AM13884-AS AM13883-SS 2138
    AD09738 AM13886-AS AM13885-SS 2148
    AD09739 AM13888-AS AM13887-SS 2157
    AD09740 AM13890-AS AM13889-SS 2209
    AD09741 AM13892-AS AM13891-SS 2320
    AD09742 AM13894-AS AM13893-SS 2357
    AD09743 AM13896-AS AM13895-SS 2361
    AD09744 AM13898-AS AM13897-SS 2696
    AD09745 AM13900-AS AM13899-SS 2701
    AD09937 AM14175-AS AM14174-SS 1963
    AD09938 AM14176-AS AM13897-SS 2696
    AD09962 AM14204-AS AM14203-SS 1964
    AD09963 AM14206-AS AM14205-SS 1965
    AD09964 AM14208-AS AM14207-SS 1967
    AD09965 AM14209-AS AM14174-SS 1963
    AD09966 AM14210-AS AM14174-SS 1963
    AD09967 AM14211-AS AM14174-SS 1963
    AD09968 AM14212-AS AM14174-SS 1963
    AD09969 AM14211-AS AM14213-SS 1963
    AD09970 AM14211-AS AM14214-SS 1963
    AD09971 AM14216-AS AM14215-SS 238
    AD09972 AM14218-AS AM14217-SS 484
    AD09973 AM14220-AS AM14219-SS 493
    AD09974 AM14222-AS AM14221-SS 497
    AD09975 AM14224-AS AM14223-SS 886
    AD09976 AM14226-AS AM14225-SS 1117
    AD09977 AM14228-AS AM14227-SS 1615
    AD09978 AM14230-AS AM14229-SS 2064
    AD09979 AM14232-AS AM14231-SS 2370
    AD09980 AM14234-AS AM14233-SS 2684
    AD09981 AM14236-AS AM14235-SS 2995
    AD09982 AM14238-AS AM14237-SS 3016
    AD09983 AM14240-AS AM14239-SS 3041
    AD09984 AM14242-AS AM14241-SS 3498
    AD09985 AM14244-AS AM14243-SS 3598
    AD09986 AM14246-AS AM14245-SS 3600
    AD09987 AM14248-AS AM14247-SS 3877
    AD09988 AM14250-AS AM14249-SS 3930
    AD09989 AM14252-AS AM14251-SS 4394
    AD09990 AM14254-AS AM14253-SS 4513
    AD09991 AM14256-AS AM14255-SS 4531
    AD09992 AM14258-AS AM14257-SS 4666
    AD09993 AM14260-AS AM14259-SS 4843
    AD09994 AM14262-AS AM14261-SS 5234
    AD09995 AM14264-AS AM14263-SS 5411
    AD10008 AM14280-AS AM13897-SS 2696
    AD10009 AM14281-AS AM13897-SS 2696
    AD10010 AM14282-AS AM13897-SS 2696
    AD10011 AM14283-AS AM13897-SS 2696
    AD10012 AM14282-AS AM14284-SS 2696
    AD10013 AM14285-AS AM14284-SS 2696
    AD10014 AM14282-AS AM14286-SS 2696
    AD10015 AM14285-AS AM14286-SS 2696
    AD10016 AM14288-AS AM14287-SS 231
    AD10017 AM14290-AS AM14289-SS 242
    AD10018 AM14292-AS AM14291-SS 1384
    AD10019 AM14293-AS AM14291-SS 1384
    AD10020 AM14292-AS AM14294-SS 1384
    AD10021 AM14296-AS AM14295-SS 1612
    AD10022 AM14297-AS AM14295-SS 1612
    AD10023 AM14298-AS AM14295-SS 1612
    AD10024 AM14299-AS AM14295-SS 1612
    AD10025 AM14299-AS AM14300-SS 1612
    AD10026 AM14301-AS AM14295-SS 1612
    AD10027 AM14299-AS AM14302-SS 1612
    AD10028 AM14299-AS AM14303-SS 1612
    AD10029 AM14304-AS AM14303-SS 1612
    AD10030 AM14305-AS AM14302-SS 1612
    AD10091 AM14383-AS AM13877-SS 1322
    AD10092 AM14384-AS AM13877-SS 1322
    AD10093 AM14385-AS AM13877-SS 1322
    AD10094 AM14384-AS AM14386-SS 1322
    AD10095 AM14385-AS AM14386-SS 1322
    AD10096 AM14387-AS AM13877-SS 1322
    AD10097 AM14388-AS AM13877-SS 1322
    AD10099 AM14391-AS AM14390-SS 263
    AD10100 AM14393-AS AM14392-SS 318
    AD10101 AM14395-AS AM14394-SS 328
    AD10102 AM14397-AS AM14396-SS 482
    AD10103 AM14399-AS AM14398-SS 857
    AD10104 AM14401-AS AM14400-SS 874
    AD10105 AM14403-AS AM14402-SS 1278
    AD10106 AM14405-AS AM14404-SS 1319
    AD10107 AM14407-AS AM14406-SS 1320
    AD10108 AM14409-AS AM14408-SS 1351
    AD10109 AM14411-AS AM14410-SS 2006
    AD10110 AM14413-AS AM14412-SS 2156
    AD10111 AM14415-AS AM14414-SS 2398
    AD10112 AM14417-AS AM14416-SS 2400
    AD10113 AM14419-AS AM14418-SS 2435
    AD10176 AM14522-AS AM13169-SS 3083
    AD10177 AM14523-AS AM13169-SS 3083
    AD10178 AM14524-AS AM13169-SS 3083
    AD10179 AM14524-AS AM14525-SS 3083
    AD10180 AM14524-AS AM14526-SS 3083
    AD10181 AM14527-AS AM13673-SS 4289
    AD10182 AM14529-AS AM14528-SS 4289
    AD10183 AM14530-AS AM14528-SS 4289
    AD10184 AM14529-AS AM14531-SS 4289
    AD10200 AM14543-AS AM13673-SS 4289
    AD10201 AM14544-AS AM13673-SS 4289
    AD10202 AM14545-AS AM13673-SS 4289
    AD10203 AM14544-AS AM14528-SS 4289
    AD10204 AM14545-AS AM14528-SS 4289
    AD10205 AM14544-AS AM14531-SS 4289
    AD10275 AM14642-AS AM13881-SS 1963
    AD10276 AM14643-AS AM13881-SS 1963
    AD10277 AM14644-AS AM13881-SS 1963
    AD10278 AM14645-AS AM13881-SS 1963
    AD10279 AM14644-AS AM14646-SS 1963
    AD10280 AM14647-AS AM14646-SS 1963
    AD10281 AM14648-AS AM14646-SS 1963
    AD10282 AM14649-AS AM14646-SS 1963
    AD10283 AM14650-AS AM14646-SS 1963
    AD10619 AM14281-AS AM14284-SS 2696
    AD10620 AM15134-AS AM14284-SS 2696
    AD10621 AM15135-AS AM14284-SS 2696
    AD10622 AM14283-AS AM14284-SS 2696
    AD10623 AM15137-AS AM15136-SS 2696
    AD10624 AM15139-AS AM15138-SS 2696
    AD10625 AM15141-AS AM15140-SS 2696
    AD10626 AM15143-AS AM15142-SS 2696
    AD10627 AM15145-AS AM15144-SS 2696
    AD10628 AM15146-AS AM14284-SS 2696
    AD10629 AM15147-AS AM14528-SS 4289
    AD10630 AM15148-AS AM14528-SS 4289
    AD10631 AM15149-AS AM14528-SS 4289
    AD10632 AM15150-AS AM14528-SS 4289
    AD10633 AM15151-AS AM14531-SS 4289
    AD10634 AM15152-AS AM14531-SS 4289
    AD10635 AM15153-AS AM14531-SS 4289
    AD10636 AM15154-AS AM14531-SS 4289
    AD10728 AM14244-AS AM15284-SS 3598
    AD10729 AM15285-AS AM15284-SS 3598
    AD10730 AM15286-AS AM15284-SS 3598
    AD10731 AM15287-AS AM15284-SS 3598
    AD10732 AM15289-AS AM15288-SS 3598
    AD10733 AM15290-AS AM15284-SS 3598
    AD10734 AM15291-AS AM15284-SS 3598
    AD10735 AM15292-AS AM15284-SS 3598
    AD10736 AM15294-AS AM15293-SS 3598
    AD10737 AM15296-AS AM15295-SS 3598
    AD10952 AM15606-AS AM14284-SS 2696
    AD10953 AM15607-AS AM15136-SS 2696
    AD10954 AM15608-AS AM14213-SS 1963
    AD10967 AM13882-AS AM14646-SS 1963
    AD10968 AM15626-AS AM14646-SS 1963
    AD10969 AM15627-AS AM14646-SS 1963
    AD12167 AM17243-AS AM17242-SS 2701
    AD12168 AM17245-AS AM17244-SS 2696
  • TABLE 5C
    XDH RNAi Agent Duplexes Showing Chemically Modified Antisense Strand and Sense Strand Sequences
    Sense SEQ SEQ
    Strand Modified ID ID
    ID: Antisense Strand (5′ → 3′) NO. Modified Sense Strand (5′ → 3′) NO.
    AD09217 usUfsgsGfaAfgGfcAfuUfcUfcAfaUf 945 (NAG37)s(invAb)sgagauugaGfAfAfugccuuccaas(invAb) 1175
    cUfsc
    AD09218 usUfsggaAfgGfCfauucUfcAfaucusc 946 (NAG37)s(invAb)sgagauuGfaGfAfAfugccuuccaas(invAb) 1176
    AD09219 asAfscsUfuGfaAfgAfaGfaAfgCfuGf 947 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguus(invAb) 1177
    aGfsg
    AD09220 asGfsasAfcUfuGfaAfgAfaGfaAfgCf 948 (NAG37)s(invAb)sgcagcuucUfUfCfuucaaguucus(invAb) 1178
    uGfsc
    AD09221 usGfsusAfgAfaCfuUfgAfaGfaAfgAf 949 (NAG37)s(invAb)sgcuucuucUfUfCfaaguucuacas(invAb) 1179
    aGfsc
    AD09222 usCfsasUfaGfgUfgAfuUfuUfcAfcCf 950 (NAG37)s(invAb)saggggugaAfAfAfucaccuaugas(invAb) 1180
    cCfsu
    AD09223 usUfsusCfaUfaGfgUfgAfuUfuUfcAf 951 (NAG37)s(invAb)sgggugaaaAfUfCfaccuaugaaas(invAb) 1181
    cCfsc
    AD09224 usCfsusUfcAfuAfgGfuGfaUfuUfuCf 952 (NAG37)s(invAb)sggugaaaaUfCfAfccuaugaagas(invAb) 1182
    aCfsc
    AD09225 usUfscsUfuCfaUfaGfgUfgAfuUfuUf 953 (NAG37)s(invAb)sgugaaaauCfAfCfcuaugaagaas(invAb) 1183
    cAfsc
    AD09226 asAfsusUfgUfgAfuAfaUfgGfcUfgGf 954 (NAG37)s(invAb)scuaccagcCfAfUfuaucacaauus(invAb) 1184
    uAfsg
    AD09227 usCfsasUfaAfaAfgGfaGfuUfgUfuCf 955 (NAG37)s(invAb)sgaagaacaAfCfUfccuuuuaugas(invAb) 1185
    uUfsc
    AD09228 usCfscsAfuAfaAfaGfgAfgUfuGfuUf 956 (NAG37)s(invAb)sgagaacaaCfUfCfcuuuuauggas(invAb) 1186
    cUfsc
    AD09229 usAfscsAfgUfgUfuAfgUfgCfuUfgUf 957 (NAG37)s(invAb)sgagacaagCfAfCfuaacacuguas(invAb) 1187
    cUfsc
    AD09230 usUfsgsUfgUfaCfaUfaCfuCfaUfgAf 958 (NAG37)s(invAb)sucgucaugAfGfUfauguacacaas(invAb) 1188
    cGfsa
    AD09231 usAfscsCfaGfuUfaUfcAfgCfaUfgUf 959 (NAG37)s(invAb)saggacaugCfUfGfauaacugiuas(invAb) 1189
    cCfsu
    AD09232 usAfsusGfaAfgCfcAfaCfcUfuGfuAf 960 (NAG37)s(invAb)sggauacaaGfGfUfuggcuucauas(invAb) 1190
    uCfsc
    AD09233 usCfsusUfcAfuGfaAfgCfcAfaCfcUf 961 (NAG37)s(invAb)sgcaagguuGfGfCfuucaugaagas(invAb) 1191
    uGfsc
    AD09234 usUfscsUfuCfaUfgAfaGfcCfaAfcCf 962 (NAG37)s(invAb)scaagguugGfCfUfucaugaagaas(invAb) 1192
    uUfsg
    AD09235 usAfsgsUfcUfuCfaUfgAfaGfcCfaAf 963 (NAG37)s(invAb)sagguuggcUfUfCfaugaagacuas(invAb) 1193
    cCfsu
    AD09236 usCfsusUfuUfuCfcAfaCfaAfuUfcUf 964 (NAG37)s(invAb)saggagaauUfGfUfuggaaaaagas(invAb) 1194
    cCfsu
    AD09237 usUfscsUfaCfuUfcAfgAfgCfaAfgCf 965 (NAG37)s(invAb)sguggcuugCfUfCfugaaguagaas(invAb) 1195
    cAfsc
    AD09238 usAfsusUfuCfuAfcUfuCfaGfaGfcAf 966 (NAG37)s(invAb)sgcuugcucUfGfAfaguagaaauas(invAb) 1196
    aGfsc
    AD09239 usGfsusCfcAfaUfaUfcAfaUfgGfcAf 967 (NAG37)s(invAb)scccugccaUfUfGfauauuigacas(invAb) 1197
    gGfsg
    AD09302 usCfsasGfaAfaAfgUfgGfaCfgAfuCf 968 (NAG37)s(invAb)scaagaucgUfCfCfacuuuucugas(invAb) 1198
    uUfsg
    AD09303 asCfsasAfcAfuUfaUfcUfgCfuUfcGf 969 (NAG37)s(invAb)sguccgaagCfAfGfauaauguugus(invAb) 1199
    gAfsc
    AD09304 usCfsasUfaAfuAfcUfcUfgAfgAfgAf 970 (NAG37)s(invAb)sgucucucuCfAfGfaguauuaugas(invAb) 1200
    gAfsc
    AD09305 usCfsusUfaUfuCfcAfaAfcUfuGfgUf 971 (NAG37)s(invAb)scccaccaaGfUfUfuggaauaagas(invAb) 1201
    gGfsg
    AD09306 usAfsgsUfaAfuCfuUfgCfuUfuAfuGf 972 (NAG37)s(invAb)scugcauaaAfGfCfaagauuacuas(invAb) 1202
    cAfsg
    AD09307 asAfsasGfaAfaUfcUfaGfaAfcAfuUf 973 (NAG37)s(invAb)sgacaauguUfCfUfagauuucuuus(invAb) 1203
    gUfsc
    AD09308 usCfsasgaaaagugGfaCfgAfuCfuUfsg 974 (NAG37)s(invAb)scaagaucgUfcCfaCfuuuucugas(invAb) 1204
    AD09309 asCfsasacauUfaUfcUfgCfuUfcggasc 975 (NAG37)s(invAb)sguccgaagCfAfGfauaauguugus(invAb) 1199
    AD09310 usCfsasUfaAfuacucUfgAfgAfgagasc 976 (NAG37)s(invAb)sgucucucuCfaGfaGfuauuaugas(invAb) 1205
    AD09311 asAfsasGfaAfaUfcUfaGfaAfcAfuUfu 977 (NAG37)s(invAb)sgaaaauguUfCfUfagauuucuuus(invAb) 1206
    Ufsc
    AD09323 usCfsasGfaAfaagugGfaCfgAfuCfuU 978 (NAG37)s(invAb)scaagaucgUfCfCfacuuuucugas(invAb) 1198
    fsg
    AD09324 usCfsasUfaAfuacucUfgAfgAfgAfgA 979 (NAG37)s(invAb)sgucucucuCfAfGfaguauuaugas(invAb) 1200
    fsc
    AD09325 usCfsusUfaUfuccaaAfcUfuGfgUfg 980 (NAG37)s(invAb)scccaccaaGfUfUfuggaauaagas(invAb) 1201
    gsg
    AD09326 usAfsgsUfaAfucuugCfuUfuAfuGfc 981 (NAG37)s(invAb)scugcauaaAfGfCfaagauuacuas(invAb) 1202
    Afsg
    AD09571 usAfsasCfuUfcacucAfuCfcAfgCfa 982 (NAG37)s(invAb)sagugcuggAfUfGfagugaaguuas(invAb) 1207
    csu
    AD09572 usCfsasAfcuucacuCfaUfcCfagcasc 983 (NAG37)s(invAb)sgugcuigaUfGfAfgugaaguugas(invAb) 1208
    AD09573 usGfscsAfacuucacUfcAfuCfcagcsa 984 (NAG37)s(invAb)sugcuggauGfAfGfugaaguuicas(invAb) 1209
    AD09598 usGfsasucauacuuGfgAfgAfgcausc 985 (NAG37)s(invAb)sgaugcucuCfcAfaGfuaugaucas(invAb) 1210
    AD09599 usCfsusuguucugcAfgAfcGfaucasc 986 (NAG37)s(invAb)sgugaucguCfuGfcAfgaacaagas(invAb) 1211
    AD09600 usGfsasucuuguucUfgCfaGfacgasc 987 (NAG37)s(invAb)sgucgucugCfaGfaAfcaagaucas(invAb) 1212
    AD09601 usAfsgsuaaaguugCfaCfuGfgcgasc 988 (NAG37)s(invAb)sgucgccagUfgCfaAfcuuuacuas(invAb) 1213
    AD09602 usAfsasCfacaaguaAfcCfuUfauccsu 989 (NAG37)s(invAb)saggauaAfgGfuUfacuuguguuas(invAb) 1214
    AD09603 usCfsasAfuugugauAfaUfgGfcuggsu 990 (NAG37)s(invAb)saccagccaUfuAfuCfacaauugas(invAb) 1215
    AD09604 usAfsgscaugauacUfgAfgAfgcuusg 991 (NAG37)s(invAb)scaagcucuCfaGfuAfucaugcuas(invAb) 1216
    AD09605 asAfscsUfugucaacCfuCfaCfucuusc 992 (NAG37)s(invAb)sgaagagugAfgGfuUfgacaaguus(invAb) 1217
    AD09606 usAfsasCfuugucaaCfcUfcAfcucusc 993 (NAG37)s(invAb)sgagagugaGfGfUfugacaaguuas(invAb) 1218
    AD09607 usAfsasCfaauucucCfuUfgUfugaasc 994 (NAG37)s(invAb)sguucaacaAfGfGfagaauuguuas(invAb) 1219
    AD09608 usCfsasuguucuguGfgUfaUfguucsc 995 (NAG37)s(invAb)sggaacaUfaCfcAfcagaacaugas(invAb) 1220
    AD09609 usAfscsUfuUfaauagAfuCfcAfuguusc 996 (NAG37)s(invAb)sgaacauggAfuCfuAfuuaaaguas(invAb) 1221
    AD09610 usGfsascuuuAfaUfaGfaUfcCfaugusc 997 (NAG37)s(invAb)sgacauggaUfcUfaUfuaaagucas(invAb) 1222
    AD09611 usGfscsauauucacCfaUfuUfaggcsa 998 (NAG37)s(invAb)sugccuaAfaUfgGfugaauaugcas(invAb) 1223
    AD09612 usGfsusUfuaagcuuCfuAfgAfgguusc 999 (NAG37)s(invAb)sgaaccucuAfGfAfagcuuaaacas(invAb) 1224
    AD09613 usUfsgsuucauuggUfuUfgAfaggcsc 1000 (NAG37)s(invAb)sggccuucaAfaCfcAfaugaacaas(invAb) 1225
    AD09614 usUfsasUfgCfuuugcUfgUfuCfauugsg 1001 (NAG37)s(invAb)sccaaugAfaCfaGfcaaagcauaas(invAb) 1226
    AD09615 usGfsusUfaugcuuuGfcUfgUfuCfausc 1002 (NAG37)s(invAb)sgaugaacaGfcAfAfagcauaacas(invAb) 1227
    AD09616 asGfsgsUfuaugcuuUfgCfuGfuucasc 1003 (NAG37)s(invAb)sgugaacagCfAfAfagcauaaccus(invAb) 1228
    AD09617 usAfsasgguuaugcUfuUfgCfuguusc 1004 (NAG37)s(invAb)sgaacagcaAfaGfcAfuaaccuuas(invAb) 1229
    AD09618 asGfsasUfucaagguUfaUfgCfuuugsc 1005 (NAG37)s(invAb)sgcaaagcaUfAfAfccuugaaucus(invAb) 1230
    AD09619 usUfscsAfauaauugAfgUfuGfguugsg 1006 (NAG37)s(invAb)sccaaccaaCfuCfaAfuuauugaas(invAb) 1231
    AD09620 asGfsusAfaaauggaUfcAfcAfggaasg 1007 (NAG37)s(invAb)scuuccuguGfAfUfccauuuuacus(invAb) 1232
    AD09621 usCfsasUfaugacagUfaAfgAfaaacsc 1008 (NAG37)s(invAb)sgguuuucuUfAfCfugucauaugas(invAb) 1233
    AD09623 usUfsgsgaaggcauUfcUfcGfaucusc 1009 (NAG37)s(invAb)sgagaucgaGfAfAfugccuuccaas(invAb) 1234
    AD09624 usCfsasUfcauugaaAfaUfgCfcagusc 1010 (NAG37)s(invAb)sgacuggcaUfUfUfucaaugaugas(invAb) 1235
    AD09625 asAfsasGfacaguuuCfaUfcAfuugasc 1011 (NAG37)s(invAb)sgucaaugaUfGfAfaacugucuuus(invAb) 1236
    AD09626 asAfscsacaaguaaCfcUfcAfuccusc 1012 (NAG37)s(invAb)sgaggaugaGfGfUfuacuuguguus(invAb) 1237
    AD09627 asGfsascaacauugUfcAfgCfuucasg 1013 (NAG37)s(invAb)scugaagcuGfAfCfaauguugucus(invAb) 1238
    AD09628 usCfsasacaucuuuGfcAfaUfaaagsc 1014 (NAG37)s(invAb)sgcuuuauuGfCfAfaagauguugas(invAb) 1239
    AD09629 asGfsasUfuagucuuAfcAfaAfuccusc 1015 (NAG37)s(invAb)sgaggauuuGfUfAfagacuaaucus(invAb) 1240
    AD09630 usCfsusUfauuccaaAfcUfuAfgucgsg 1016 (NAG37)s(invAb)sccgacuaaGfUfUfuggaauaagas(invAb) 1241
    AD09631 usCfsasGfaaaagaaAfgUfgUfgaagsc 1017 (NAG37)s(invAb)sgcuucacaCfUfUfucuuuucugas(invAb) 1242
    AD09632 usAfsgsAfguuugucUfcAfaAfgcugsc 1018 (NAG37)s(invAb)sgcagcuuuGfAfGfacaaacucuas(invAb) 1243
    AD09633 usUfsgsUfuaagcagUfcAfaUfuUfcusc 1019 (NAG37)s(invAb)sgagaaauuGfAfCfugcuuaacaas(invAb) 1244
    AD09634 usUfsgsGfaaaucugGfaUfaCfuacgsg 1020 (NAG37)s(invAb)sccguaguaUfCfCfagauuuccaas(invAb) 1245
    AD09635 usCfsusUfgaaaaugCfcAfuCfcugcsu 1021 (NAG37)s(invAb)sagcaggauGfGfCfauuuucaagas(invAb) 1246
    AD09636 asUfsgsAfuuuggauCfaCfaAfuugusc 1022 (NAG37)s(invAb)sgacaauugUfGfAfuccaaaucaus(invAb) 1247
    AD09637 usAfsgsAfauuacucAfaAfaCfugccsa 1023 (NAG37)s(invAb)suggcaguuUfUfGfaguaauucuas(invAb) 1248
    AD09638 usGfsasucaaAfAfauGfgAfcUfcagasc 1024 (NAG37)s(invAb)sgucugaguCfCfAfuuuuugaucas(invAb) 1249
    AD09639 usAfsasGfaaagcauGfcAfgAfucuasg 1025 (NAG37)s(invAb)scuagaucuGfCfAfugcuuucuuas(invAb) 1250
    AD09640 usCfsasgauauaagCfuCfuCfugaasg 1026 (NAG37)s(invAb)scuucagagAfGfCfuuauaucugas(invAb) 1251
    AD09650 usAfsusGfaagccaaCfcUfuGfuAfucsc 1027 (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252
    AD09651 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252
    AD09652 usAfsusGfaagCuNAcaaCfcUfuGfuau 1029 (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252
    csc
    AD09653 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacAfaGfgUfugicuucauas(invAb) 1253
    AD09654 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacAfaGfgUfuigcuucauas(invAb) 1254
    AD09655 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacAfaGfgUfugguuucauas(invAb) 1255
    AD09656 usAfsusGfaagucaaCfcUfuGfuaucsc 1030 (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252
    AD09657 usAfsusGfaagcuaaCfcUfuGfuaucsc 1031 (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252
    AD09658 cPrpusAfsusGfaagccaaCfcUfuGfua 1032 (NAG37)s(invAb)sggauacAfaGfgUfuggcuucauas(invAb) 1252
    ucsc
    AD09659 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacaaGfGfUfuggcuucauas(invAb) 1190
    AD09660 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacaaGfgUfUfggcuucauas(invAb) 1256
    AD09661 usAfsusGfaagccaaCfcUfuGfuaucsc 1028 (NAG37)s(invAb)sggauacaaGfgUfuGfgcuucauas(invAb) 1257
    AD09662 usCfsusUfcaugaagCfcAfaCfcuugsc 1028 (NAG37)s(invAb)sgcaagguuGfGfCfuucaugaagas(invAb) 1191
    AD09663 cPrpusCfsusUfcaugaagCfcAfaCfcu 1034 (NAG37)s(invAb)sgcaagguuGfGfCfuucaugaagas(invAb) 1191
    ugsc
    AD09664 usCfsusUfcaugaagCfcAfaCfcuugsc 1033 (NAG37)s(invAb)sgcaagguuGfgCfuUfcaugaagas(invAb) 1258
    AD09665 usCfsusUfcaUuNAgaagCfcAfaCfcuu 1035 (NAG37)s(invAb)sgcaagguuGfgCfuUfcaugaagas(invAb) 1258
    gsc
    AD09724 usGfsgsAfuCfugcauUfuUfuCfuC 1036 (NAG37)s(invAb)sguggagaaAfAfAfugcaiauccas(invAb) 1259
    fcasc
    AD09725 usCfscsAfaAfaggguUfgUfcUfcU 1037 (NAG37)s(invAb)succagagaCfAfAfcucuuuuggas(invAb) 1260
    fggsa
    AD09726 usAfsgsAfcGfaucauAfcUfuGfgA 1038 (NAG37)s(invAb)scucuccaaGfUfAfugauciucuas(invAb) 1261
    fgasg
    AD09727 usCfscsUfaUfuccuuCfcAfcAfgU 1039 (NAG37)s(invAb)sgcaacuguGfGfAfaggaauaggas(invAb) 1262
    fugsc
    AD09728 usAfscsAfuAfcucauGfaCfgAfuG 1040 (NAG37)s(invAb)suggcaucgUfCfAfugaguauguas(invAb) 1263
    fccsa
    AD09729 usCfsasCfaGfauuucCfuUfgGfaA 1041 (NAG37)s(invAb)sgccuuccaAfGfGfaaaucuguias(invAb) 1264
    fggsc
    AD09730 usGfsasAfcUfucaucUfcAfaUfgC 1042 (NAG37)s(invAb)sguggcauuGfAfGfaugaaguucas(invAb) 1265
    fcasc
    AD09731 asGfscsAfuAfuucuuGfaAfcUfuC 1043 (NAG37)s(invAb)sga_2NugaaguUfCfAfagaauaugcus 1266
    fausc (invAb)
    AD09732 usCfsasUfaGfgaaacAfgCfaUfaU 1044 (NAG37)s(invAb)sggaauaugCfUfGfuuuccuaugas(invAb) 1267
    fucsc
    AD09733 usGfsgsAfuCfucuauGfgAfgAfgC 1045 (NAG37)s(invAb)sgcugcucuCfCfAfuagaiauccas(invAb) 1268
    fagsc
    AD09734 usUfsusGfaAfugcugAfgAfaAfuA 1046 (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269
    fcusc
    AD09735 usCfsusAfuGfgacuuGfaUfcUfuG 1047 (NAG37)s(invAb)scgccaagaUfCfAfaguccauagas(invAb) 1270
    fgcsg
    AD09736 asUfsgsAfaAfcaaacAfaAfcCfcU 1048 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaus(invAb) 1271
    fggsa
    AD09737 usGfsgsUfaGfuucuuCfaUfaGfgU 1049 (NAG37)s(invAb)sgucaccuaUfGfAfagaacuaccas(invAb) 1272
    fgasc
    AD09738 usGfsasUfaAfuggcuGfgUfaGfuU 1050 (NAG37)s(invAb)sgagaacuaCfCfAfgccauuaucas(invAb) 1273
    fcusc
    AD09739 usCfsusCfaAfuugugAfuAfaUfgG 1051 (NAG37)s(invAb)scagccauuAfUfCfacaauugagas(invAb) 1274
    fcusg
    AD09740 usCfsusUfuCfucgauCfuUfcAfgC 1052 (NAG37)s(invAb)sugagcugaAfGfAfucgagaaagas(invAb) 1275
    fucsa
    AD09741 usUfsusGfgAfacagcAfaUfgGfuG 1053 (NAG37)s(invAb)scugcaccaUfUfGfcuguuccaaas(invAb) 1276
    fcasg
    AD09742 usGfsusAfgAfcacaaAfgAfgCfuC 1054 (NAG37)s(invAb)sguggagcuCfUfUfuguguuuacas(invAb) 1277
    fcasc
    AD09743 usCfsusGfuGfuagacAfcAfaAfgA 1055 (NAG37)s(invAb)sagcucuuuGfUfGfucuacacaias(invAb) 1278
    fgcsu
    AD09744 usUfscsCfaUfaauacUfcUfgAfgA 1056 (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279
    fgasg
    AD09745 usCfsusCfgUfuccauAfaUfaCfuC 1057 (NAG37)s(invAb)sgcagaguaUfUfAfuggaacgaias(invAb) 1280
    fugsc
    AD09937 cPrpusUfsgsAfaAfcaaacAfaAfc 1058 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaas(invAb) 1281
    CfcUfggsa
    AD09938 cPrpusUfscsCfaUfaauacUfcUfg 1059 (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279
    AfgAfgasg
    AD09962 asAfsusGfaaacaaaCfaAfaCfccugsg 1060 (NAG37)s(invAb)sccaggguuUfGfUfuuguuucauus(invAb) 1282
    AD09963 asAfsasUfgaaacaaAfcAfaAfcccusg 1061 (NAG37)s(invAb)scaggguuuGfUfUfuguuucauuus(invAb) 1283
    AD09964 usGfsasAfaugaaacAfaAfcAfaaccsc 1062 (NAG37)s(invAb)sggguuuguUfUfGfuuucauuucas(invAb) 1284
    AD09965 usUfsgsAfaAfcaaacAfaAfcCfcUfg 1063 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaas(invAb) 1281
    gsa
    AD09966 cPrpusUfsgsAfaacaaacAfaAfcCfcug 1064 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaas(invAb) 1281
    gsa
    AD09967 cPrpuUfgAfaacaaacAfaAfcCfcuggsa 1065 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaas(invAb) 1281
    AD09968 cPrpuUfgAfaacaaacAfaAfcCfcugs 1066 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaas(invAb) 1281
    gsa
    AD09969 cPrpuUfgAfaacaaacAfaAfcCfcuggsa 1065 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaas(invAb) 1285
    AD09970 cPrpuUfgAfaacaaacAfaAfcCfcuggsa 1065 (NAG37)s(invAb)succaggguUfuGfUfuuguuucaas(invAb) 1286
    AD09971 asGfsasCfgaucauaCfuUfgGfagagsc 1067 (NAG37)s(invAb)sgcucuccaAfGfUfaugauciucus(invAb) 1287
    AD09972 asAfsgsGfcauucucAfaUfcUfccucsc 1068 (NAG37)s(invAb)sggaggagaUfUfGfagaauiccuus(invAb) 1288
    AD09973 usUfsusCfcuuggaaGfgCfaUfucucsg 1069 (NAG37)s(invAb)scgagaaugCfCfUfuccaaggaaas(invAb) 1289
    AD09974 usAfsgsAfuuuccuuGfgAfaGfgcausc 1070 (NAG37)s(invAb)sgaugccuuCfCfAfaggaaaucuas(invAb) 1290
    AD09975 asUfsasGfgaaacagCfaUfaUfucuusg 1071 (NAG37)s(invAb)sca_2NagaauaUfGfCfuguuuccuaus 1291
    (invAb)
    AD09976 usUfsgsAfugauguuCfcCfuCfcaacsg 1072 (NAG37)s(invAb)scguuggagGfGfAfacaucaucaas(invAb) 1292
    AD09977 usAfsgsAfacuugaaGfaAfgAfagcusg 1073 (NAG37)s(invAb)scagcuucuUfCfUfucaaguucuas(invAb) 1293
    AD09978 usAfscsCfaaugauaUfgCfcCfaacasc 1074 (NAG37)s(invAb)sguguugggCfAfUfaucauugguas(invAb) 1294
    AD09979 usCfsasUfggUuNAguucUfgUfgUfaga 1075 (NAG37)s(invAb)scgucuacaCfAfGfaacaccaugas(invAb) 1295
    csg
    AD09980 usUfsgsAfgagagauCfcUfgGfgugusc 1076 (NAG37)s(invAb)sgacacccaGfGfAfucucuuucaas(invAb) 1296
    AD09981 usCfsasUfgauacugAfgAfgCfuugcsu 1077 (NAG37)s(invAb)sagcaagcuCfUfCfaguaucaugas(invAb) 1297
    AD09982 usUfsgsUfcaaccucAfcUfcUfuccgsa 1078 (NAG37)s(invAb)sucggaagaGfUfGfagguugacaas(invAb) 1298
    AD09983 usUfsusCfcaacaauUfcUfcCfuugusc 1079 (NAG37)s(invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) 1299
    AD09984 usUfsgsAfguuagucUfcAfaAfgcugsc 1080 (NAG37)s(invAb)sgcagcuuuGfAfGfacuaacucaas(invAb) 1300
    AD09985 asUfsgsAfcaauaucUfgUfgCfggagsg 1081 (NAG37)s(invAb)sccuccgcaCfAfGfauauugucaus(invAb) 1301
    AD09986 usCfsasUfgacaauaUfcUfgUfgcggsa 1082 (NAG37)s(invAb)succgcacaGfAfUfauugucaugas(invAb) 1302
    AD09987 usCfsasAfagaagauAfgAfaGfcagcsc 1083 (NAG37)s(invAb)sggcugcuuCfUfAfucuucuuugas(invAb) 1303
    AD09988 usCfsasCfguuauuaCfcUfgUfgugcsu 1084 (NAG37)s(invAb)sagcacacaGfGfUfaauaacguias(invAb) 1304
    AD09989 usAfsgsAfacuugagGfuUfaUfacagsg 1085 (NAG37)s(invAb)sccuguauaAfCfCfucaaguucuas(invAb) 1305
    AD09990 asUfsgsCfuuugcugUfuCfaUfuggusc 1086 (NAG37)s(invAb)sgaccaaugAfAfCfagcaaagcaus(invAb) 1306
    AD09991 usAfsgsUfauagauuCfaAfgGfuuausg 1087 (NAG37)s(invAb)sca_2NuaaccuUfGfAfaucuauacuas 1307
    (invAb)
    AD09992 asGfsasGfuaaucuuGfcUfuUfaugcsc 1088 (NAG37)s(invAb)sggcauaaaGfCfAfagauuacucus(invAb) 1308
    AD09993 asUfsasGfcaucauuUfcUfaGfguggsa 1089 (NAG37)s(invAb)succaccuaGfAfAfaugaugcuaus(invAb) 1309
    AD09994 asGfsasCfagaagagAfcAfgAfgcuasg 1090 (NAG37)s(invAb)scuagcucuGfUfCfucuucuiucus(invAb) 1310
    AD09995 asGfsusAfagaaaacCfaAfgCfcuuasg 1091 (NAG37)s(invAb)scua_2NaggcuUfGfGfuuuucuuacus 1311
    (invAb)
    AD10008 usUfscsCfauaauacUfcUfgAfgagasg 1092 (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279
    AD10009 cPrpusUfscsCfauaauacUfcUfgAfga 1093 (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279
    gasg
    AD10010 cPrpuUfcCfauaauacUfcUfgAfgagasg 1094 (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279
    AD10011 cPrpuUfcCfauaauacUfcUfgAfgags 1095 (NAG37)s(invAb)scucucucaGfAfGfuauuauggaas(invAb) 1279
    asg
    AD10012 cPrpuUfcCfauaauacUfcUfgAfgagasg 1094 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    AD10013 cPrpuUfccauaaUfacUfcUfgAfgagasg 1096 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    AD10014 cPrpuUfcCfauaauacUfcUfgAfgagasg 1094 (NAG37)s(invAb)scucucucaGfaGfUfauuauggaas(invAb) 1313
    AD10015 cPrpuUfccauaaUfacUfcUfgAfgagasg 1096 (NAG37)s(invAb)scucucucaGfaGfUfauuauggaas(invAb) 1313
    AD10016 usAfsusAfcuuggagAfgCfaUfcacusg 1097 (NAG37)s(invAb)scagugaugCfUfCfuccaaguauas(invAb) 1314
    AD10017 usUfsgsCfagacgauCfaUfaCfuuggsc 1098 (NAG37)s(invAb)sgccaaguaUfGfAfucgucuicaas(invAb) 1315
    AD10018 usUfsgsAfaUfaaaacUfcUfcAfugccsa 1099 (NAG37)s(invAb)suggcaugaGfAfGfuuuuauucaas(invAb) 1316
    AD10019 cPrpusUfsgsAfaUfaaaacUfcUfcAfug 1100 (NAG37)s(invAb)suggcaugaGfAfGfuuuuauucaas(invAb) 1316
    ccsa
    AD10020 usUfsgsAfaUfaaaacUfcUfcAfugccsa 1099 (NAG37)s(invAb)suggcaugaGfAfGfuuuua_2Nuucaas 1317
    (invAb)
    AD10021 usAfscsUfuGfaAfgAfaGfaAfgCfuGf 1101 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguas(invAb) 1318
    aGfsg
    AD10022 usAfscsUfugaagaaGfaAfgCfugagsg 1102 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguas(invAb) 1318
    AD10023 cPrpusAfscsUfugaagaaGfaAfgCfug 1103 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguas(invAb) 1318
    agsg
    AD10024 cPrpuAfcUfugaagaaGfaAfgCfugagsg 1104 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguas(invAb) 1318
    AD10025 cPrpuAfcUfugaagaaGfaAfgCfugagsg 1104 (NAG37)s(invAb)sccucagcuUfCfUfucuuuaaguas(invAb) 1319
    AD10026 cPrpuAfcUfugaagaaGfaAfgCfugas 1105 (NAG37)s(invAb)sccucagcuUfCfUfucuucaaguas(invAb) 1318
    gsg
    AD10027 cPrpuAfcUfugaagaaGfaAfgCfugagsg 1104 (NAG37)s(invAb)sccucagcuUfcUfUfcuucaaguas(invAb) 1320
    AD10028 cPrpuAfcUfugaagaaGfaAfgCfugagsg 1104 (NAG37)s(invAb)sccucagcuUfcUfuCfuucaaguas(invAb) 1321
    AD10029 cPrpuAfcuugAfagaaGfaAfgCfugagsg 1106 (NAG37)s(invAb)sccucagcuUfcUfuCfuucaaguas(invAb) 1321
    AD10030 cPrpuAfcuugaaGfaaGfaAfgCfugagsg 1107 (NAG37)s(invAb)sccucagcuUfcUfUfcuucaaguas(invAb) 1320
    AD10091 cPrpusUfsusGfaAfugcugAfgAfaAfuA 1108 (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269
    fcusc
    AD10092 cPrpusUfsusGfaaugcugAfgAfaAfua 1109 (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269
    cusc
    AD10093 cPrpusUfsusgaaUfgcugAfgAfaAfua 1110 (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269
    cusc
    AD10094 cPrpusUfsusGfaaugcugAfgAfaAfua 1109 (NAG37)s(invAb)sgaguauuuCfuCfAfgcauucaaas(invAb) 1322
    cusc
    AD10095 cPrpusUfsusgaaUfgcugAfgAfaAfua 1110 (NAG37)s(invAb)sgaguauuuCfuCfAfgcauucaaas(invAb) 1322
    cusc
    AD10096 cPrpuUfuGfaaugcugAfgAfaAfuacusc 1111 (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269
    AD10097 cPrpuUfuGfaaugcugAfgAfaAfuacsusc 1112 (NAG37)s(invAb)sgaguauuuCfUfCfagcauucaaas(invAb) 1269
    AD10099 asGfsasAfaAfguggaCfgAfuCfuUfgusc 1113 (NAG37)s(invAb)sgacaagauCfGfUfccacuuuucus(invAb) 1323
    AD10100 usAfsgsUfuGfucacuGfcAfaCfaUfggsu 1114 (NAG37)s(invAb)saccauguuGfCfAfgugacaacuas(invAb) 1324
    AD10101 asUfsusCfcUfuccacAfgUfuGfuCfacsc 1115 (NAG37)s(invAb)sggugacaaCfUfGfuggaaggaaus(invAb) 1325
    AD10102 usGfscsAfuUfcucaaUfcUfcCfuCfcasc 1116 (NAG37)s(invAb)sguggaggaGfAfUfugagaaugcas(invAb) 1326
    AD10103 usUfscsAfaUfgccaaUfcUfcCfgUfgusc 1117 (NAG37)s(invAb)sgacacggaGfAfUfuggcauugaas(invAb) 1327
    AD10104 asUfsasUfuCfuugaaCfuUfcAfuCfucsg 1118 (NAG37)s(invAb)scgagaugaAfGfUfucaagaaua_2Nus 1328
    (invAb)
    AD10105 usUfsasUfgGfagagcAfgUfaUfcUfccsu 1119 (NAG37)s(invAb)saggagauaCfUfGfcucuccauaas(invAb) 1329
    AD10106 usAfsasUfgCfugagaAfaUfaCfuCfccsc 1120 (NAG37)s(invAb)sggggaguaUfUfUfcucagcauuas(invAb) 1330
    AD10107 usGfsasAfuGfcugagAfaAfuAfcUfccsc 1121 (NAG37)s(invAb)sgggaguauUfUfCfucagcauucas(invAb) 1331
    AD10108 usCfsasAfuGfucaucUfuCfuCfuCfcgsg 1122 (NAG37)s(invAb)sccggagagAfAfGfaugacauugas(invAb) 1332
    AD10109 asCfsasAfaUfuccagUfuAfuGfuUfacsc 1123 (NAG37)s(invAb)sgguaacauAfAfCfuggaauuugus(invAb) 1333
    AD10110 usUfscsAfaUfugugaUfaAfuGfgCfugsg 1124 (NAG37)s(invAb)sccagccauUfAfUfcacaauugaas(invAb) 1334
    AD10111 asAfscsAfuUfuuugcAfaCfaAfaGfcusc 1125 (NAG37)s(invAb)sgagcuuugUfUfGfcaaaaauguus(invAb) 1335
    AD10112 usCfsasAfcAfuuuuuGfcAfaCfaAfagsc 1126 (NAG37)s(invAb)sgcuuuguuGfCfAfaaaauguugas(invAb) 1336
    AD10113 usUfsusCfaCfucgaaCfcAfcAfaUfccsg 1127 (NAG37)s(invAb)scggauuguGfGfUfucgagugaaas(invAb) 1337
    AD10176 cPrpusCfsusUfaUfuccaaAfcUfuGfgU 1128 (NAG37)s(invAb)scccaccaaGfUfUfuggaauaagas(invAb) 1201
    fggsg
    AD10177 cPrpuCfuUfaUfuccaaAfcUfuGfgUfg 1129 (NAG37)s(invAb)scccaccaaGfUfUfuggaauaagas(invAb) 1201
    gsg
    AD10178 cPrpuCfuuauucCfaaAfcUfuGfguggsg 1130 (NAG37)s(invAb)scccaccaaGfUfUfuggaauaagas(invAb) 1201
    AD10179 cPrpuCfuuauucCfaaAfcUfuGfguggsg 1130 (NAG37)s(invAb)scccaccaaGfuUfuGfgaauaagas(invAb) 1338
    AD10180 cPrpuCfuuauucCfaaAfcUfuGfguggsg 1130 (NAG37)s(invAb)scccaccaaGfuUfUfggaauaagas(invAb) 1339
    AD10181 cPrpuGfcauauucacCfaUfuUfaggcsa 1131 (NAG37)s(invAb)sugccuaAfaUfgGfugaauaugcas(invAb) 1223
    AD10182 cPrpuGfcauaUfucacCfaUfuUfaggcsa 1132 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    AD10183 cPrpuGfcauauuCfacCfaUfuUfaggcsa 1133 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    AD10184 cPrpuGfcauaUfucacCfaUfuUfaggcsa 1132 (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341
    AD10200 usGfscauauucacCfaUfuUfaggcsa 1134 (NAG37)s(invAb)sugccuaAfaUfgGfugaauaugcas(invAb) 1223
    AD10201 usGfscauaUfucacCfaUfuUfaggcsa 1135 (NAG37)s(invAb)sugccuaAfaUfgGfugaauaugcas(invAb) 1223
    AD10202 usGfscauauuCfacCfaUfuUfaggcsa 1136 (NAG37)s(invAb)sugccuaAfaUfgGfugaauaugcas(invAb) 1223
    AD10203 usGfscauaUfucacCfaUfuUfaggcsa 1135 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    AD10204 usGfscauauuCfacCfaUfuUfaggcsa 1136 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    AD10205 usGfscauaUfucacCfaUfuUfaggcsa 1135 (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341
    AD10275 cPrpasUfsgsAfaacaaacAfaAfcCfc 1137 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaus(invAb) 1271
    uggsa
    AD10276 cPrpasUfsgsAfaacaaacAfaAfcCfc 1138 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaus(invAb) 1271
    ugsgsa
    AD10277 cPrpasUfsgAfaacaaacAfaAfcCfcu 1139 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaus(invAb) 1271
    gsgsa
    AD10278 cPrpaUfgAfaacaaacAfaAfcCfcugs 1140 (NAG37)s(invAb)succaggguUfUfGfuuuguuucaus(invAb) 1271
    gsa
    AD10279 cPrpasUfsgAfaacaaacAfaAfcCfcu 1139 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    gsgsa
    AD10280 cPrpasUfsgaaacaAfacAfaAfcCfcu 1141 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    gsgsa
    AD10281 cPrpasUfsgaaaCfaaacAfaAfcCfcu 1142 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    gsgsa
    AD10282 cPrpasUfsgaAfacaaacAfaAfcCfcu 1143 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    gsgsa
    AD10283 cPrpasUfsgAfaaCfaAfacAfaAfcCf 1144 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    cugsgsa
    AD10619 cPrpusUfscsCfauaauacUfcUfgAfg 1093 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    agasg
    AD10620 cPrpusUfscCfauaauacUfcUfgAfga 1145 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    gasg
    AD10621 cPrpusUfscCfauaauacUfcUfgAfga 1146 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    gsasg
    AD10622 cPrpuUfcCfauaauacUfcUfgAfgags 1095 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    asg
    AD10623 cPrpuUfcCfauaauacUfcUfgAfgagasc 1147 (NAG37)s(invAb)sgucucucaGfaGfuAfuuauggaas(invAb) 1343
    AD10624 cPrpuUfcCfauaauacUfcUfgAfgaggsg 1148 (NAG37)s(invAb)scccucucaGfaGfuAfuuauggaas(invAb) 1344
    AD10625 cPrpuUfcCfauaauacUfcUfgAfgaggsc 1149 (NAG37)s(invAb)sgccucucaGfaGfuAfuuauggaas(invAb) 1345
    AD10626 cPrpuUfcCfauaauacUfcUfgAfgaggsu 1150 (NAG37)s(invAb)saccucucaGfaGfuAfuuauggaas(invAb) 1346
    AD10627 cPrpuUfcCfauaauacUfcUfgAfgaggsa 1151 (NAG37)s(invAb)succucucaGfaGfuAfuuauggaas(invAb) 1347
    AD10628 cPrpuUfccauAfauacUfcUfgAfgagasg 1152 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    AD10629 cPrpusGfscsauauuCfacCfaUfuUfagg 1153 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    csa
    AD10630 cPrpusGfscauauuCfacCfaUfuUfagg 1154 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    csa
    AD10631 cPrpusGfscauauuCfacCfaUfuUfagg 1155 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    scsa
    AD10632 cPrpuGfcauauuCfacCfaUfuUfaggs 1156 (NAG37)s(invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) 1340
    csa
    AD10633 cPrpusGfscsauaUfucacCfaUfuUfa 1157 (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341
    ggcsa
    AD10634 cPrpusGfscauaUfucacCfaUfuUfag 1158 (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341
    gcsa
    AD10635 cPrpusGfscauaUfucacCfaUfuUfag 1159 (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341
    gscsa
    AD10636 cPrpuGfcauaUfucacCfaUfuUfaggs 1160 (NAG37)s(invAb)sugccuaaaUfgGfUfgaauaugcas(invAb) 1341
    csa
    AD10728 asUfsgsAfcaauaucUfgUfgCfggagsg 1081 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    AD10729 asUfsgsacaAfuaucUfgUfgCfggagsg 1161 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    AD10730 asUfsgsacaauAfucUfgUfgCfggagsg 1162 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    AD10731 cPrpasUfsgsacaauAfucUfgUfgCfgg 1163 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    agsg
    AD10732 cPrpusUfsgsacaauAfucUfgUfgCfgg 1164 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaas(invAb) 1349
    agsg
    AD10733 cPrpaUfgacaauAfucUfgUfgCfggag 1165 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    sg
    AD10734 cPrpaUfgacaauAfucUfgUfgCfggasg 1166 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    sg
    AD10735 cPrpasUfsgacaauAfucUfgUfgCfgga 1167 (NAG37)s(invAb)sccuccgcaCfaGfaUfauugucaus(invAb) 1348
    sgsg
    AD10736 cPrpasUfsgsacaauAfucUfgUfgCfgg 1168 (NAG37)s(invAb)scuccgcaCfaGfaUfauugucaus(invAb) 1350
    asg
    AD10737 cPrpasUfsgsacaauAfucUfgUfgCfgg 1169 (NAG37)s(invAb)succgcaCfaGfaUfauugucaus(invAb) 1351
    sa
    AD10952 cPrpusUfsccauaaUfacUfcUfgAfgag 1170 (NAG37)s(invAb)scucucucaGfaGfuAfuuauggaas(invAb) 1312
    sasg
    AD10953 cPrpusUfscCfauaauacUfcUfgAfgag 1171 (NAG37)s(invAb)sgucucucaGfaGfuAfuuauggaas(invAb) 1343
    sasc
    AD10954 cPrpusUfsgaaaCfaaacAfaAfcCfcug 1172 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaas(invAb) 1285
    sgsa
    AD10967 asUfsgsAfaAfcaaacAfaAfcCfcUfgg 1048 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    sa
    AD10968 asUfsgAfaAfcaaacAfaAfcCfcUfgsg 1173 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    sa
    AD10969 asUfsgAfaacaaacAfaAfcCfcugsgsa 1174 (NAG37)s(invAb)succaggguUfuGfuUfuguuucaus(invAb) 1342
    AD12167 asCfsucgUfuccauaaUfaCfucugasgsa 1672 (NAG37)suscagagUfaUfUfAfuggaacgagus(invAb) 1676
    AD12168 asUfsccaUfaauacucUfgAfgagagsasu 1673 (NAG37)scsucucuCfaGfAfGfuauuauggaus(invAb) 1677
  • In some aspects, an XDH RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. The RNAi agents described herein, upon delivery to a cell expressing an XDH gene, inhibit or knockdown expression of one or more XDH genes in vivo and/or in vitro.
    • Targeting Ligands or Groups, Linking Groups, and Delivery Vehicles
  • In some aspects, an XDH RNAi agent is conjugated to one or more non-nucleotide groups including, but not limited to, a targeting group, a linking group, a targeting ligand, a delivery polymer, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery or attachment of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6. The non-nucleotide group can be covalently linked to the 3′ and/or 5′ end of either the sense strand and/or the antisense strand. In some aspects, an XDH RNAi agent contains a non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand. In some aspects, a non-nucleotide group is linked to the 5′ end of an XDH RNAi agent sense strand. A non-nucleotide group may be linked directly or indirectly to the RNAi agent via a linker/linking group. In some aspects, a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.
  • In some aspects, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the RNAi agent or conjugate. In some aspects, a non-nucleotide group enhances endocytosis of the RNAi agent.
  • Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate or RNAi agent to which they are attached to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the conjugate or RNAi agent. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency for the target to which it is directed. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules.
  • In some aspects, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) residues, which can in some instances serve as linkers. In some aspects, a targeting ligand comprises a galactose-derivative cluster.
  • The XDH RNAi agents described herein can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine), at the 5′-terminus and/or the 3′-terminus. The reactive group can be used subsequently to attach a targeting moiety using methods typical in the art.
  • In some aspects, a targeting group comprises an asialoghycoprotein receptor ligand. As used herein, an asialoglycoprotein receptor ligand is a ligand that contains a moiety having affinity for the asialoglycoprotein receptor. As noted herein, the asialoglycoprotein receptor is highly expressed on hepatocytes. In some aspects, an asialoglycoprotein receptor ligand includes or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor that is equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosammne, and N-iso-butanoylgalactos-amine (see for example: S. T. Iobst and K. Drickamer, J. B. C., 1996, 271, 6686). Galactose derivatives, and clusters of galactose derivatives, that are useful for in vivo targeting of oligonucleotides and other molecules to the liver are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).
  • Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to the asialoglycoprotein receptor(s) facilitates cell-specific targeting to hepatocytes and endocytosis of the molecule into hepatocytes. Asialoglycoprotein receptor ligands can be monomeric (e.g., having a single galactose derivative, also referred to as monovalent or monodentate) or multimeric (e.g., having multiple galactose derivatives). The galactose derivative or galactose derivative cluster can be attached to the 3′ or 5′ end of the sense or antisense strand of the RNAi agent using methods known in the art. The preparation of targeting ligands, such as galactose derivative clusters, is described in, for example, International Patent Application Publication No. WO 2018/044350 to Arrowhead Pharmaceuticals, Inc., and International Patent Application Publication No. WO 2017/156012 to Arrowhead Pharmaceuticals, Inc., the contents of both of which are incorporated by reference herein in their entirety.
  • As used herein, a galactose derivative cluster comprises a molecule having two to four terminal galactose derivatives. A terminal galactose derivative is attached to a molecule through its C-1 carbon. In some aspects, the galactose derivative cluster is a galactose derivative trimer (also referred to as tri-antennary galactose derivative or tri-valent galactose derivative). In some aspects, the galactose derivative cluster comprises N-acetyl-galactosamine moieties. In some aspects, the galactose derivative cluster comprises three N-acetyl-galactosamine moieties. In some aspects, the galactose derivative cluster is a galactose derivative tetramer (also referred to as tetra-antennary galactose derivative or tetra-valent galactose derivative). In some aspects, the galactose derivative cluster comprises four N-acetyl-galactosamine moieties.
  • As used herein, a galactose derivative trimer contains three galactose derivatives, each linked to a central branch point. As used herein, a galactose derivative tetramer contains four galactose derivatives, each linked to a central branch point. The galactose derivatives can be attached to the central branch point through the C-1 carbons of the saccharides. In some aspects, the galactose derivatives are linked to the branch point via linkers or spacers. In some aspects, the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, e.g., U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546). In some aspects, the PEG spacer is a PEG3 spacer. The branch point can be any small molecule which permits attachment of three galactose derivatives and further permits attachment of the branch point to the RNAi agent. An example of branch point group is a di-lysine or di-glutamate. Attachment of the branch point to the RNAi agent can occur through a linker or spacer. In some aspects, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some aspects, the linker comprises a rigid linker, such as a cyclic group. In some aspects, a galactose derivative comprises or consists of N-acetyl-galactosamine. In some aspects, the galactose derivative cluster is comprised of a galactose derivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.
  • Certain aspects of the present disclosure include pharmaceutical compositions for delivering an XDH RNAi agent to a liver cell in vivo. Such pharmaceutical compositions can include, for example, an XDH RNAi agent conjugated to a galactose derivative cluster. In some aspects, the galactose derivative cluster is comprised of a galactose derivative trimer, which can be, for example, an N-acetyl-galactosamine trimer, or galactose derivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.
  • A targeting ligand or targeting group can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of an XDH RNAi agent disclosed herein.
  • Targeting ligands include, but are not limited to (NAG37) and (NAG37)s as defined in Table 6. Other targeting groups and targeting ligands, including galactose cluster targeting ligands, are known in the art.
  • In some aspects, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the agent to a targeting group, delivery polymer, or delivery vehicle. The linking group can be linked to the 3′ and/or the 5′ end of the RNAi agent sense strand or antisense strand. In some aspects, the linking group is linked to the RNAi agent sense strand. In some aspects, the linking group is conjugated to the 5′ or 3′ end of an RNAi agent sense strand. In some aspects, a linking group is conjugated to the 5′ end of an RNAi agent sense strand. Examples of linking groups, can include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), and/or PEG groups.
  • In some aspects, a targeting group is linked internally to a nucleotide on the sense strand and/or the antisense strand of the RNAi agent. In some aspects, a targeting group is linked to the RNAi agent via a linker.
  • A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer can further add flexibility and/or length to the linkage. Spacers include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.
  • In some aspects, when two or more RNAi agents are included in a single composition, each of the RNAi agents may be linked to the same targeting group or two a different targeting groups (i.e., targeting groups having different chemical structure). In some aspects, targeting groups are linked to the XDH RNAi agents disclosed herein without the use of an additional linker. In some aspects, the targeting group itself is designed having a linker or other site to facilitate conjugation readily present. In some aspects, when two or more XDH RNAi agents are included in a single molecule, each of the RNAi agents may utilize the same linker or different linkers (i.e., linkers having different chemical structures).
  • Any of the XDH RNAi agent nucleotide sequences listed in Tables 2, 3, 4, or 5C, whether modified or unmodified, can contain 3′ and/or 5′ targeting group(s) or linking group(s). Any of the XDH RNAi agent sequences listed in Table 3 or 4, or are otherwise described herein, which contain a 3′ or 5′ targeting group or linking group, can alternatively contain no 3′ or 5′ targeting group or linking group, or can contain a different 3′ or 5′ targeting group or linking group including, but not limited to, those depicted in Table 6. Any of the XDH RNAi agent duplexes listed in Tables 5A, 5B and 5C, whether modified or unmodified, can further comprise a targeting group or linking group, including, but not limited to, those depicted in Table 6, and the targeting group or linking group can be attached to the 3′ or 5′ terminus of either the sense strand or the antisense strand of the XDH RNAi agent duplex.
  • Examples of targeting groups and linking groups (which when combined can form targeting ligands) are provided in Table 6. Table 4 and Table 5C provide several aspects of XDH RNAi agent sense strands having a targeting group or linking group linked to the 5′ or 3′ end.
  • TABLE 6
    Structures Representing Various Modified Nucleotides, Targeting Ligands or Targeting Groups, Capping Residues, and Linking Groups
    Figure US20230139166A1-20230504-C00002
    cPrpus
    Figure US20230139166A1-20230504-C00003
    cPrpu
    Figure US20230139166A1-20230504-C00004
    cPrpas
    Figure US20230139166A1-20230504-C00005
    cPrpa
    Figure US20230139166A1-20230504-C00006
    a_2N
    Figure US20230139166A1-20230504-C00007
    a_2Ns
    Figure US20230139166A1-20230504-C00008
    AUNA
    Figure US20230139166A1-20230504-C00009
    AUNAS
    Figure US20230139166A1-20230504-C00010
    CUNA
    Figure US20230139166A1-20230504-C00011
    CUNAS
    Figure US20230139166A1-20230504-C00012
    GUNA
    Figure US20230139166A1-20230504-C00013
    GUNAS
    Figure US20230139166A1-20230504-C00014
    UUNA
    Figure US20230139166A1-20230504-C00015
    UUNAS
    When positioned internally:
    Figure US20230139166A1-20230504-C00016
    (invAb)
    When positioned internally:
    Figure US20230139166A1-20230504-C00017
    (invAb)s
    When positioned at the 3′ terminal end:
    Figure US20230139166A1-20230504-C00018
    (invAb)
    Figure US20230139166A1-20230504-C00019
    (NAG37)
    Figure US20230139166A1-20230504-C00020
    (NAG37)s
    Figure US20230139166A1-20230504-C00021
    N-[tris(GalNAc-alkyl)-amidododecanoyl)]-4-hydroxyprolinol (Hyp-GalNac-alky)3)
  • In each of the above structures in Table 6, NAG comprises an N-acetyl-galactosamine or another galactose derivative, as would be understood by a person of ordinary skill in the art to be attached in view of the structures above and description provided herein. Other linking groups known in the art may be used.
  • In some aspects, a delivery vehicle can be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine. In some aspects, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO 2008/0022309. WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors, or other delivery systems suitable for nucleic acid or oligonucleotide delivery as known and available in the art.
    • Pharmaceutical Compositions and Formulations
  • The XDH RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “medicaments”). In some aspects, pharmaceutical compositions include at least one XDH RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism.
  • The pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from reduction in the level of the target XDH mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease, disorder, symptom, or condition that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering an XDH RNAi agent linked to a targeting ligand as described herein, to a subject to be treated. In some aspects, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions that include an XDH RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.
  • The pharmaceutical compositions that include an XDH RNAi agent and methods disclosed herein decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, organ, or subject, including by administering to the subject a therapeutically effective amount of a herein described XDH RNAi agent, thereby inhibiting the expression of XDH mRNA in the subject. In some aspects, the subject has been previously identified as having a pathogenic upregulation of the target gene in hepatocytes. In some aspects, the subject has been previously identified or diagnosed as having gout or hyperuricemia. In some aspects, the subject has been suffering from symptoms associated with gout or hyperuricemia. In some aspects, the subject would benefit from a reduction of XDH gene expression in the subject's liver.
  • In some aspects, the described pharmaceutical compositions including an XDH RNAi agent are used for treating or managing clinical presentations associated with gout or hyperuricemia. In some aspects, a therapeutically (including prophylactically) effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment. In some aspects, administration of any of the disclosed XDH RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
  • The described pharmaceutical compositions that include an XDH RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of XDH mRNA and/or a reduction in serum uric acid levels. Measuring serum uric acid levels can be conducted in accordance with established methods known in the art.
  • In some aspects, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions that include an XDH RNAi agent thereby treating the symptom. In other aspects, the subject is administered a prophylactically effective amount of one or more XDH RNAi agents, thereby preventing or inhibiting the at least one symptom.
  • The route of administration is the path by which an XDH RNAi agent is brought into contact with the body. In general, methods of administering drugs and oligonucleotides and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The XDH RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some aspects, the herein described pharmaceutical compositions are administered via subcutaneous injection.
  • The pharmaceutical compositions including an XDH RNAi agent described herein can be delivered to a cell, group of cells, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain aspects, the compositions are administered by subcutaneous or intravenous infusion or injection.
  • In some aspects, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.
  • As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical Ingredient (API, therapeutic product, e.g., XDH RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients can act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.
  • Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water. Cremophor® ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Suitable carriers should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • In some aspects, pharmaceutical formulations that include the XDH RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in an aqueous sodium phosphate buffer (e.g., the XDH RNAi agent formulated in 0.5 mM sodium phosphate monobasic, 0.5 mM sodium phosphate dibasic, in water). In some aspects, pharmaceutical formulations that include the XDH RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in water for injection (sterile water). XDH RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in isotonic saline (0.9%).
  • Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
  • Formulations suitable for oral administration of the XDH RNAi agents disclosed herein can also be prepared. In some aspects, the XDH RNAi agents disclosed herein are administered orally. In some aspects, the XDH RNAi agents disclosed herein are formulated in a capsule for oral administration.
  • The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • The XDH RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, analgesics, antihistamines, or anti-inflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine, etc.). It is also envisioned that cells, tissues, or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.
  • In some aspects, the methods disclosed herein further comprise the step of administering a second therapeutic or treatment in addition to administering an RNAi agent disclosed herein. In some aspects, the second therapeutic is another XDH RNAi agent (e.g., an XDH RNAi agent that targets a different sequence within the XDH target). In other aspects, the second therapeutic can be a small molecule drug, an antibody, an antibody fragment, or an aptamer.
  • In some aspects, the described XDH RNAi agent(s) are optionally combined with one or more additional therapeutics. The XDH RNAi agent and additional therapeutic(s) can be administered in a single composition or they can be administered separately. In some aspects, the one or more additional therapeutics is administered separately in separate dosage forms from the RNAi agent (e.g., the XDH RNAi agent is administered by subcutaneous injection, while the additional therapeutic involved in the method of treatment dosing regimen is administered orally) In some aspects, the described XDH RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered orally, which together provide for a treatment regimen for diseases and conditions associated with gout or hyperuricemia. In some aspects, the described XDH RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered via a separate subcutaneous injection. In some aspects, the XDH RNAi agent and one or more additional therapeutics are combined into a single dosage form (e.g., a “cocktail” formulated into a single composition for subcutaneous injection). The XDH RNAi agents, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.
  • Generally, an effective amount of an XDH RNAi agent will be in the range of from about 0.1 to about 100 mg/kg of body weight/dose, e.g., from about 1.0 to about 50 mg/kg of body weight/dose. In some aspects, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose. In some aspects, an effective amount of an active ingredient will be in the range of from about 0.5 to about 4 mg/kg of body weight per dose. In some aspects, an effective amount of an XDH RNAi agent may be a fixed dose. In some aspects, the fixed dose is in the range of from about 5 mg to about 1,000 mg of XDH RNAi agent. In some aspects, the fixed does is in the range of 50 to 400 mg of XDH RNAi agent. Dosing may be weekly, bi-weekly, monthly, quarterly, or at any other interval depending on the dose of XDH RNAi agent administered, the activity level of the particular XDH RNAi agent, and the desired level of inhibition for the particular subject. The Examples herein show suitable levels for inhibition in certain animal species. The amount administered will depend on such variables as the overall health status of the patient or subject, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
  • For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including an XDH RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, peptide and/or an aptamer.
  • The described XDH RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers. The pharmaceutical compositions described herein may be packaged in pre-filled syringes, pen injectors, autoinjectors, infusion bags/devices, or vials.
    • Methods of Treatment and Inhibition of Expression
  • The XDH RNAi agents disclosed herein can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of the RNAi agent. In some aspects, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from reduction and/or inhibition in expression of XDH mRNA and/or XDH protein levels, which can lead to a reduction in serum uric acid levels in, for example, a subject that has been diagnosed with or is suffering from symptoms related to gout or hyperuricemia.
  • In some aspects, the subject is administered a therapeutically effective amount of any one or more XDH RNAi agents. Treatment of a subject can include therapeutic and/or prophylactic treatment. The subject is administered a therapeutically effective amount of any one or more XDH RNAi agents described herein. The subject can be a human, patient, or human patient. The subject may be an adult, adolescent, child, or infant. Administration of a pharmaceutical composition described herein can be to a human being or animal.
  • The XDH RNAi agents described herein can be used to treat at least one symptom in a subject having an XDH-related disease or disorder, or having a disease or disorder that is mediated at least in part by XDH gene expression. In some aspects, the XDH RNAi agents are used to treat or manage a clinical presentation of a subject with a disease or disorder that would benefit from or be mediated at least in part by a reduction in XDH mRNA. The subject is administered a therapeutically effective amount of one or more of the XDH RNAi agents or XDH RNAi agent-containing compositions described herein. In some aspects, the methods disclosed herein comprise administering a composition comprising an XDH RNAi agent described herein to a subject to be treated. In some aspects, the subject is administered a prophylactically effective amount of any one or more of the described XDH RNAi agents, thereby treating the subject by preventing or inhibiting the at least one symptom.
  • In certain aspects, the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by XDH gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the XDH RNAi agents described herein.
  • In some aspects, the RNAi agent comprises an antisense strand comprising an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882. AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • In some aspects, the XDH RNAi agent comprises an antisense strand comprising a modified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising a modified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • In some aspects, the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of UUCCAUAAUACUCUGAGAGAG (SEQ ID NO:1448) and a sense strand comprising a nucleic acid sequence of CUCUCUCAGAGUAUUAUGGAA (SEQ ID NO:1603). In some aspects, a nucleic acid sequence of the antisense strand comprises a nucleic acid sequence of cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146) and a nucleic acid sequence of the sense strand comprises a nucleic acid sequence of cucucucaGfaGfuAfuuauggaa (SEQ ID NO: 1663) or (invAb)scucucucaGfaGfuAfuuauggaas(invAb) (SEQ ID NO:1680).
  • In some aspects, the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of AUGACAAUAUCUGUGCGGAGG (SEQ ID NO:1468) and a sense strand comprising a nucleic acid sequence of CCUCCGCACAGAUAUUGUCAU (SEQ ID NO:1623). In some aspects, a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and a nucleic acid sequence of the sense strand comprises a nucleic acid sequence of ccuccgcaCfAfGfauauugucau (SEQ ID NO:1664) or (invAb)sccuccgcaCfAfGfauauugucaus(invAb) (SEQ ID NO:1681).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UGCAUAUUCACCAUUUAGGCA (SEQ ID NO:1397) and a sense strand comprising a nucleic acid sequence of UGCCUAAAUGGUGAAUAUGCA (SEQ ID NO:1551). In some aspects, a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO: 1155) and a nucleic acid sequence of the sense strand comprises ugccuaaaUfgGfuGfaauaugca (SEQ ID NO:1665) or (invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) (SEQ ID NO:1682).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AUGAAACAAACAAACCCUGGA (SEQ ID NO:1440) and a sense strand comprising a nucleic acid sequence of UCCAGGGUUUGUUUGUUUCAU (SEQ ID NO:1595). In some aspects, a nucleic acid sequence of the antisense strand comprises asUfsgsAfaAfcaaacAfaAfcCfcUfggsa (SEQ ID NO:1048) and a nucleic acid sequence of the sense strand comprises uccaggguUfUfGfuuuguuucau (SEQ ID NO:1666) or (invAb)succaggguUfUfGfuuuguuucaus(invAb) (SEQ ID NO: 1683).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AGACGAUCAUACUUGGAGAGC (SEQ ID NO:1454) and a sense strand comprising a nucleic acid sequence of GCUCUCCAAGUAUGAUCIUCU (SEQ ID NO:1609). In some aspects, a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises gcucuccaAfGfUfaugauciucu (SEQ ID NO:1667) or (invAb)sgcucuccaAfGfUfaugauciucus(invAb) (SEQ ID NO:1684).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUGAAUGCUGAGAAAUACUC (SEQ ID NO:1438) and a sense strand comprising a nucleic acid sequence of GAGUAUUUCUCAGCAUUCAAA (SEQ ID NO:1593). In some aspects, a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and a nucleic acid sequence of the sense strand comprises gaguauuuCfUfCfagcauucaaa (SEQ ID NO:1668) or (invAb)sgaguauuuCfUfCfagcauucaaas(invAb) (SEQ ID NO:1685).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUCCAACAAUUCUCCUUGUC (SEQ ID NO:1466) and a sense strand comprising a nucleic acid sequence of GACAAGGAGAAUUGUUGGAAA (SEQ ID NO:1621). In some aspects, a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and a nucleic acid sequence of the sense strand comprises gacaaggaGfAfAfuuguuggaaa (SEQ ID NO:1669) or (invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) (SEQ ID NO:1686).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUGUCAACCUCACUCUUCCGA (SEQ ID NO:1465) and a sense strand comprising a nucleic acid sequence of UCGGAAGAGUGAGGUUGACAA (SEQ ID NO:1620). In some aspects, a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and a nucleic acid sequence of the sense strand comprises ucggaagaGfUfGfagguugacaa (SEQ ID NO:1670) or (invAb)sucggaagaGfUfGfagguugacaas(invAb) (SEQ ID NO:1687).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UCAUGAUACUGAGAGCUUGCU (SEQ ID NO:1464) and a sense strand comprising a nucleic acid sequence of AGCAAGCUCUCAGUAUCAUGA (SEQ ID NO:1619). In some aspects, a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and a nucleic acid sequence of the sense strand comprises agcaagcuCfUfCfaguaucauga (SEQ ID NO:1671) or (invAb)sagcaagcuCfUfCfaguaucaugas(invAb) (SEQ ID NO: 1688).
  • In some aspects, the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37)s.
  • In some aspects, the gene expression level and/or mRNA level of an XDH gene in a subject to whom a described XDH RNAi agent is administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the XDH RNAi agent or to a subject not receiving the XDH RNAi agent. The gene expression level and/or mRNA level in the subject may be reduced in a cell, group of cells, and/or tissue of the subject. In some aspects, the XDH gene expression is inhibited by at least about 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, or greater than 65% in the cytoplasm of hepatocytes relative to the subject prior to being administered the XDH RNAi agent or to a subject not receiving the XDH RNAi agent.
  • In some aspects, the XDH protein expression level in a subject to whom a described XDH RNAi agent has been administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the XDH RNAi agent or to a subject not receiving the XDH RNAi agent. The protein expression level in the subject may be reduced in a cell, group of cells, tissue, blood, and/or other fluid of the subject.
  • A reduction in XDH mRNA expression levels and XDH protein expression levels can be assessed by any methods known in the art. As used herein, a reduction or decrease in XDH mRNA level and/or protein level are collectively referred to herein as a reduction or decrease in XDH or inhibiting or reducing the gene expression of XDH. The Examples set forth herein illustrate known methods for assessing inhibition of XDH gene expression. The person of ordinary skill in the art would further know suitable methods for assessing inhibition of XDH gene expression in vivo and/or in vitro.
  • In some aspects, disclosed herein are methods of treatment (including prophylactic or preventative treatment) of diseases, disorders, or symptoms caused by caused by gout and/or hyperuricemia, wherein the methods include administering to a subject in need thereof a therapeutically effective amount of an XDH RNAi agent that includes an antisense strand that is at least partially complementary to the portion of the XDH mRNA having the sequence in Table 1. In some aspects, disclosed herein are methods of treatment (including prophylactic or preventative treatment) of diseases or symptoms caused by caused by gout or hyperuricemia, wherein the methods include administering to a subject in need thereof a therapeutically effective amount of an XDH RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Tables 2, 3 or 5C, and a sense strand that comprises any of the sequences in Tables 2, 4, or 5C that is at least partially complementary to the antisense strand. In some aspects, disclosed herein are methods of treatment (including prophylactic or preventative treatment) of diseases or symptoms caused by gout or hyperuricemia, wherein the methods include administering to a subject in need thereof a therapeutically effective amount of an XDH RNAi agent that includes a sense strand that comprises any of the sequences in Tables 2, 4, or 5C, and an antisense strand comprising the sequence of any of the sequences in Tables 2, 3, or 5C that is at least partially complementary to the sense strand.
  • In some aspects, the RNAi agent comprises an antisense strand comprising an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • In some aspects, The RNAi agent comprises an antisense strand comprising a modified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216. AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising a modified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • In some aspects, the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of UUCCAUAAUACUCUGAGAGAG (SEQ ID NO:1448) and a sense strand comprising a nucleic acid sequence of CUCUCUCAGAGUAUUAUGGAA (SEQ ID NO:1603). In some aspects, a nucleic acid sequence of the antisense strand comprises cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146) and a nucleic acid sequence of the sense strand comprises cucucucaGfaGfuAfuuauggaa (SEQ ID NO:1663) or (invAb)scucucucaGfaGfuAfuuauggaas(invAb) (SEQ ID NO:1680).
  • In some aspects, the RNAi agent comprises an antisense strand comprising a nucleic acid sequence of AUGACAAUAUCUGUGCGGAGG (SEQ ID NO:1468) and a sense strand comprising a nucleic acid sequence of CCUCCGCACAGAUAUUGUCAU (SEQ ID NO:1623). In some aspects, a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and a nucleic acid sequence of the sense strand comprises ccuccgcaCfAfGfauauugucau (SEQ ID NO:1664) or (invAb)sccuccgcaCfAfGfauauugucaus(invAb) (SEQ ID NO:1681).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UGCAUAUUCACCAUUUAGGCA (SEQ ID NO:1397) and a sense strand comprising a nucleic acid sequence of UGCCUAAAUGGUGAAUAUGCA (SEQ ID NO:1551). In some aspects, a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO:1155) and a nucleic acid sequence of the sense strand comprises ugccuaaaUfgGfuGfaauaugca (SEQ ID NO:1665) or (invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) (SEQ ID NO:1682).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AUGAAACAAACAAACCCUGGA (SEQ ID NO:1440) and a sense strand comprising a nucleic acid sequence of UCCAGGGUUUGUUUGUUUCAU (SEQ ID NO:1595). In some aspects, a nucleic acid sequence of the antisense strand comprises asUfsgsAfaAfcaaacAfaAfcCfcUfggsa (SEQ ID NO:1048) and a nucleic acid sequence of the sense strand comprises uccaggguUfUfGfuuuguuucau (SEQ ID NO: 1666) or (invAb)succaggguUfUfGfuuuguuucaus(invAb) (SEQ ID NO:1683).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of AGACGAUCAUACUUGGAGAGC (SEQ ID NO:1454) and a sense strand comprising a nucleic acid sequence of GCUCUCCAAGUAUGAUCIUCU (SEQ ID NO:1609). In some aspects, a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises gcucuccaAfGfUfaugauciucu (SEQ ID NO:1667) or (invAb)sgcucuccaAfGfUfaugauciucus(invAb) (SEQ ID NO: 1684).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUGAAUGCUGAGAAAUACUC (SEQ ID NO:1438) and a sense strand comprising a nucleic acid sequence of GAGUAUUUCUCAGCAUUCAAA (SEQ ID NO:1593). In some aspects, a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and a nucleic acid sequence of the sense strand comprises gaguauuuCfUffagcauucaaa (SEQ ID NO:1668) or (invAb)sgaguauuuCfUfCfagcauucaaas(invAb) (SEQ ID NO:1685).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUUCCAACAAUUCUCCUUGUC (SEQ ID NO:1466) and a sense strand comprising a nucleic acid sequence of GACAAGGAGAAUUGUUGGAAA (SEQ ID NO:1621). In some aspects, a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and a nucleic acid sequence of the sense strand comprises gacaaggaGfAfAfuuguuggaaa (SEQ ID NO:1669) or (invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) (SEQ ID NO:1686).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UUGUCAACCUCACUCUUCCGA (SEQ ID NO:1465) and a sense strand comprising a nucleic acid sequence of UCGGAAGAGUGAGGUUGACAA (SEQ ID NO:1620). In some aspects, a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and a nucleic acid sequence of the sense strand comprises ucggaagaGfUfGfagguugacaa (SEQ ID NO:1670) or (invAb)sucggaagaGfUfGfagguugacaas(invAb) (SEQ ID NO:1687).
  • In some aspects, the RNAi agent comprises an antisense sequence comprising a nucleic acid sequence of UCAUGAUACUGAGAGCUUGCU (SEQ ID NO:1464) and a sense strand comprising a nucleic acid sequence of AGCAAGCUCUCAGUAUCAUGA (SEQ ID NO:1619). In some aspects, a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and a nucleic acid sequence of the sense strand comprises agcaagcuCfUfCfaguaucauga (SEQ ID NO:1671) or (invAb)sagcaagcuCfUfCfaguaucaugas(invAb) (SEQ ID NO:1688).
  • In some aspects, the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37)s.
  • In some aspects, disclosed herein are methods for inhibiting expression of an XDH gene in a cell, wherein the methods include administering to the cell an XDH RNAi agent that includes an antisense strand that is at least partially complementary to the portion of the XDH mRNA having the sequence in Table 1. In some aspects, disclosed herein are methods of inhibiting expression of an XDH gene in a cell, wherein the methods include administering to a cell an XDH RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Tables 2, 3, or 5C and a sense strand that comprises any of the sequences in Tables 2, 4, or 5C that is at least partially complementary to the antisense strand. In some aspects, disclosed herein are methods of inhibiting expression of an XDH gene in a cell, wherein the methods include administering an XDH RNAi agent that includes a sense strand that comprises any of the sequences in Tables 2, 4, or 5C, and an antisense strand that includes the sequence of any of the sequences in Tables 2, 3, or 5C that is at least partially complementary to the sense strand.
  • In some aspects, the XDH RNAi agents are administered to a subject in need thereof as a first line therapy. In some aspects, the XDH RNAi agents are administered to a subject in need thereof as a second line therapy. In certain aspects, the XDH RNAi agents are administered as a second line therapy to patients who have failed one or more first line standard of care therapies. In certain aspects, the XDH RNAi agents are administered as a maintenance therapy following the administration of one or more prior therapies. In certain aspects, the XDH RNAi agents administered as a maintenance therapy following the administration of one or more standard of care therapies. In some aspects, the XDH RNAi agents administered in combination with one or more additional therapies. In some aspects, the one or more additional therapies is a standard of care therapy. In some aspects, the one or more additional therapies is an oral therapy.
  • Provided herein are methods for treating gout using the XDH RNAi agents described herein, for example, RNAi agent comprising an antisense strand comprising an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a sense strand comprising an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235. In some aspects, the gout is uncontrolled gout. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered as a second line therapy to patients who have failed allopurinol and/or febuxostat. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered prior to KRYSTEXXA. In some aspects, the oligonucleotide, composition, or pharmaceutical composition described herein is administered as a maintenance therapy following the administration of KRYSTEXXA.
  • The use of XDH RNAi agents provides methods for therapeutic (including prophylactic) treatment of diseases/disorders associated with gout, hyperuricemia, elevated serum uric acid levels, or elevated XDH gene expression. The described XDH RNAi agents mediate RNA interference to inhibit the expression of one or more genes necessary for production of XDH protein. XDH RNAi agents can also be used to treat or prevent various diseases, disorders, or conditions, including gout. Furthermore, compositions for delivery of XDH RNAi agents to liver cells, and specifically to hepatocytes, in vivo, are described.
    • Cells, Tissues, Organs, and Non-Human Organisms
  • Cells, tissues, organs, and non-human organisms that include at least one of the XDH RNAi agents described herein are contemplated. The cell, tissue, organ, or non-human organism is made by delivering the RNAi agent to the cell, tissue, organ or non-human organism.
    • Illustrative Embodiments
  • Provided here are illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached hereto.
  • Embodiment 1. An RNAi agent for inhibiting expression of an XDH gene, comprising:
  • an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3, nucleotides from any one of the sequences antisense strand sequences disclosed in Table 2, Table 3, or Table 5C; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.
  • Embodiment 2. An RNAi agent for inhibiting expression of an XDH gene, comprising:
  • a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from a stretch of the same length of nucleotides of SEQ ID NO: 1; and an antisense strand comprising a nucleotide sequences that is at least partially complementary to the sense strand.
  • Embodiment 3. The RNAi agent of embodiment 1, wherein the antisense strand comprises nucleotides at positions 2-18 of any one of the antisense strand sequences of Table 2, Table 3, or Table 5C.
  • Embodiment 4. The RNAi agent of embodiment 1 or embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides differing by 0 or 1 nucleotide from any one of the sense strand sequences of Table 2, Table 4, or Table 5C, and wherein the sense strand has a region of at least 85% complementarity over the 15 contiguous nucleotides to the antisense strand.
  • Embodiment 5. The RNAi agent of any one of embodiments 1-4, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.
  • Embodiment 6. The RNAi agent of any one of aspects 1-5, wherein all or substantially all of the nucleotides of the sense and/or antisense strand of the RNAi agent are modified nucleotides.
  • Embodiment 7. The RNAi agent of any one of aspects 5-6, wherein the modified nucleotide is selected from the group consisting of: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide, 2′,3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide, 2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxy nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3′-O-methyl nucleotide.
  • Embodiment 8. The RNAi agent of embodiment 7, wherein all or substantially all of the modified nucleotides are 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.
  • Embodiment 9. The RNAi agent of any one of aspects 1-8, wherein the antisense strand comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3 or Table 5C.
  • Embodiment 10. The RNAi agent of any one of aspects 1-9, wherein the sense strand comprises the nucleotide sequence of any of the modified sense strand sequences of Table 4 or Table 5C.
  • Embodiment 11. The RNAi agent of embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences of Table 5C and the sense strand comprises the nucleotide sequence of any one of the modified sequences of Table 5C.
  • Embodiment 12. The RNAi agent of any one of aspects 1-11, wherein the RNAi agent is linked to a targeting ligand.
  • Embodiment 13. The RNAi agent of embodiment 12, wherein the targeting ligand comprises n-acetyl-galactosamine.
  • Embodiment 14. The RNAi agent of embodiment 12 or 13, wherein the targeting ligand comprises the structure of (NAG37) or (NAG37)s.
  • Embodiment 15. The RNAi agent of any one of aspects 11-14, wherein the targeting ligand is linked to the sense strand.
  • Embodiment 16. The RNAi agent of embodiment 15, wherein the targeting ligand is linked to the 5′ terminal end of the sense strand.
  • Embodiment 17. The RNAi agent of any one of aspects 1-16, wherein the sense strand is between 15 and 30 nucleotides in length, and the antisense strand is between 18 and 30 nucleotides in length.
  • Embodiment 18. The RNAi agent of embodiment 17, wherein the sense strand and the antisense strand are each between 18 and 27 nucleotides in length.
  • Embodiment 19. The RNAi agent of embodiment 18, wherein the sense strand and the antisense strand are each between 18 and 24 nucleotides in length.
  • Embodiment 20. The RNAi agent of embodiment 19, wherein the sense strand and the antisense strand are each 21 nucleotides in length.
  • Embodiment 21. The RNAi agent of any one of aspects 17-20, wherein the RNAi agent has two blunt ends.
  • Embodiment 22. The RNAi agent of any one of aspects 1-21, wherein the sense strand comprises one or two terminal caps.
  • Embodiment 23. The RNAi agent of any one of aspects 1-22, wherein the sense strand comprises one or two inverted abasic residues.
  • Embodiment 24. The RNAi agent of embodiment 1, wherein the RNAi agent is comprised of a sense strand and an antisense strand that form a duplex sequence of any one of the duplexes as listed in Table 5A, Table 5B, or Table 5C.
  • Embodiment 25. The RNAi agent of any one of aspects 1-23, wherein the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
  • Embodiment 26. The RNAi agent of embodiment 1, comprising an antisense strand that comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the antisense strand nucleotide sequences of Table 3 or Table 5C, wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, cytidine, guanosine, and uridine, respectively; cPrpa and cPrpu represent 5′-cyclopropyl phosphonate-2′-O-methyl adenosine and 5′-cyclopropyl phosphonate-2′-O-methyl uridine, respectively; CUNA and UUNA represent 2′,3′-seco-cytidine and 2′,3′-seco-uridine, respectively; s represents a phosphorothioate linkage; and wherein all or substantially all of the nucleotides on the sense strand are modified nucleotides.
  • Embodiment 27. The RNAi agent of embodiment 1, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the nucleotide sequences of Table 4 or Table 5C, wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, cytidine, guanosine, and uridine, respectively, a_2N represents 2′-O-methyl-2-aminoadenosine; s represents a phosphorothioate linkage; and wherein all or substantially all of the nucleotides on the antisense strand are modified nucleotides.
  • Embodiment 28. The RNAi agent of any one of aspects 24-27, wherein the sense strand includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
  • Embodiment 29. The RNAi agent of any one of aspects 24-28, wherein the sense strand of the RNAi agent is linked to a targeting ligand.
  • Embodiment 30. The RNAi agent of embodiment 29, wherein the targeting ligand has affinity for the asialoglycoprotein receptor.
  • Embodiment 31. The RNAi agent of embodiment 30, wherein the targeting ligand comprises N-acetyl-galactosamine.
  • Embodiment 32. The RNAi agent of embodiment 1, wherein the targeting ligand comprises:
  • Figure US20230139166A1-20230504-C00022
  • Embodiment 33. The RNAi agent of embodiment 1, wherein the antisense strand consists of a modified nucleotide sequence of Table 3 or Table 5C and the sense strand consists of a modified nucleotide sequence of Table 4 or Table 5C, wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, cytidine, guanosine, and uridine, respectively; cPrpa and cPrpu represent 5′-cyclopropyl phosphonate-2′-O-methyl adenosine and 5′-cyclopropyl phosphonate-2′-O-methyl uridine, respectively; a_2N represents 2′-O-methyl-2-aminoadenosine; CUNA and UUNA represent 2′,3′-seco-cytidine and 2′,3′-seco-uridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted abasic deoxyribose residue; and (NAG37)s has the following chemical structure:
  • Figure US20230139166A1-20230504-C00023
  • Embodiment 34. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises an unmodified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a nucleic acid sequence of the sense strand comprises an unmodified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877, AD14239, AD14237, or AD14235.
  • Embodiment 35. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises a modified nucleic acid sequence of AM15135, AM14244, AM15149, AM13882, AM14216, AM14387, AM14240, AM14238, or AM14236, and a nucleic acid sequence of the sense strand comprises a modified nucleic acid sequence of AM14284, AM14243, AM14528, AM13881, AM14215, AM13877. AD14239, AD14237, or AD14235.
  • Embodiment 36. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUCCAUAAUACUCUGAGAGAG (SEQ ID NO:1448) and a nucleic acid sequence of the sense strand comprises CUCUCUCAGAGUAUUAUGGAA (SEQ ID NO:1603).
  • Embodiment 37. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146) and a nucleic acid sequence of the sense strand comprises cucucucaGfaGfuAfuuauggaa (SEQ ID NO:1663), wherein lower case (n)=2′-O-Me; Nf=2′-F; cPrpn=5′-cyclopropyl phosphonate-2′-O-methyl; (and s=phosphorothioate backbone modification.
  • Embodiment 38. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises cPrpusUfscCfauaauacUfcUfgAfgagsasg (SEQ ID NO:1146) and the sense strand comprises (invAb)scucucucaGfaGfuAfuuauggaas(invAb) (SEQ ID NO:1680) wherein lower case (n)=2′-O-Me; Nf=2′-F; cPrpn=5′-cyclopropyl phosphonate-2′-O-methyl; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 39. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises AUGACAAUAUCUGUGCGGAGG (SEQ ID NO:1468) and a nucleic acid sequence of the sense strand comprises CCUCCGCACAGAUAUUGUCAU (SEQ ID NO:1623).
  • Embodiment 40. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and a nucleic acid sequence of the sense strand comprises ccuccgcaCfAfGfauauugucau (SEQ ID NO:1664), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 41. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO:1081) and the sense strand comprises (invAb)sccuccgcaCfAfGfauauugucaus(invAb) (SEQ ID NO:1681) wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 42. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UGCAUAUUCACCAUUUAGGCA (SEQ ID NO:1397) and a nucleic acid sequence of the sense strand comprises UGCCUAAAUGGUGAAUAUGCA (SEQ ID NO:1551).
  • Embodiment 43. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO:1155) and a nucleic acid sequence of the sense strand comprises ugccuaaaUfgGfuGfaauaugca (SEQ ID NO:1665), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 44. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises cPrpusGfscauauuCfacCfaUfuUfaggscsa (SEQ ID NO: 1155) and the sense strand comprises (invAb)sugccuaaaUfgGfuGfaauaugcas(invAb) (SEQ ID NO:1682), wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 45. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises AUGAAACAAACAAACCCUGGA (SEQ ID NO:1440) and a nucleic acid sequence of the sense strand comprises UCCAGGGUUUGUUUGUUUCAU (SEQ ID NO:1595).
  • Embodiment 46. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises asUfsgsAfaAfcaaacAfaAfcCfcUfggsa (SEQ ID NO:1048) and a nucleic acid sequence of the sense strand comprises uccaggguUfUfGfuuuguuucau (SEQ ID NO:1666), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 47. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises asUfsgsAfaAfcaaacAfaAfcCfcUfggsa (SEQ ID NO:1048) and the sense strand comprises (invAb)succaggguUfUfGfuuuguuucaus(invAb)(SEQ ID NO:1683), wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 48. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises AGACGAUCAUACUUGGAGAGC (SEQ ID NO:1454) and a nucleic acid sequence of the sense strand comprises GCUCUCCAAGUAUGAUCIUCU (SEQ ID NO:1609).
  • Embodiment 49. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and a nucleic acid sequence of the sense strand comprises gcucuccaAfGfUfaugauciucu (SEQ ID NO:1667), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 50. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises asGfsasCfgaucauaCfuUfgGfagagsc (SEQ ID NO:1067) and the sense strand comprises (invAb)sgcucuccaAfGfUfaugauciucus(invAb) (SEQ ID NO:1684), wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 51. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUUGAAUGCUGAGAAAUACUC (SEQ ID NO:1438) and a nucleic acid sequence of the sense strand comprises GAGUAUUUCUCAGCAUUCAAA (SEQ ID NO:1593).
  • Embodiment 52. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and a nucleic acid sequence of the sense strand comprises gaguauuuCfUfCfagcauucaaa (SEQ ID NO:1668), wherein lower case (n)=2′-O-Me; Nf=2′-F; cPrpn=5′-cyclopropyl phosphonate-2′-O-methyl; and s=phosphorothioate backbone modification.
  • Embodiment 53. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises cPrpuUfuGfaaugcugAfgAfaAfuacusc (SEQ ID NO:1111) and the sense strand comprises (invAb)sgaguauuuCfUfCfagcauucaaas(invAb) (SEQ ID NO:1685), wherein lower case (n)=2′-O-Me; Nf=2′-F; cPrpn=5′-cyclopropyl phosphonate-2′-O-methyl; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 54. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUUCCAACAAUUCUCCUUGUC (SEQ ID NO:1466) and a nucleic acid sequence of the sense strand comprises GACAAGGAGAAUUGUUGGAAA (SEQ ID NO:1621).
  • Embodiment 55. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and a nucleic acid sequence of the sense strand comprises gacaaggaGfAfAfuuguuggaaa (SEQ ID NO:1669), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 56. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises usUfsusCfcaacaauUfcUfcCfuugusc (SEQ ID NO:1079) and the sense strand comprises (invAb)sgacaaggaGfAfAfuuguuggaaas(invAb) (SEQ ID NO:1686), wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 57. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UUGUCAACCUCACUCUUCCGA (SEQ ID NO:1465) and a nucleic acid sequence of the sense strand comprises UCGGAAGAGUGAGGUUGACAA (SEQ ID NO:1620).
  • Embodiment 58. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and a nucleic acid sequence of the sense strand comprises ucggaagaGfUfGfagguugacaa (SEQ ID NO:1670), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 59. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises usUfsgsUfcaaccucAfcUfcUfuccgsa (SEQ ID NO:1078) and the sense strand comprises (invAb)sucggaagaGfUfGfagguugacaas(invAb) (SEQ ID NO:1687), wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 60. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises UCAUGAUACUGAGAGCUUGCU (SEQ ID NO:14(4) and a nucleic acid sequence of the sense strand comprises AGCAAGCUCUCAGUAUCAUGA (SEQ ID NO:1619).
  • Embodiment 61. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and a nucleic acid sequence of the sense strand comprises agcaagcuCfUfCfaguaucauga (SEQ ID NO:1671), wherein lower case (n)=2′-O-Me; Nf=2′-F; and s=phosphorothioate backbone modification.
  • Embodiment 62. The RNAi agent of any one of embodiments 1-3, wherein a nucleic acid sequence of the antisense strand comprises usCfsasUfgauacugAfgAfgCfuugcsu (SEQ ID NO:1077) and the sense strand comprises (invAb)sagcaagcuCfUfCfaguaucaugas(invAb) (SEQ ID NO:1688), wherein lower case (n)=2′-O-Me; Nf=2′-F; (invAb)=inverted abasic residue; and s=phosphorothioate backbone modification.
  • Embodiment 63. The RNAi agent of any one of embodiments 31-62, wherein the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37) or (NAG37)s.
  • Embodiment 64. The RNAi agent of any one of embodiments 31-62, wherein the 5′ end of the sense strand is coupled to a targeting ligand comprising the structure of (NAG37)s.
  • Embodiment 65. The RNAi agent of any one of embodiments 31-64, wherein RNAi agent is a pharmaceutically acceptable salt.
  • Embodiment 66. A composition comprising the RNAi agent of any one of embodiments 1-65, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • Embodiment 67. A method for inhibiting expression of an XDH gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of embodiments 1-66 or the composition of embodiment 66.
  • Embodiment 68. The method of embodiment 67, wherein the cell is within a subject.
  • Embodiment 69. The method of embodiment 68, wherein the subject is a human subject.
  • Embodiment 70. The method of any one of embodiments 67-69, wherein the XDH gene expression is inhibited by at least about 30%.
  • Embodiment 71. The method of any one of embodiments 67-70, wherein the XDH activity is reduced by at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.
  • Embodiment 72. A method of treating an XDH-related disease, disorder, or symptom, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the composition of embodiment 66.
  • Embodiment 73. The method of embodiment 72, wherein the disease is gout.
  • Embodiment 74. The method of any one of embodiments 67-73, wherein the RNAi agent is administered at a dose of about 0.05 mg/kg to about 5.0 mg/kg of body weight of the human subject.
  • Embodiment 75. The method of any one of embodiments 67-74, wherein the RNAi agent is administered in two or more doses.
  • Embodiment 76. A single-stranded antisense compound for inhibiting an XDH gene, comprising an antisense nucleotide sequence having at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides, wherein the nucleotides are complementary to any of the target nucleotide sequences of Table 1.
  • Embodiment 77. A single-stranded antisense compound for inhibiting an XDH gene, comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides of any of the antisense strand sequences disclosed in Table 2, Table 3, or Table 5C.
  • The above provided embodiments and items are now illustrated with the following, non-limiting examples.
    • Examples Example 1. Synthesis of XDH RNAi Agents
  • XDH RNAi agent duplexes shown in Tables 5A, 5B, and 5C, above, were synthesized in accordance with the following general procedures:
    • A. Synthesis.
  • The sense and antisense strands of the RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Such standard synthesis is generally known in the art. Depending on the scale, either a MerMade96E® (Bioautomation), a MerMade121, (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston. Pa., USA). The monomer positioned at the 3′ end of the respective strand was attached to the solid support as a starting point for synthesis. All RNA and 2′-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, Wis., USA) or Hongene Biotech (Shanghai, PRC). The 2′-O-methyl phosphoramidites included the following: (5′-O-dimethoxytrityl-N-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphommidite, 5′-O-dimethoxy-trityl-N4-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5′-O-dimethoxytrityl-N2-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2′-deoxy-2′-fluoro-phosphoramidites carried the same protecting groups as the 2′-O-methyl amidites, 5′-(4,4′-Dimethoxytrityl)-2′,3′-seco-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was also purchased from Thermo Fisher Scientific or Hongene Biotech. 5′-dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia) or Hongene Biotech. The cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017/214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)). The inverted abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, Mass., USA) or SAFC (St Louis, Mo., USA). 5′-O-dimethoxytrityl-N2,N6-(phenoxvacetate)-2′-O-methyl-diaminopurine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were obtained from ChemGenes or Hongene Biotech.
  • Targeting ligand-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), or anhydrous dimethylformamide and molecular sieves (3 Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 12 min (RNA), 15 min (targeting ligand), 90 sec (2′-OMe), and 60 sec (2′-F). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, Mass., USA) in anhydrous Acetonitrile was employed. Unless specifically identified as a “naked” RNAi agent having no targeting ligand present, each of the XDH RNAi agent duplexes synthesized and tested in the following Examples utilized N-acetyl-galactosamine as “NAG” in the targeting ligand chemical structures represented in Table 6. (NAG37) and (NAG37)s targeting ligand phosphoramidite compounds can be synthesized in accordance with International Patent Application Publication No. WO 2018/044350 to Arrowhead Pharmaceuticals, Inc.
    • B. Cleavage and Deprotection of Support Bound Oligomer
  • After finalization of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt. % methylamine in water and 28% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below).
    • C. Purification.
  • Crude oligomers were purified by anionic exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 26/40 column packed with Sephadex G-25 fine with a running buffer of filtered DI water or 100 mM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile.
    • D. Annealing.
  • Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1× Phosphate-Buffered Saline (Corning, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at −15 to −25° C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 Phosphate-Buffered Saline. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the duplex concentration. The conversion factor used was either 0.050 mg/(mL-cm) or was calculated from an experimentally determined extinction coefficient.
    • Example 2. XDH-GLuc AAV Mouse Model
  • To evaluate certain XDH RNAi agents, an XDH-GLuc (Gaussia Luciferase) AAV (Adeno-associated virus) mouse model was used. Six- to eight-week-old male C57BL/6 mice were transduced with XDH-GLuc AAV serotype 8, administered at least 14 days prior to administration of an XDH RNAi agent or control. Two types of XDH-GLuc AAV were used. The genome of the first XDH-GLuc AAV contains the 80-2899 region of the human XDH cDNA sequence (GenBank NM_000379.4 (SEQ ID NO:1)) inserted into the 3′ UTR of the GLuc reporter gene sequence. The genome of the second XDH-GLuc AAV contains the 2820-5715 region of the human XDH cDNA sequence (GenBank NM_000379.4 (SEQ ID NO:1)) inserted into the 3′ UTR of the GLuc reporter gene sequence. 5E12 to 1E13 GC/kg of the respective virus in PBS in a total volume of 10 mL/kg animal's body weight was injected into mice via the tail vein to create XDH-GLuc AAV model mice. Inhibition of expression of XDH by an XDH RNAi agent results in concomitant inhibition of GLuc expression, which is measured. Prior to administration of a treatment (between day −7 and day 1 pre-dose), GLuc expression levels in serum were measured by the Pierce™ Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific), and the mice were grouped according to average GLuc levels.
  • Mice were anesthetized with 2-3% isoflurane and blood samples were collected from the submandibular area into serum separation tubes (Sarstedt AG & Co., Nümbrecht, Germany). Blood was allowed to coagulate at ambient temperature for 20 min. The tubes were centrifuged at 8,000×g for 3 min to separate the serum and stored at 4° C. Serum was collected and measured by the Pierce™ Gaussia Luciferase Glow Assay Kit according to the manufacturer's instructions. Serum GLuc levels for each animal can be normalized to the control group of mice injected with vehicle control in order to account for the non-treatment related shift in XDH expression with this model. To do so, first, the GLuc level for each animal at a time point was divided by the pre-treatment level of expression in that animal (Day 1) in order to determine the ratio of expression “normalized to pre-treatment”. Expression at a specific time point was then normalized to the control group by dividing the “normalized to pre-treatment” ratio for an individual animal by the average “normalized to pre-treatment” ratio of all mice in the normal vehicle control group. Alternatively, the serum GLuc levels for each animal was assessed by normalizing to pre-treatment levels only.
    • Example 3. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 7.
  • TABLE 7
    Targeted Positions and Dosing Groups of Example 3
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 488 2.0 mg/kg AD09218 Single injection on day 1
    3 122 2.0 mg/kg AD09724 Single injection on day 1
    4 249 2.0 mg/kg AD09599 Single injection on day 1
    5 252 2.0 mg/kg AD09600 Single injection on day 1
    6 1285 2.0 mg/kg AD09733 Single injection on day 1
    7 2209 2.0 mg/kg AD09740 Single injection on day 1
    8 1963 2.0 mg/kg AD09736 Single injection on day 1
    9 1963 2.0 mg/kg AD09937 Single injection on day 1
    10 2696 2.0 mg/kg AD09744 Single injection on day 1
    11 2696 2.0 mg/kg AD09938 Single injection on day 1
    12 2616 2.0 mg/kg AD09663 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agent AD09218 (Group 2) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 488 of the gene; the XDH RNAi agent AD09724 (Group 3) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 122 of the gene; the XDH RNAi agent AD09599 (Group 4) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 249 of the gene; the XDH RNAi agent AD09600 (Group 5) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 252 of the gene; the XDH RNAi agent AD09733 (Group 6) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1285 of the gene; the XDH RNAi agent AD09740 (Group 7) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2209 of the gene; the XDH RNAi agents AD09736 (Group 8) and AD09937 (Group 9) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene; the XDH RNAi agents AD09744 (Group 10) and AD09938 (Group 11) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2696 of the gene; and the XDH RNAi agent AD09663 (Group 12) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2616 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • While it has been previously reported that an RNAi agent targeting position 488 of the XDH gene can be active in vitro and in vivo in mice and in rats, the nucleotide sequence of an RNAi agent targeting this position is compromised and unsuitable for therapeutic use. More specifically, the seed region (2 to 7 nt) of the RNAi agent targeting position 488 matches perfectly with that of a known human microRNA (miRNA), thus this agent is expected to result in undesired silencing of hundreds of potential off-targets mimicking the known miRNA (See, e.g., Kamola et al., The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects, 11(12) PLoS Comput Biol (2015)). In addition, the core 17-mer sequence (nucleotides located at positions 2-18 of the antisense strand (5′→3′)) of the RNAi agent targeting position 488 is complementary to transcripts of four human genes with only one mismatch, hence bearing an additional risk of reducing the expression of these four genes through a different off-target mechanism. Thus, the RNAi agent of Group 2 is not a viable candidate for human therapeutic treatment.
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment are shown in the following Table 8:
  • TABLE 8
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 3
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1  1.000 0.105 1.000 0.020 1.000 0.096
    (Saline vehicle)
    Group 2  0.601 0.094 0.505 0.085 0.531 0.103
    (2.0 mg/kg AD09218)
    Group 3  1.115 0.149 0.890 0.095 0.964 0.208
    (2.0 mg/kg AD09724)
    Group 4  1.009 0.088 0.872 0.096 0.991 0.092
    (2.0 mg/kg AD09599)
    Group 5  0.874 0.292 0.865 0.415 0.927 0.348
    (2.0 mg/kg AD09600)
    Group 6  1.024 0.054 0.896 0.129 1.209 0.262
    (2.0 mg/kg AD09733)
    Group 7  0.963 0.083 0.793 0.103 1.132 0.084
    (2.0 mg/kg AD09740)
    Group 8  0.607 0.154 0.521 0.111 0.809 0.135
    (2.0 mg/kg AD09736)
    Group 9  0.673 0.148 0.593 0.120 0.748 0.108
    (2.0 mg/kg AD09937)
    Group 10 0.679 0.084 0.694 0.078 0.934 0.163
    (2.0 mg/kg AD09744)
    Group 11 0.552 0.076 0.478 0.076 0.711 0.095
    (2.0 mg/kg AD09938)
    Group 12 0.826 0.102 0.849 0.435 1.246 0.895
    (2.0 mg/kg AD09663)

    As shown in Table 8, above, as expected the RNAi agent of Group 2 (targeting position 488) was active and showed reductions of approximately 49.5% on day 15 (0.505). The RNAi agents of Group 8 (AD09736) and Group 9 (AD09937), both of which target the XDH gene at position 1963, showed generally comparable reductions of XDH (reductions of 47.9% and 40.7% on day 15, respectively) with Group 2. Similarly, the RNAi agents of Group 10 (AD09744) and Group 11 (AD09938), both of which target the XDH gene at position 2696, showed generally comparable reductions of XDH (showing reductions of 30.6% and 52.2%) with Group 2.
    • Example 4. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 9.
  • TABLE 9
    Targeted Positions and Dosing Groups of Example 4
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 1963 2.0 mg/kg AD09736 Single injection on day 1
    3 1963 2.0 mg/kg AD09965 Single injection on day 1
    4 1963 2.0 mg/kg AD09937 Single injection on day 1
    5 1963 2.0 mg/kg AD09966 Single injection on day 1
    6 1963 2.0 mg/kg AD09967 Single injection on day 1
    7 1963 2.0 mg/kg AD09968 Single injection on day 1
    8 1963 2.0 mg/kg AD09969 Single injection on day 1
    9 1963 2.0 mg/kg AD09970 Single injection on day 1
    10 1964 2.0 mg/kg AD09962 Single injection on day 1
    11 1965 2.0 mg/kg AD09963 Single injection on day 1
    12 1967 2.0 mg/kg AD09964 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents AD09736 (Group 2), AD09965 (Group 3), AD09937 (Group 4), AD09966 (Group 5), AD09967 (Group 6), AD09%8 (Group 7), AD09969 (Group 8), and AD09970 (Group 9) all included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene; the XDH RNAi agent AD09962 (Group 10) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1964 of the gene; the XDH RNAi agent AD09963 (Group 11) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1965 of the gene; and the XDH RNAi agent AD0964 (Group 12) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1967 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment are shown in the following Table 10:
  • TABLE 10
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 4
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.136 1.000 0.205 1.000 0.110
    Group 2 (2.0 mg/kg AD09218) 0.625 0.146 0.603 0.078 0.642 0.066
    Group 3 (2.0 mg/kg AD09965) 0.812 0.143 0.623 0.182 0.670 0.198
    Group 4 (2.0 mg/kg AD09937) 0.502 0.045 0.581 0.183 0.528 0.099
    Group 5 (2.0 mg/kg AD09966) 0.486 0.093 0.469 0.173 0.502 0.207
    Group 6 (2.0 mg/kg AD09967) 0.644 0.065 0.490 0.141 0.483 0.084
    Group 7 (2.0 mg/kg AD09968) 0.551 0.244 0.599 0.234 0.554 0.168
    Group 8 (2.0 mg/kg AD09969) 0.603 0.105 0.573 0.078 0.611 0.118
    Group 9 (2.0 mg/kg AD09970) 0.659 0.228 0.618 0.230 0.621 0.110
    Group 10 (2.0 mg/kg AD09962) 0.820 0.161 0.818 0.132 0.744 0.093
    Group 11 (2.0 mg/kg AD09963) 0.793 0.061 0.743 0.065 0.722 0.095
    Group 12 (2.0 mg/kg AD09664) 0.836 0.088 0.783 0.146 0.683 0.058
  • As shown in Table 10, above, the RNAi agents of Groups 2-9, which all included nucleotide sequences targeting position 1963 of the XDH gene, reported substantial inhibitory activity, with certain XDH RNAi agents achieving greater than 50% inhibition in vivo. Further, the XDH RNAi agents of each of Groups 2-9, all of which target position 1963 of the XDH gene, generally showed an increase in inhibition of XDH gene expression of approximately 20-35% compared to sequences targeting neighboring positions of an XDH gene, shown in Groups 10-12 (Compare, for example, Group 5 (AD09600) at day 15 showing 53.1% inhibition (0.469) with Groups 10-12 at day 15 showing 18.2% inhibition (0.818); 25.7% inhibition (0.743); and 21.7% inhibition (0.783), respectively).
    • Example 5. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 11.
  • TABLE 11
    Targeted Positions and Dosing Groups of Example 5
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 2696 2.0 mg/kg AD09744 Single injection on day 1
    3 2696 2.0 mg/kg AD09938 Single injection on day 1
    4 2696 2.0 mg/kg AD10008 Single injection on day 1
    5 2696 2.0 mg/kg AD10009 Single injection on day 1
    6 2696 2.0 mg/kg AD10010 Single injection on day 1
    7 2696 2.0 mg/kg AD10011 Single injection on day 1
    8 2696 2.0 mg/kg AD10012 Single injection on day 1
    9 2696 2.0 mg/kg AD10013 Single injection on day 1
    10 2696 2.0 mg/kg AD10014 Single injection on day 1
    11 2696 2.0 mg/kg AD10015 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents of Groups 2-11 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2696 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8 (and planned to be collected on days 15, and day 22), and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 22 are shown in the following Table 12:
  • TABLE 12
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 5
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.183 1.000 0.274 1.000 0.213
    Group 2 (2.0 mg/kg AD09744) 0.818 0.161 0.615 0.092 0.800 0.255
    Group 3 (2.0 mg/kg AD09938) 0.669 0.120 0.606 0.099 0.699 0.128
    Group 4 (2.0 mg/kg AD10008) 0.786 0.140 0.627 0.248 0.744 0.102
    Group 5 (2.0 mg/kg AD10009) 0.671 0.364 0.457 0.133 0.550 0.241
    Group 6 (2.0 mg/kg AD10010) 0.591 0.134 0.535 0.103 0.494 0.105
    Group 7 (2.0 mg/kg AD10011) 0.589 0.280 0.432 0.169 0.546 0.144
    Group 8 (2.0 mg/kg AD10012) 0.362 0.077 0.295 0.055 0.369 0.029
    Group 9 (2.0 mg/kg AD10013) 0.393 0.073 0.482 0.054 0.577 0.061
    Group 10 (2.0 mg/kg AD10014) 0.423 0.055 0.426 0.082 0.548 0.100
    Group 11 (2.0 mg/kg AD10015) 0.502 0.034 0.477 0.056 0.535 0.077

    As shown in Table 12, each of the RNAi agents of Groups 2-11, which all included nucleotide sequences targeting position 2696 of the XDH gene, reported substantial inhibitory activity of XDH gene expression.
    • Example 6. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 13.
  • TABLE 13
    Targeted Positions and Dosing Groups of Example 6
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 488 2.0 mg/kg AD09218 Single injection on day 1
    3 231 2.0 mg/kg AD10016 Single injection on day 1
    4 242 2.0 mg/kg AD10017 Single injection on day 1
    5 1322 2.0 mg/kg AD09734 Single injection on day 1
    6 1322 2.0 mg/kg AD10091 Single injection on day 1
    7 1322 2.0 mg/kg AD10092 Single injection on day 1
    8 1322 2.0 mg/kg AD10093 Single injection on day 1
    9 1322 2.0 mg/kg AD10094 Single injection on day 1
    10 1322 2.0 mg/kg AD10095 Single injection on day 1
    11 1322 2.0 mg/kg AD10096 Single injection on day 1
    12 1322 2.0 mg/kg AD10097 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agent AD09218 (Group 2) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 488 of the gene; the XDH RNAi agent AD10016 (Group 3) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 231 of the gene; the XDH RNAi agent AD10017 (Group 4) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 242 of the gene; and the XDH RNAi agents AD09734 (Group 5), AD10091 (Group 6), AD10092 (Group 7), AD10093 (Group 8), AD10094 (Group 9), AD10095 (Group 10), AD10096 (Group 11), and AD10097 (Group 12) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1322 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • As noted in Example 3, above, the RNAi agent targeting position 488 of the XDH gene (Group 2), while previously reported to be active in vivo in mice and rats, includes a compromised nucleotide sequence and is unsuitable for therapeutic use due to toxicity concerns.
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8 (and planned for days 15 and day 22), and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 8 are shown in the following Table 14:
  • TABLE 14
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 6
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.069 1.000 0.046 1.000 0.058
    Group 2 (2.0 mg/kg AD09218) 0.550 0.223 0.489 0.204 0.461 0.116
    Group 3 (2.0 mg/kg AD10016) 0.652 0.098 0.700 0.115 0.620 0.092
    Group 4 (2.0 mg/kg AD10017) 0.645 0.085 0.640 0.154 0.632 0.064
    Group 5 (2.0 mg/kg AD09734) 0.718 0.059 0.705 0.119 0.632 0.087
    Group 6 (2.0 mg/kg AD10091) 0.673 0.112 0.757 0.157 0.673 0.100
    Group 7 (2.0 mg/kg AD10092) 0.757 0.031 0.694 0.085 0.633 0.089
    Group 8 (2.0 mg/kg AD10093) 0.717 0.039 0.752 0.117 0.634 0.082
    Group 9 (2.0 mg/kg AD10094) 0.728 0.071 0.727 0.219 0.664 0.106
    Group 10 (2.0 mg/kg AD10095) 0.805 0.193 0.776 0.110 0.767 0.170
    Group 11 (2.0 mg/kg AD10096) 0.536 0.044 0.587 0.147 0.561 0.093
    Group 12 (2.0 mg/kg AD10097) 0.839 0.383 0.952 0.450 1.033 0.632
    • Example 7. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 15.
  • TABLE 15
    Targeted Positions and Dosing Groups of Example 7
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 3083 2.0 mg/kg AD09325 Single injection on day 1
    3 2995 2.0 mg/kg AD09981 Single injection on day 1
    4 3016 2.0 mg/kg AD09982 Single injection on day 1
    5 3041 2.0 mg/kg AD09983 Single injection on day 1
    6 3498 2.0 mg/kg AD09984 Single injection on day 1
    7 3598 2.0 mg/kg AD09985 Single injection on day 1
    8 3877 2.0 mg/kg AD09987 Single injection on day 1
    9 4394 2.0 mg/kg AD09989 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agent AD09325 (Group 2) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3083 of the gene; the XDH RNAi agent AD09981 (Group 3) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 2995 of the gene; the XDH RNAi agent AD09982 (Group 4) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3016 of the gene; the XDH RNAi agent AD09983 (Group 5) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3041 of the gene; the XDH RNAi agent AD09984 (Group 6) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3498 of the gene; the XDH RNAi agent AD09985 (Group 7) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3598 of the gene; the XDH RNAi agent AD09987 (Group 8) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3877 of the gene; and the XDH RNAi agent AD09989 (Group 9) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4394 of the gene. (See, e.g., SEQ ID NO: 1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment are shown in the following Table 16:
  • TABLE 16
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 7
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.375 1.000 0.397 1.000 0.397
    Group 2 (2.0 mg/kg AD09325) 0.513 0.078 0.823 0.154 0.823 0.154
    Group 3 (2.0 mg/kg AD09981) 0.600 0.040 0.681 0.129 0.681 0.129
    Group 4 (2.0 mg/kg AD09982) 0.592 0.058 0.631 0.137 0.631 0.137
    Group 5 (2.0 mg/kg AD09983) 0.596 0.066 0.574 0.087 0.574 0.087
    Group 6 (2.0 mg/kg AD09984) 0.724 0.043 0.941 0.221 0.941 0.221
    Group 7 (2.0 mg/kg AD09985) 0.472 0.076 0.449 0.092 0.449 0.092
    Group 8 (2.0 mg/kg AD09987) 0.691 0.225 0.751 0.149 0.751 0.149
    Group 9 (2.0 mg/kg AD09989) 0.585 0.076 0.757 0.120 0.757 0.120

    As shown in Table 16, each of the RNAi agents of Groups 2-9, reported inhibition of XDH gene expression.
    • Example 8. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 17.
  • TABLE 17
    Targeted Positions and Dosing Groups of Example 8
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 3083 2.0 mg/kg AD09325 Single injection on day 1
    3 3600 2.0 mg/kg AD09986 Single injection on day 1
    4 3930 2.0 mg/kg AD09988 Single injection on day 1
    5 4513 2.0 mg/kg AD09990 Single injection on day 1
    6 4531 2.0 mg/kg AD09991 Single injection on day 1
    7 4666 2.0 mg/kg AD09992 Single injection on day 1
    8 4843 2.0 mg/kg AD09993 Single injection on day 1
    9 5234 2.0 mg/kg AD09994 Single injection on day 1
    10 5411 2.0 mg/kg AD09995 Single injection on day 1
    11 4136 2.0 mg/kg AD09608 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-11 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at the specific positions of the gene as set forth in Table 17, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8 (and planned to be collected on days 15, and day 22), and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 8 are shown in the following Table 18:
  • TABLE 18
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 8
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.119 1.000 0.059 1.000 0.177
    Group 2 (2.0 mg/kg AD09325) 0.650 0.022 0.628 0.083 0.548 0.143
    Group 3 (2.0 mg/kg AD09986) 0.999 0.145 0.628 0.090 0.625 0.086
    Group 4 (2.0 mg/kg AD09988) 0.616 0.163 0.746 0.284 0.756 0.149
    Group 5 (2.0 mg/kg AD09990) 0.617 0.190 0.901 0.197 0.971 0.283
    Group 6 (2.0 mg/kg AD09991) 0.883 0.154 0.782 0.134 0.728 0.156
    Group 7 (2.0 mg/kg AD09992) 1.020 0.074 0.808 0.039 0.788 0.074
    Group 8 (2.0 mg/kg AD09993) 0.961 0.048 0.775 0.122 0.831 0.169
    Group 9 (2.0 mg/kg AD09994) 1.334 0.237 1.005 0.121 1.193 0.357
    Group 10 (2.0 mg/kg AD09995) 0.795 0.095 0.729 0.120 0.777 0.137
    Group 11 (2.0 mg/kg AD09608) 0.993 0.103 0.744 0.267 0.435 0.088
    • Example 9. In Vivo Testing of XDH RNAi Agents in Wild-Type Mice
  • Certain XDH RNAi agents have sufficient homology with the mouse XDH gene transcript that they are suitable to be examined for XDH gene expression inhibitory activity in wild-type mice. At day 1, six- to eight-week-old female C57bl/6 mice were given a single subcutaneous administration of 200 μl/20 g animal weight containing 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 19.
  • TABLE 19
    Targeted Positions and Dosing Groups of Example 9
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 488 1.0 mg/kg AD09217 Single injection on day 1
    3 488 1.0 mg/kg AD09218 Single injection on day 1
    4 1612 1.0 mg/kg AD09219 Single injection on day 1
    5 1614 1.0 mg/kg AD09220 Single injection on day 1
    6 1617 1.0 mg/kg AD09221 Single injection on day 1
    7 2128 1.0 mg/kg AD09222 Single injection on day 1
    8 2130 1.0 mg/kg AD09223 Single injection on day 1
    9 2131 1.0 mg/kg AD09224 Single injection on day 1
    10 2132 1.0 mg/kg AD09225 Single injection on day 1
    11 2153 1.0 mg/kg AD09226 Single injection on day 1
    12 2185 1.0 mg/kg AD09227 Single injection on day 1
    13 2186 1.0 mg/kg AD09228 Single injection on day 1
    14 3272 1.0 mg/kg AD09229 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 51B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-14 each included nucleotide sequences that, while also being homologous to the mouse XDH gene transcript, were designed to inhibit expression of an XDH gene at the specific positions of the human XDH gene as set forth in Table 19, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Mice were euthanized on study day 10, and total RNA was isolated from both livers following collection and homogenization. Mouse XDH mRNA expression was quantitated by probe-based quantitative PCR, normalized to mouse beta-actin expression, and expressed as fraction of vehicle control group (geometric mean, +/−95% confidence interval).
  • TABLE 20
    Average Relative Mouse XDH
    mRNA at Sacrifice (Day 10) in Example 9
    Average Relative Low High
    Group ID mXDH mRNA (error) (error)
    Group 1 (No Treatment) 1.000 0.197 0.246
    Group 2 (1.0 mg/kg AD09217) 0.600 0.100 0.119
    Group 3 (1.0 mg/kg AD09218) 0.628 0.132 0.167
    Group 4 (1.0 mg/kg AD09219) 0.649 0.071 0.080
    Group 5 (1.0 mg/kg AD09220) 0.943 0.157 0.188
    Group 6 (1.0 mg/kg AD09221) 1.174 0.205 0.249
    Group 7 (1.0 mg/kg AD09222) 1.098 0.242 0.310
    Group 8 (1.0 mg/kg AD09223) 1.196 0.191 0.228
    Group 9 (1.0 mg/kg AD09224) 1.348 0.179 0.207
    Group 10 (1.0 mg/kg AD09225) 1.663 0.241 0.281
    Group 11 (1.0 mg/kg AD09226) 1.711 0.126 0.136
    Group 12 (1.0 mg/kg AD09227) 0.912 0.047 0.050
    Group 13 (1.0 mg/kg AD09228) 0.983 0.114 0.128
    Group 14 (1.0 mg/kg AD09229) 1.023 0.117 0.132
  • The data were normalized to the non-treatment group (Group 1). As noted above in, for example, Example 3, the RNAi agent targeting position 488 of the XDH gene of Group 2 (AD09217) and Group 3 (AD09218), while being previously identified as having activity in mice and rats in vivo, includes a compromised nucleotide sequence and is unsuitable for therapeutic use due to toxicity concerns. As shown in Table 20, above, the XDH RNAi agent AD09219 (Group 4), which targets position 1612 of the XDH gene transcript, showed mRNA reductions of approximately 35.1% (0.649) in mice, which was generally comparable to the reductions exhibited by the XDH RNAi agents of Group 2 (40% inhibition (0.600)) and Group 3 (37.2% inhibition; (0.628)), which both included RNAi agents having sequences targeting position 488 of the XDH gene which as noted above has toxicity concerns.
    • Example 10. In Vivo Testing of XDH RNAi Agents in Wild-Type Mice
  • Certain XDH RNAi agents have sufficient homology with the mouse XDH gene transcript that they are suitable to be examined for XDH gene expression inhibitory activity in wild-type mice. At day 1 six- to eight-week-old male C57bl/6 mice were given a single subcutaneous administration of 200 μl/20 g animal weight containing 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 21.
  • TABLE 21
    Targeted Positions and Dosing Groups of Example 10
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 1612 1.0 mg/kg AD09219 Single injection on day 1
    3 1612 1.0 mg/kg AD10021 Single injection on day 1
    4 1612 1.0 mg/kg AD10022 Single injection on day 1
    5 1612 1.0 mg/kg AD10023 Single injection on day 1
    6 1612 1.0 mg/kg AD10024 Single injection on day 1
    7 1612 1.0 mg/kg AD10025 Single injection on day 1
    8 1612 1.0 mg/kg AD10026 Single injection on day 1
    9 1612 1.0 mg/kg AD10027 Single injection on day 1
    10 1612 1.0 mg/kg AD10028 Single injection on day 1
    11 1612 1.0 mg/kg AD10029 Single injection on day 1
    12 1612 1.0 mg/kg AD10030 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-14 each included nucleotide sequences that, while also being homologous to the mouse XDH gene transcript, were designed to inhibit expression of an XDH gene at positions 1612 of the human XDH gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Mice were euthanized on study day 8, and total RNA was isolated from both livers following collection and homogenization. Mouse XDH mRNA expression was quantitated by probe-based quantitative PCR, normalized to mouse beta-actin expression, and expressed as fraction of vehicle control group (geometric mean, +/−95% confidence interval).
  • TABLE 22
    Average Relative Mouse XDH mRNA at Sacrifice (Day 8) in Example 10
    Average Relative Low High
    Group ID mXDH mRNA (error) (error)
    Group 1 (No Treatment) 1.000 0.242 0.319
    Group 2 (1.0 mg/kg AD09219) 0.607 0.044 0.048
    Group 3 (1.0 mg/kg AD10021) 0.653 0.139 0.177
    Group 4 (1.0 mg/kg AD10022) 0.711 0.055 0.060
    Group 5 (1.0 mg/kg AD10023) 0.609 0.067 0.076
    Group 6 (1.0 mg/kg AD10024) 0.703 0.116 0.139
    Group 7 (1.0 mg/kg AD10025) 0.659 0.083 0.095
    Group 8 (1.0 mg/kg AD10026) 0.561 0.093 0.111
    Group 9 (1.0 mg/kg AD10027) 0.540 0.090 0.108
    Group 10 (1.0 mg/kg AD10028) 0.631 0.054 0.059
    Group 11 (1.0 mg/kg AD10029) 0.440 0.042 0.046
    Group 12 (1.0 mg/kg AD10030) 0.550 0.118 0.150
  • The data were normalized to the non-treatment group (Group 1). As shown in Table 22, above, each of the XDH RNAi agents targeting position 1612 (Groups 2-12) showed mouse mRNA reductions.
    • Example 11. In Vivo Testing of XDH RNAi Agents in Wild-Type Rats
  • Certain XDH RNAi agents have sufficient homology with the rat XDH gene transcript that they are suitable to be examined for XDH gene expression inhibitory activity in wild-type rats. At day 1, male Sprague Dawley rats were given a single subcutaneous administration of 4 mL/1 kg animal weight containing a dose of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 23.
  • TABLE 23
    Targeted Positions and Dosing Groups of Example 11
    Targeted
    Gene Position
    (within SEQ RNAi Agent
    Group ID NO: 1) and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 488 10.0 mg/kg AD09218 Single injection on day 1
    3 488  3.0 mg/kg AD09218 Single injection on day 1
    4 488  1.0 mg/kg AD09218 Single injection on day 1
    5 488  0.3 mg/kg AD09218 Single injection on day 1
    6 2612 10.0 mg/kg AD09651 Single injection on day 1
    7 2612  3.0 mg/kg AD09651 Single injection on day 1
    8 2612  1.0 mg/kg AD09651 Single injection on day 1
    9 2612  0.3 mg/kg AD09651 Single injection on day 1
    10 2616 10.0 mg/kg AD09663 Single injection on day 1
    11 2616  3.0 mg/kg AD09663 Single injection on day 1
    12 2616  1.0 mg/kg AD09663 Single injection on day 1
    13 2616  0.3 mg/kg AD09663 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agent in Groups 2-5 (AD09218) included nucleotide sequences that, while also being homologous to the rat XDH gene transcript, were designed to inhibit expression of an XDH gene at position 488 of the human XDH gene; the XDH RNAi agent in Groups 6-9 (AD09651) included nucleotide sequences that, while also being homologous to the rat XDH gene transcript, were designed to inhibit expression of an XDH gene at position 2612 of the human XDH gene; and the XDH RNAi agents in Groups 10-13 (AD09663) included nucleotide sequences that, while also being homologous to the rat XDH gene transcript, were designed to inhibit expression of an XDH gene at position 2616 of the human XDH gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) rats in each group were tested (n=4). Rats were euthanized on study day 10, and total RNA was isolated from both livers following collection and homogenization. Rat XDH mRNA expression was quantitated by probe-based quantitative PCR, normalized to rat beta-actin expression, and expressed as fraction of vehicle control group (geometric mean, +/−95% confidence interval).
  • TABLE 24
    Average Relative Mouse XDH
    mRNA at Sacrifice (Day 10) in Example 11
    Average Relative Low High
    Group ID rXDH mRNA (error) (error)
    Group 1 (No Treatment) 1.000 0.164 0.197
    Group 2 (10.0 mg/kg AD09218) 0.207 0.079 0.128
    Group 3 (3.0 mg/kg AD09218) 0.295 0.105 0.163
    Group 4 (1.0 mg/kg AD09218) 0.369 0.061 0.072
    Group 5 (0.3 mg/kg AD09218) 0.556 0.086 0.102
    Group 6 (10.0 mg/kg AD09651) 0.209 0.056 0.076
    Group 7 (3.0 mg/kg AD09651) 0.271 0.045 0.054
    Group 8 (1.0 mg/kg AD09651) 0.625 0.111 0.135
    Group 9 (0.3 mg/kg AD09651) 0.828 0.114 0.132
    Group 10 (10.0 mg/kg AD09663) 0.122 0.045 0.072
    Group 11 (3.0 mg/kg AD09663) 0.213 0.060 0.083
    Group 12 (1.0 mg/kg AD09663) 0.428 0.094 0.120
    Group 13 (0.3 mg/kg AD09663) 0.481 0.112 0.146
  • The data were normalized to the non-treatment group (Group 1). As noted above in, for example, Example 3, the RNAi agent targeting position 488 of the XDH gene of Groups 2-5 (AD09218), while being previously identified as having activity in rats in vivo, includes a compromised nucleotide sequence and is unsuitable for therapeutic use due to toxicity concerns. As shown in Table 24, the XDH RNAi agent AD09651 (Groups 6-9), which targets position 2612 of the XDH gene transcript, and the XDH RNAi agent AD09663 (Groups 10-13), which targets position 2616, both showed dose-dependent mRNA reductions that were comparable to AD09218 (targeting position 488 of the XDH gene).
    • Example 12. In Vivo Testing of XDH RNAi Agents in Cynomolgus Monkeys
  • XDH RNAi agents AD09325 and AD09307 were evaluated in cynomolgus monkeys (cynos). On day 1, three male cynos for each group (n=3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3.0 mg/kg (10 mg/mL) of the respective XDH RNAi agent, formulated in isotonic saline.
  • TABLE 25
    Targeted Positions and Dosing Groups of Example 12
    Targeted
    Gene Position
    (within SEQ RNAi Agent Dosing Regimen
    Group ID NO: 1) and Dose (on day 1)
    A 3083 3.0 mg/kg AD09325 Single
    subcutaneous injection
    B 4725 3.0 mg/kg AD09307 Single
    subcutaneous injection
  • The XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agent in Groups A (AD09325) included nucleotide sequences that were designed to inhibit expression of a human XDH gene at position 3083; and the XDH RNAi agent in Group B (AD09307) included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4725. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • On days −8 (8 days before dose) and 15, survival liver biopsies were taken. On the date of each biopsy collection, cynos were anesthetized and laparoscopy was used to extract two liver tissue samples approximately 80 mg to 120 mg each, and aliquots of approximately 50 mg were snap-frozen and stored at −70° C. until analysis. On day 29, cynos were euthanized and aliquots of approximately 50 mg liver samples were collected. The biopsy samples were then homogenized, and levels of cyno XDH (cXDH) mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day −8) cXDH mRNA measurements. The resulting mRNA data are reflected in the following Table 26:
  • TABLE 26
    Cyno XDH mRNA Levels Normalized to Pre-Dose
    (Day −8) from Example 12 for each Group (n = 3)
    Relative Relative
    cXDH eXDH
    mRNA Low High mRNA Low High
    Expression Error Error Expression Error Error
    Day −8 Day 15
    Group A: AD09325 1.000 0.211 0.268 0.609 0.097 0.115
    Group B: AD09307 1.000 0.339 0.512 1.139 0.316 0.437
    Day 29
    Group A: AD09325 1.178 0.286 0.378
    Group B: AD09307 1.591 0.509 0.748

    As shown in Table 26, XDH RNAi agent AD09325, which was designed to target position 3083 of the XDH gene, showed 39% inhibition of cXDH mRNA at Day 15 and returned to baseline by day 29. XDH RNAi agent AD09307, which was designed to target position 4725 of the XDH gene, showed no inhibitory activity at either of the time points measured.
    • Example 13. In Vivo Testing of XDH RNAi Agents in Cynomolgus Monkeys
  • XDH RNAi agents AD09734, AD09651. AD09663, and AD09611 were evaluated in cynomolgus monkeys (cynos). On days 1 and 31, three male cynos for each group (n=3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3.0 mg/kg (10 mg/mL) of the respective XDH RNAi agent, formulated in isotonic saline.
  • TABLE 27
    Targeted Positions and Dosing Groups of Example 13
    Targeted
    Gene Position
    (within SEQ RNAi Agent Dosing Regimen
    Group ID NO: 1) and Dose (on days 1 and 31)
    1 1322 3.0 mg/kg AD09734 Two subcutaneous injections
    2 2612 3.0 mg/kg AD09651 Two subcutaneous injections
    3 2616 3.0 mg/kg AD09663 Two subcutaneous injections
    4 4289 3.0 mg/kg AD09611 Two subcutaneous injections
  • The XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 27, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • On days −14 or −7 (pre-dose), 15, 43, and 80 (for Group 4 only) liver biopsies were taken. On the date of each biopsy collection, cynos were anesthetized and laparoscopy was used to extract two liver tissue samples approximately 80 mg to 120 mg each, and aliquots of approximately 50 mg were snap-frozen and stored at −70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day −14 or −7, depending on the animals) cXDH mRNA measurements. Batch analysis of samples across timepoints was performed where applicable. The resulting mRNA data are reflected in the following Table 28:
  • TABLE 28
    Cyno XDH mRNA Levels Normalized to Pre-Dose
    (Day −14 or −7) from Example 13 for each Group (n = 3)
    Relative Relative
    eXDH eXDH
    mRNA Low High Low High mRNA
    Expression Error Error Error Error Expression
    Pre-Dose (Day −14 or Day −7) Day 15
    Group 1: 1.000 0.127 0.145 0.351 0.028 0.031
    AD09734
    Group 2: 1.000 0.170 0.205 0.433 0.131 0.188
    AD09651
    Group 3: 1.000 0.374 0.597 0.621 0.274 0.489
    AD09663
    Group 4: 1.000 0.202 0.254 0.570 0.122 0.156
    AD09611
    Day 43 Day 80
    Group 1: 0.434 0.134 0.194
    AD09734
    Group 2: 0.342 0.074 0.094
    AD09651
    Group 3: 0.605 0.316 0.662
    AD09663
    Group 4: 0.239 0.015 0.016 0.493 0.090 0.110
    AD09611

    As shown in Table 28, each of the XDH RNAi agents showed inhibition of XDH gene expression.
  • XDH Activity Assay. Using RNAScope (see, e.g., RNAscope, A Novel in Situ RNA Analysis Platform for Formalin-Fixed, Paraffin-Embedded Tissues, J Mol Diagn. 2012 January; 14(1): 22-29), it was determined that XDH mRNA transcripts are partitioned between both nuclear and cytosolic compartments. As translation to XDH protein only occurs in the cytoplasm, inhibition of cytoplasmic mRNA transcripts is considered therapeutically relevant. Measurement of XDH mRNA transcripts using q-PCR from whole liver homogenate, as explained in Table 28, is therefore not necessarily reflective of determining therapeutically relevant XDH inhibition as it measures the presence of XDH mRNA in both the cytosolic and nucleic compartments. Thus, to obtain a more accurate assessment of the inhibitory activity of the various XDH RNAi agents disclosed herein, an XDH activity assay was developed capable of indirectly measuring the amount of XDH protein inhibited by the XDH RNAi agents through the RNA interference mechanism.
  • More specifically, XDH activity was assessed using the following method: frozen cyno liver biopsy samples were homogenized in buffer containing 100 mM oxonic acid to inhibit endogenous uricase activity which is known to degrade uric acid. Liver homogenates were purified using Sephadex G25 spin columns, and protein concentrations adjusted to 0.5 μg/μl total protein (lysate). XDH activity was measured by liquid-chromatography mass spectrometry (LCMS) as the conversion of xanthine to uric acid at 37° C. within a 30-minute timeframe. The amount of uric acid generated over time is an indirect measure of the amount of cXDH protein present in the lysate; accordingly, the less uric acid identified, the less cXDH protein was present in lysate, thereby indicating a more potent XDH RNAi agent for reducing XDH protein. The resulting XDH activity data (normalized to pre-dose) are shown in Table 29.
  • TABLE 29
    Cyno XDH Activity Levels Normalized to Pre-Dose
    (Day −14 or −7) from Example 13 for each Group (n = 3)
    Pre-Dose
    (Day −14 or Day −7) Day 15 Day 43 Day 80
    Relative Std Relative Std Relative Std Relative Std
    XDH Dev XDH Dev XDH Dev XDH Dev
    Activity (+/−) Activity (+/−) Activity (+/−) Activity (+/−)
    Group 1: 1.000 0.042 0.363 0.056 0.240 0.056
    AD09734
    Group 2: 1.000 0.026 0.511 0.121 0.289 0.053
    AD09651
    Group 3: 1.000 0.003 0.412 0.219 0.247 0.164
    AD09663
    Group 4: 1.000 0.025 0.555 0.115 0.226 0.057 0.268 0.082
    AD09611
  • As shown in Table 29, through day 43 each of the RNAi agents tested above showed XDH activity reductions of greater than 70%. Further, RNAi agent AD09611 showed substantial reductions of XDH activity that were maintained for seven weeks post the last dose (day 31).
    • Example 14. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 30.
  • TABLE 30
    Targeted Positions and Dosing Groups of Example 14
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 4289 2.0 mg/kg AD09611 Single injection on day 1
    3 4289 2.0 mg/kg AD10183 Single injection on day 1
    4 4289 2.0 mg/kg AD10629 Single injection on day 1
    5 4289 2.0 mg/kg AD10630 Single injection on day 1
    6 4289 2.0 mg/kg AD10631 Single injection on day 1
    7 4289 2.0 mg/kg AD10632 Single injection on day 1
    8 4289 2.0 mg/kg AD10184 Single injection on day 1
    9 4289 2.0 mg/kg AD10633 Single injection on day 1
    10 4289 2.0 mg/kg AD10634 Single injection on day 1
    11 4289 2.0 mg/kg AD10635 Single injection on day 1
    12 4289 2.0 mg/kg AD10636 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-12 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4289 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, and day 15, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 22 are shown in Table 31:
  • TABLE 31
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 14
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.164 1.000 0.044 1.000 0.085
    Group 2 (2.0 mg/kg AD09611) 0.877 0.113 0.710 0.100 0.629 0.148
    Group 3 (2.0 mg/kg AD10183) 0.585 0.084 0.402 0.082 0.432 0.098
    Group 4 (2.0 mg/kg AD10629) 0.548 0.119 0.443 0.127 0.501 0.195
    Group 5 (2.0 mg/kg AD10630) 0.708 0.076 0.609 0.130 0.497 0.045
    Group 6 (2.0 mg/kg AD10631) 0.523 0.035 0.398 0.090 0.477 0.080
    Group 7 (2.0 mg/kg AD10632) 0.679 0.248 0.583 0.125 0.574 0.314
    Group 8 (2.0 mg/kg AD10184) 0.573 0.051 0.501 0.029 0.529 0.070
    Group 9 (2.0 mg/kg AD10633) 0.686 0.153 0.544 0.080 0.562 0.111
    Group 10 (2.0 mg/kg AD10634) 0.680 0.136 0.572 0.088 0.615 0.092
    Group 11 (2.0 mg/kg AD10635) 0.764 0.178 0.678 0.105 0.674 0.083
    Group 12 (2.0 mg/kg AD10636) 0.555 0.068 0.440 0.091 0.488 0.126
    • Example 15. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 1.0 mg/kg (mpk) or 3.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 32.
  • TABLE 32
    Targeted Positions and Dosing Groups of Example 15
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 1963 3.0 mg/kg AD09736 Single injection on day 1
    3 1963 1.0 mg/kg AD09736 Single injection on day 1
    4 1963 3.0 mg/kg AD09937 Single injection on day 1
    5 1963 1.0 mg/kg AD09937 Single injection on day 1
    6 1963 3.0 mg/kg AD09967 Single injection on day 1
    7 1963 1.0 mg/kg AD09967 Single injection on day 1
    8 1963 3.0 mg/kg AD10278 Single injection on day 1
    9 1963 1.0 mg/kg AD10278 Single injection on day 1
    10 1963 3.0 mg/kg AD10281 Single injection on day 1
    11 1963 1.0 mg/kg AD10281 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents of Groups 2-11 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 22 are shown in Table 33:
  • TABLE 33
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 15
    Day 8 Day 22
    Avg Std Dev Avg Std Dev
    Group ID XHD (+/−) XHD (+/−)
    Group 1 (Saline vehicle) 1.000 0.202 1.000 0.112
    Group 2 (3.0 mg/kg AD09736) 0.587 0.080 0.682 0.182
    Group 3 (1.0 mg/kg AD09736) 1.100 0.292 1.063 0.212
    Group 4 (3.0 mg/kg AD09937) 0.554 0.211 0.547 0.214
    Group 5 (1.0 mg/kg AD09937) 0.914 0.175 0.851 0.175
    Group 6 (3.0 mg/kg AD09967) 0.638 0.035 0.696 0.139
    Group 7 (1.0 mg/kg AD09967) 0.838 0.103 0.790 0.149
    Group 8 (3.0 mg/kg AD10278) 0.518 0.036 0.678 0.112
    Group 9 (1.0 mg/kg AD10278) 1.209 0.116 0.940 0.266
    Group 10 (3.0 mg/kg AD10281) 0.769 0.184 0.762 0.145
    Group 11 (1.0 mg/kg AD10281) 1.224 0.172 0.995 0.160
    • Example 16. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used. At day 1 each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 34.
  • TABLE 34
    Targeted Positions and Dosing Groups of Example 16
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 4289 4.0 mg/kg AD09611 Single injection on day 1
    3 4289 2.0 mg/kg AD09611 Single injection on day 1
    4 4289 1.0 mg/kg AD09611 Single injection on day 1
    5 4289 4.0 mg/kg AD10183 Single injection on day 1
    6 4289 2.0 mg/kg AD10183 Single injection on day 1
    7 4289 1.0 mg/kg AD10183 Single injection on day 1
    8 4289 4.0 mg/kg AD10631 Single injection on day 1
    9 4289 2.0 mg/kg AD10631 Single injection on day 1
    10 4289 1.0 mg/kg AD10631 Single injection on day 1
    11 4289 4.0 mg/kg AD10184 Single injection on day 1
    12 4289 2.0 mg/kg AD10184 Single injection on day 1
    13 4289 1.0 mg/kg AD10184 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-13 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 4289 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2. Data from the experiment through day 22 are shown in Table 35:
  • TABLE 35
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 16
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.167 1.000 0.099 1.000 0.048
    Group 2 (4.0 mg/kg AD09611) 0.808 0.086 0.810 0.089 0.958 0.118
    Group 3 (2.0 mg/kg AD09611) 1.100 0.224 0.998 0.383 1.245 0.476
    Group 4 (1.0 mg/kg AD09611) 0.917 0.198 0.941 0.224 0.780 0.544
    Group 5 (4.0 mg/kg AD10183) 0.636 0.140 0.642 0.044 0.797 0.112
    Group 6 (2.0 mg/kg AD10183) 0.768 0.059 0.672 0.206 0.870 0.079
    Group 7 (1.0 mg/kg AD10183) 0.841 0.111 0.792 0.266 0.938 0.122
    Group 8 (4.0 mg/kg AD10631) 0.755 0.110 0.677 0.094 0.664 0.126
    Group 9 (2.0 mg/kg AD10631) 0.852 0.066 0.755 0.103 0.869 0.149
    Group 10 (1.0 mg/kg AD10631) 0.884 0.153 0.954 0.128 1.060 0.071
    Group 11 (4.0 mg/kg AD10184) 0.640 0.079 0.663 0.055 0.680 0.068
    Group 12 (2.0 mg/kg AD10184) 0.729 0.049 0.746 0.126 0.811 0.116
    Group 13 (1.0 mg/kg AD10184) 0.807 0.069 0.730 0.090 0.796 0.119
    • Example 17. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 200 μl/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 36.
  • TABLE 36
    Targeted Positions and Dosing Groups of Example 17
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 3598 4.0 mg/kg AD09985 Single injection on day 1
    3 3598 2.0 mg/kg AD09985 Single injection on day 1
    4 3598 1.0 mg/kg AD09985 Single injection on day 1
    5 3598 4.0 mg/kg AD10729 Single injection on day 1
    6 3598 2.0 mg/kg AD10729 Single injection on day 1
    7 3598 1.0 mg/kg AD10729 Single injection on day 1
    8 3598 4.0 mg/kg AD10730 Single injection on day 1
    9 3598 2.0 mg/kg AD10730 Single injection on day 1
    10 3598 1.0 mg/kg AD10730 Single injection on day 1
    11 3598 4.0 mg/kg AD10734 Single injection on day 1
    12 3598 2.0 mg/kg AD10734 Single injection on day 1
    13 3598 1.0 mg/kg AD10734 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C. and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-13 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 3598 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 22 are shown in Table 37:
  • TABLE 37
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 17
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Groupd ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.294 1.000 0.350 1.000 0.156
    Group 2 (4.0 mg/kg AD09985) 0.342 0.061 0.340 0.052 0.320 0.074
    Group 3 (2.0 mg/kg AD09985) 0.464 0.065 0.443 0.076 0.457 0.108
    Group 4 (1.0 mg/kg AD09985) 0.527 0.163 0.509 0.075 0.487 0.094
    Group 5 (4.0 mg/kg AD10729) 0.393 0.081 0.379 0.074 0.359 0.045
    Group 6 (2.0 mg/kg AD10729) 0.504 0.176 0.447 0.132 0.394 0.176
    Group 7 (1.0 mg/kg AD10729) 0.480 0.168 0.535 0.279 0.486 0.205
    Group 8 (4.0 mg/kg AD10730) 0.322 0.035 0.316 0.046 0.244 0.064
    Group 9 (2.0 mg/kg AD10730) 0.467 0.076 0.397 0.052 0.360 0.113
    Group 10 (1.0 mg/kg AD10730) 0.560 0.114 0.540 0.079 0.536 0.068
    Group 11 (4.0 mg/kg AD10734) 0.369 0.048 0.340 0.074 0.278 0.025
    Group 12 (2.0 mg/kg AD10734) 0.574 0.338 0.467 0.255 0.432 0.299
    Group 13 (1.0 mg/kg AD10734) 0.616 0.198 0.617 0.086 0.389 0.076
    • Example 18. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 and 2820-5715 regions of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 200 μl/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 38.
  • TABLE 38
    Targeted Positions and Dosing Groups of Example 18
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 2696 Saline (no RNAi agent) Single injection on day 1
    2 2696 4.0 mg/kg AD09744 Single injection on day 1
    3 2696 2.0 mg/kg AD09744 Single injection on day 1
    4 2696 1.0 mg/kg AD09744 Single injection on day 1
    5 2696 4.0 mg/kg AD10621 Single injection on day 1
    6 2696 2.0 mg/kg AD10621 Single injection on day 1
    7 2696 1.0 mg/kg AD10621 Single injection on day 1
    8 1963 4.0 mg/kg AD09736 Single injection on day 1
    9 1963 2.0 mg/kg AD09736 Single injection on day 1
    10 1963 1.0 mg/kg AD09736 Single injection on day 1
    11 1963 4.0 mg/kg AD09937 Single injection on day 1
    12 1963 2.0 mg/kg AD09937 Single injection on day 1
    13 1963 1.0 mg/kg AD09937 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C. and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-13 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at positions 2696 and 1963 of the gene. (See, e.g. SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2. Data from the experiment through day 22 are shown in Table 39:
  • TABLE 39A
    Average XDH Normalized to Pre-Treatment
    in XDH-GLUC AAV Mice from Example 18
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.183 0.346 1.164 0.468 1.448 0.573
    Group 2 (4.0 mg/kg AD09744) 0.538 0.113 0.404 0.106 0.601 0.062
    Group 3 (2.0 mg/kg AD09744) 0.704 0.210 0.663 0.165 0.950 0.214
    Group 4 (1.0 mg/kg AD09744) 0.903 0.100 0.842 0.154 1.101 0.249
    Group 5 (4.0 mg/kg AD10621) 0.406 0.226 0.366 0.293 0.650 0.532
    Group 6 (2.0 mg/kg AD10621) 0.521 0.261 0.411 0.225 0.640 0.343
    Group 7 (1.0 mg/kg AD10621) 0.580 0.202 0.467 0.227 0.669 0.361
    Group 8 (4.0 mg/kg AD09736) 0.870 0.117 0.732 0.045 1.084 0.195
    Group 9 (2.0 mg/kg AD09736) 0.867 0.088 0.809 0.100 1.187 0.254
    Group 10 (1.0 mg/kg AD09736) 1.313 0.177 1.199 0.185 1.344 0.185
    Group 11 (4.0 mg/kg AD09937) 0.540 .0164 0.588 0.268 0.780 0.257
    Group 12 (2.0 mg/kg AD09937) 0.636 0.249 0.812 0.480 0.846 0.312
    Group 13 (1.0 mg/kg AD09937) 0.927 0.215 0.932 0.127 1.011 0.057
  • TABLE 39B
    Average XDH Normalized to Pre-Treatment & Control in XDH-GLUC AAV Mice from Example 18
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.292 1.000 0.403 1.000 0.396
    Group 2 (4.0 mg/kg AD09744) 0.455 0.095 0.347 0.091 0.415 0.043
    Group 3 (2.0 mg/kg AD09744) 0.595 0.178 0.570 0.142 0.656 0.147
    Group 4 (1.0 mg/kg AD09744) 0.763 0.084 0.724 0.132 0.760 0.172
    Group 5 (4.0 mg/kg AD10621) 0.343 0.191 0.315 0.252 0.449 0.367
    Group 6 (2.0 mg/kg AD10621) 0.441 0.220 0.353 0.193 0.442 0.237
    Group 7 (1.0 mg/kg AD10621) 0.491 0.171 0.402 0.195 0.462 0.249
    Group 8 (4.0 mg/kg AD09736) 0.736 0.099 0.629 0.039 0.748 0.135
    Group 9 (2.0 mg/kg AD09736) 0.733 0.075 0.696 0.086 0.820 0.175
    Group 10 (1.0 mg/kg AD09736) 1.110 0.150 1.031 0.159 0.928 0.128
    Group 11 (4.0 mg/kg AD09937) 0.457 0.139 0.505 0.230 0.538 0.171
    Group 12 (2.0 mg/kg AD09937) 0.538 0.210 0.698 0.413 0.584 0.216
    Group 13 (1.0 mg/kg AD09937) 0.783 0.182 0.801 0.109 0.698 0.039
    • Example 19. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 200 μl/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 40.
  • TABLE 40
    Targeted Positions and Dosing Groups of Example 19
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 1963 Saline (no RNAi agent) Single injection on day 1
    2 1963 4.0 mg/kg AD09736 Single injection on day 1
    3 1963 2.0 mg/kg AD09736 Single injection on day 1
    4 1963 4.0 mg/kg AD10967 Single injection on day 1
    5 1963 2.0 mg/kg AD10967 Single injection on day 1
    6 1963 4.0 mg/kg AD10968 Single injection on day 1
    7 1963 2.0 mg/kg AD10968 Single injection on day 1
    8 1963 4.0 mg/kg AD10969 Single injection on day 1
    9 1963 2.0 mg/kg AD10969 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-9 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 1963 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2. Data from the experiment through day 22 are shown in Table 41:
  • TABLE 41
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 19
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.158 1.000 0.166 1.000 0.077
    Group 2 (4.0 mg/kg AD09736) 0.607 0.088 0.704 0.077 0.635 0.230
    Group 3 (2.0 mg/kg AD09736) 0.738 0.199 0.742 0.085 0.991 0.061
    Group 4 (4.0 mg/kg AD10967) 0.468 0.115 0.542 0.083 0.714 0.131
    Group 5 (2.0 mg/kg AD10967) 0.746 0.099 0.826 0.047 0.940 0.203
    Group 6 (4.0 mg/kg AD10968) 0.520 0.131 0.488 0.149 0.685 0.176
    Group 7 (2.0 mg/kg AD10968) 0.534 0.148 0.597 0.135 0.827 0.155
    Group 8 (4.0 mg/kg AD10969) 0.614 0.194 0.617 0.211 0.758 0.264
    Group 9 (2.0 mg/kg AD10969) 0.728 0.274 0.711 0.244 0.984 0.440
    • Example 20. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 2820-5715 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 200 μl/20 g animal weight containing either 4.0 mg/kg (mpk), 2.0 mg/kg (mpk), 1.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 42.
  • TABLE 42
    Targeted Positions and Dosing Groups of Example 20
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 4289 4.0 mg/kg AD09611 Single injection on day 1
    3 4289 2.0 mg/kg AD09611 Single injection on day 1
    4 4289 1.0 mg/kg AD09611 Single injection on day 1
    5 4289 4.0 mg/kg AD10631 Single injection on day 1
    6 4289 2.0 mg/kg AD10631 Single injection on day 1
    7 4289 1.0 mg/kg AD10631 Single injection on day 1
    8 3598 4.0 mg/kg AD09985 Single injection on day 1
    9 3598 2.0 mg/kg AD09985 Single injection on day 1
    10 3598 1.0 mg/kg AD09985 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-10 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at positions 4289 and 3598 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2. Data from the experiment through day 22 are shown in Table 43:
  • TABLE 43
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 20
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.209 1.000 0.098 1.000 0.222
    Group 2 (4.0 mg/kg AD09611) 0.892 0.047 0.777 0.181 0.829 0.213
    Group 3 (2.0 mg/kg AD09611) 0.703 0.168 0.699 0.159 0.789 0.209
    Group 4 (1.0 mg/kg AD09611) 0.868 0.183 0.843 0.071 0.729 0.136
    Group 5 (4.0 mg/kg AD10631) 0.642 0.082 0.651 0.058 0.644 0.153
    Group 6 (2.0 mg/kg AD10631) 0.660 0.192 0.594 0.082 0.557 0.102
    Group 7 (1.0 mg/kg AD10631) 0.626 0.060 0.649 0.089 0.720 0.143
    Group 8 (4.0 mg/kg AD09985) 0.600 0.360 0.600 0.341 0.586 0.209
    Group 9 (2.0 mg/kg AD09985) 0.576 0.119 0.519 0.025 0.619 0.088
    Group 10 (1.0 mg/kg AD09985) 0.710 0.163 0.641 0.086 0.631 0.136
    • Example 21. In Vivo Testing of XDH RNAi Agents in Cynomolgus Monkeys
  • XDH RNAi agent AD09611, which was previously evaluated in cynomolgus monkeys (cynos) in the study described in Example 13, was further evaluated in cynomolgus monkeys (cynos). On days 1, 15, and 29, three male cynos for each group (n=3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3.0 mg/kg (10 mg/mL) of the respective XDH RNAi agent, formulated in isotonic saline.
  • TABLE 44
    Targeted Positions and Dosing Groups of Example 21
    Targeted
    Gene
    Position
    (within SEQ RNAi Agent Dosing Regimen
    Group ID NO: 1) and Dose (on days 1, 15, and 29)
    1 4289 3.0 mg/kg AD09611 Three subcutaneous injections
    2 4289 3.0 mg/kg AD09611 Three subcutaneous injections
  • The XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 43, above. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • On days −14, 29, 57, and 85, liver biopsies were taken from Group 1 animals. On days −7, 43, 71, and 99, liver biopsies were taken from Group 2 animals. On the date of each biopsy collection, cynos were sedated and Menghini technique was used to extract two liver tissue samples, and aliquots of approximately 10 mg were snap-frozen and stored at −70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day −14 or −7, depending on the animals) cXDH mRNA measurements. The resulting mRNA data are reflected in the following Table 45:
  • TABLE 45
    Cyno XDH mRNA Levels Normalized to Pre-Dose
    (Day −14 or −7) from Example 21 for each Group (n = 3)
    Relative Relative
    cXDH cXDH
    mRNA Low High mRNA Low High
    Expression Error Error Expression Error Error
    Pre-Dose (Day −14 or Day −7) Day 29
    Group 1: 1.000 0.177 0.215 0.595 0.097 0.116
    AD09611
    Group 2: 1.000 0.083 0.091 NA NA NA
    AD09611
    Day 43 Day 57
    Group 1: 0.429 0.105 0.138
    AD09611
    Group 2: 0.604 0.060 0.067
    AD09611
    Day 71 Day 85
    Group 1: 0.560 0.079 0.092
    AD09611
    Group 2: 0.758 0.121 0.144
    AD09611
    Day 99
    Group 1:
    AD09611
    Group 2: 0.950 0.066 0.071
    AD09611
  • Additionally. XDH activity was assessed using the XDH Activity Assay method described in Example 13. The resulting XDH activity data are shown in Table 46.
  • TABLE 46
    Cyno XDH Activity Levels Normalized to Pre-Dose
    (Day −14 or −7) from Example 21 for each Group (n = 3)
    Relative Std Relative Std Relative Std Relative Std
    XDH Dev XDH Dev XDH Dev XDH Dev
    Activity (+/−) Activity (+/−) Activity (+/−) Activity (+/−)
    Pre-Dose (Day −14 or Day −7) Day 29 Day 43 Day 57
    Group 1: 1.000 0.01 0.290 0.004 0.391 0.15
    AD09611
    Group 2: 1.000 0.012 0.394 0.066
    AD09611
    Day 71 Day 85 Day 99
    Group 1: 0.341 0.079
    AD09611
    Group 2: 0.357 0.098 0.465 0.067
    AD09611

    As shown in Table 46, AD09611 showed XDH activity reductions of up to 70% as measured on day 29, and reductions were maintained at greater than 50% through day 99.
    • Example 22. In Vivo Testing of XDH RNAi Agents in Cynomolgus Monkeys
  • XDH RNAi agents AD10631, AD09736, AD10621, and AD09985 were evaluated in cynomolgus monkeys (cynos). On days 1, 15, and 29, three male cynos for each group (n=3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3.0 mg/kg (10 mg/mL) of the respective XDH RNAi agent, formulated in isotonic saline.
  • TABLE 47
    Targeted Positions and Dosing Groups of Example 22
    Targeted
    Gene
    Position
    (within SEQ RNAi Agent Dosing Regimen
    Group ID NO: 1) and Dose (on days 1, 15, and 29)
    1 4289 3.0 mg/kg AD10631 Three subcutaneous injections
    2 1963 3.0 mg/kg AD09736 Three subcutaneous injections
    3 2696 3.0 mg/kg AD10621 Three subcutaneous injections
    4 3598 3.0 mg/kg AD09985 Three subcutaneous injections
  • The XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 47. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced). As noted herein. AD10631 was designed to target position 4289 and was comprised of a chemically modified nucleotide sequence similar to AD09611, but included a 5′-cyclopropyl-phosphonate modified nucleotide at the 5′ terminal end of the antisense strand.
  • On days −7, 43, 71, and 99, liver biopsies were taken. On the date of each biopsy collection, cynos were sedated and 3.5 mm×310 mm clamshell biopsy forceps were used to extract one liver tissue sample approximately 160 mg to 240 mg, and aliquots of approximately 50 mg were snap-frozen and stored at −70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day −7) cXDH mRNA measurements. The resulting mRNA data are reflected in Table 48:
  • TABLE 48
    Cyno XDH mRNA Levels Normalized to Pre-Dose
    (Day −7) from Example 22 for each Group (n = 3)
    Relative Relative
    cXDH cXDH
    mRNA Low High mRNA Low High
    Expression Error Error Expression Error Error
    Pre-Dose (Day −7) Day 43
    Group 1: 1.000 0.093 0.102 0.459 0.062 0.072
    AD10631
    Group 2: 1.000 0.120 0.136 0.420 0.076 0.092
    AD09736
    Group 3: 1.000 0.113 0.127 0.373 0.025 0.027
    AD10621
    Group 4: 1.000 0.084 0.091 0.413 0.081 0.101
    AD09985
    Day 71 Day 99
    Group 1: 0.413 0.036 0.040 0.595 0.055 0.060
    AD10631
    Group 2: 0.478 0.072 0.085 0.502 0.126 0.168
    AD09736
    Group 3: 0.397 0.029 0.031 0.477 0.038 0.042
    AD10621
    Group 4: 0.339 0.047 0.055 0.459 0.107 0.140
    AD09985
  • Additionally. XDH activity was assessed using the XDH Activity Assay method described in Example 13. The resulting XDH activity data are shown in Table 49.
  • TABLE 49
    Cyno XDH Activity Levels Normalized to Pre-Dose
    (Day −7) from Example 22 for each Group (n = 3)
    Pre-Dose (Day −7) Day 43 Day 71 Day 98
    Relative Std Relative Std Relative Std Relative Std
    XDH Dev XDH Dev XDH Dev XDH Dev
    Activity (+/−) Activity (+/−) Activity (+/−) Activity (+/−)
    Group 1: 1 0.000 0.268 0.060 0.273 0.049 0.553 0.135
    AD10631
    Group 2: 1 0.000 0.091 0.019 0.130 0.036 0.232 0.069
    AD09736
    Group 3: 1 0.000 0.052 0.004 0.161 0.063 0.186 0.080
    AD10621
    Group 4: 1 0.000 0.074 0.011 0.146 0.044 0.199 0.149
    AD09985
  • As noted above, each of AD09736 (Group 2), AD010621 (Group 3), and AD09985 (Group 4) obtained 90% or greater reductions in XDH activity, indicating these are highly potent XDH RNAi agents capable of reducing XDH protein expression by 90% in liver cells (hepatocytes). AD10631 was reported to have a 74% reduction in XDH activity, which is similar to what was seen with the XDH activity assay performed on cyno liver biopsy samples administered AD09611 (which targeted the same position on the XDH gene as AD10631) as reported in Example 13 and Example 21.
    • Example 23. In Vitro Testing of XDH RNAi Agents
  • Candidate sequence duplexes shown below in Table 50 were tested in vitro. The XDH RNAi agents were prepared in accordance with the procedures set forth in Example 1. The XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand).
  • TABLE 50
    XDH RNAi Agents Tested for In Vitro
    Free Uptake Assay in Primary Human
    and Cynomolgus Monkey Hepatocytes
    Targeted Gene
    Position (within
    RNAi Agent SEQ ID NO: 1)
    AD09218 488
    AD09744 2696
    AD10012 2696
    AD10621 2696
    AD09736 1963
    AD09937 1963
    AD10278 1963
    AD09218 488
    AD09985 3598
    AD10731 3598
    AD09611 4289
    AD10184 4289
    AD10631 4289
  • Evaluation of XDH RNAi agents in vitro was performed by seeding primary human or cynomolgus monkey hepatocytes cells. Cells were seeded at 25,000 cells per well in 50 μL culture medium in 96-well collagen coated plate. Cells were treated with each of the XDH RNAi agent duplexes shown in Table 50 immediately after cells were seeded by adding 50 μL per well at 2× the final concentration, followed by gentle mixing and incubation at 37° C., 5% CO2, for 48 hours without disturbing the cells. Isolation and purification of RNA was completed using a commercially available kit according to the manufacturer's instructions (Zymo Quick-RNA Miniprep Kit (Zymo Research, Irvine, Calif.)). Relative expression of each of the XDH RNAi agents was determined by qRT-PCR by comparing the expression levels of XDH mRNA to an endogenous control (PPIA).
  • A serial dilution of the RNAi agents was performed and the data curve fit to calculate the dose (concentration) required to knock down gene expression by 50% (“EC50,” or effective concentration estimated to reduce gene expression by 50%). Residual XDH gene activity and EC50 of the XDH RNAi agents are shown below in Tables 51 and 52. Thus, for example, for RNAi agent AD10012, in primary human hepatocytes, at 1 nM, results in 0.2485 residual XDH gene relative expression, or 75.15% XDH gene knockdown. As further provided in Table 51, AD10012 was reported to have an EC50 of 0.012 nM (6 point repeat with free uptake in primary human hepatocytes), meaning AD10012 achieves 50% XDH gene knockdown at 0.012 nM concentration.
  • TABLE 51
    In vitro inhibition of XDH RNAi Agents by free uptake in primary human hepatocytes
    RNAi Agent Concentration EC50
    RNAi 0.01 nM EC50
    Agent Avg SD 0.1 nM SD 1 nM SD 10 nM SD 100 nM SD 1000 nM SD (nM)
    AD09218 1.1908 0.4415 0.7427 0.2296 0.3515 0.1042 0.3459 0.1794 0.2624 0.0653 0.3672 0.1138 0.073
    AD09744 1.1048 0.2004 0.6711 0.0780 0.6122 0.0581 0.1599 0.0769 0.3055 0.0624 0.3551 0.1225 0.098
    AD10012 0.8735 0.0804 0.3435 0.0463 0.2485 0.0293 0.1707 0.1403 0.1840 0.0354 0.2882 0.1552 0.012
    AD10621 0.6962 0.1486 0.3373 0.0537 0.2388 0.0516 0.1614 0.0148 0.1714 0.0338 0.1947 0.0297 0.033
    AD09736 0.6916 0.0306 0.3905 0.0993 0.2970 0.0661 0.1534 0.0956 0.2394 0.0955 0.1572 0.0313 0.059
    AD09937 0.7534 0.1915 0.3373 0.0449 0.1919 0.0562 0.2224 0.0745 0.1309 0.0274 0.1282 0.0160 0.029
    AD10278 0.8245 0.1510 0.3776 0.0823 0.2635 0.0463 0.2347 0.0524 0.1359 0.0275 0.1295 0.0362 0.036
    AD09218 0.7578 0.4480 0.4888 0.0416 0.4312 0.1623 0.2016 0.0565 0.1651 0.0731 0.2039 0.0753 0.035
    AD09985 0.9439 0.0347 0.7353 0.1957 0.3808 0.1059 0.2642 0.0402 0.2657 0.0527 0.2820 0.1093 0.190
    AD10731 0.9885 0.0470 0.5503 0.0816 0.3282 0.0367 0.3649 0.1127 0.2777 0.0134 0.2634 0.0412 0.042
    AD09611 0.9968 0.0629 1.0892 0.2769 0.9445 0.0773 0.7137 0.1343 0.4735 0.0527 0.3751 0.0702 9.607
    AD10184 0.9568 0.1924 0.6296 0.0664 0.3272 0.0500 0.2448 0.0108 0.1962 0.0357 0.1766 0.0323 0.117
    AD10631 0.9386 0.0626 0.4900 0.1062 0.3561 0.0780 0.3252 0.1326 0.2606 0.0450 0.1594 0.0271 0.040
  • TABLE 52
    In vitro inhibition of XDH RNAi Agents by free uptake in primary cynomolgus monkey hepatocytes
    EC50,
    number
    of
    repeat
    RNAi Agent Concentration points
    RNAi 0.06 0.49 3.91 31.25 250 2000 EC50
    Agent nM SD nM SD nM SD nM SD nM SD nM SD (nM), 6x
    AD09218 0.7835 0.1158 0.5673 0.0789 0.5559 0.1965 0.3457 0.1295 0.3402 0.0323 0.3044 0.0532 3.767
    AD09744 0.7400 0.0417 0.5543 0.0606 0.4657 0.0987 0.3451 0.0901 0.3667 0.0903 0.3446 0.1180 0.5439
    AD10012 0.6654 0.0098 0.4408 0.1139 0.3365 0.0168 0.2600 0.0335 0.2525 0.0334 0.2234 0.0236 0.3707
    AD10621 0.5571 0.1315 0.4494 0.1035 0.3046 0.1092 0.3036 0.0667 0.2430 0.0764 0.1819 0.0379 1.03
    AD09736 0.5093 0.0602 0.3650 0.0643 0.2476 0.0325 0.2683 0.0184 0.1759 0.0188 0.2002 0.0517 0.4216
    AD09937 0.5609 0.0444 0.3441 0.0388 0.2705 0.0203 0.2531 0.0565 0.1845 0.0197 0.1933 0.0394 0.2249
    AD10278 0.4772 0.0029 0.3957 0.0457 0.2929 0.0667 0.2837 0.0210 0.1784 0.0163 0.2003 0.0536 1.918
    AD09218 0.8383 0.2444 0.6405 0.1284 0.5279 0.0812 0.3616 0.0964 0.2885 0.0710 0.3272 0.0644 2.04
    AD09985 0.8656 0.0630 0.5815 0.0823 0.5065 0.0684 0.4399 0.0955 0.2934 0.0512 0.2938 0.0481 0.4581
    AD10731 0.7837 0.1459 0.4582 0.1026 0.3867 0.1169 0.4410 0.1221 0.2709 0.0683 0.2992 0.0018 0.09407
    AD09611 0.6219 0.0679 0.8340 0.1089 0.3923 0.1597 0.5281 0.1568 0.4321 0.0247 0.3780 0.0137 20.19
    AD10184 0.6263 0.0080 0.4306 0.0235 0.4214 0.0468 0.3293 0.0610 0.2743 0.0341 0.1787 0.0679 0.5228
    AD10631 0.5973 0.0231 0.5815 0.0713 0.5537 0.1817 0.5543 0.1779 0.3033 0.0283 0.3341 0.0497 77.08
    • Example 24. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to Table 53.
  • TABLE 53
    Targeted Positions and Dosing Groups of Example 24
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 2696 Saline (no RNAi agent) Single injection on day 1
    2 2696 2.0 mg/kg AD09744 Single injection on day 1
    3 2696 2.0 mg/kg AD10012 Single injection on day 1
    4 2696 2.0 mg/kg AD10619 Single injection on day 1
    5 2696 2.0 mg/kg AD10620 Single injection on day 1
    6 2696 2.0 mg/kg AD10621 Single injection on day 1
    7 2696 2.0 mg/kg AD10622 Single injection on day 1
    8 2696 2.0 mg/kg AD10623 Single injection on day 1
    9 2696 2.0 mg/kg AD10624 Single injection on day 1
    10 2696 2.0 mg/kg AD10625 Single injection on day 1
    11 2696 2.0 mg/kg AD10626 Single injection on day 1
    12 2696 2.0 mg/kg AD10627 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents in Groups 2-12 each included nucleotide sequences that were designed to inhibit expression of an XDH gene at position 26% of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2. Data from the experiment through day 22 are shown in Table 54:
  • TABLE 54
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 24
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.107 1.000 0.172 1.000 0.233
    Group 2 (2.0 mg/kg AD09744) 0.585 0.079 0.616 0.024 0.659 0.088
    Group 3 (2.0 mg/kg AD10012) 0.336 0.034 0.305 0.014 0.343 0.018
    Group 4 (2.0 mg/kg AD10619) 0.397 0.034 0.415 0.011 0.415 0.046
    Group 5 (2.0 mg/kg AD10620) 0.394 0.049 0.326 0.046 0.306 0.053
    Group 6 (2.0 mg/kg AD10621) 0.403 0.038 0.312 0.049 0.348 0.026
    Group 7 (2.0 mg/kg AD10622) 0.382 0.068 0.317 0.061 0.338 0.065
    Group 8 (2.0 mg/kg AD10623) 0.280 0.124 0.268 0.053 0.258 0.137
    Group 9 (2.0 mg/kg AD10624) 0.302 0.069 0.362 0.091 0.376 0.174
    Group 10 (2.0 mg/kg AD10625) 0.341 0.048 0.342 0.096 0.412 0.079
    Group 11 (2.0 mg/kg AD10626) 0.436 0.078 0.394 0.063 0.415 0.035
    Group 12 (2.0 mg/kg AD10627) 0.317 0.041 0.325 0.023 0.322 0.041
    • Example 25. In Vivo Testing of XDH RNAi Agents in Cynomolgus Monkeys
  • XDH RNAi agents AD10621 and AD09985 were evaluated in cynomolgus monkeys (cynos). On day 1, three male cynos for each group (n=3) were administered a subcutaneous injection of 0.3 mL/kg (approximately 1.5 mL volume, depending on animal mass) containing 3 mg/kg or 1 mg/kg of the respective XDH RNAi agent, formulated in isotonic saline.
  • TABLE 55
    Targeted Positions and Dosing Groups of Example 22
    Targeted Gene
    Position (within RNAi Agent
    Group SEQ ID NO: 1) and Dose Dosing Regimen (on day 1)
    1 2696 3.0 mg/kg AD10621 Single subcutaneous injection
    2 3598 3.0 mg/kg AD09985 Single subcutaneous injection
    3 2696 1.0 mg/kg AD10621 Single subcutaneous injection
    4 3598 1.0 mg/kg AD09985 Single subcutaneous injection
  • The XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents included nucleotide sequences that were designed to inhibit expression of a human XDH gene at the specific positions as shown in Table 55. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • On days −6 (day −3 for one of the animals), 29, 55, and 99 (day 100 for one of the animals), liver biopsies were taken. On the date of each biopsy collection, cynos were sedated and needles were used to extract two liver tissue samples approximately 20 mg each. Samples were weighed, snap-frozen and stored at −70° C. until analysis. The biopsy samples were then homogenized, and levels of cXDH mRNA in the cyno livers were measured by RT-qPCR using a housekeeping gene as reference. Resulting values were then normalized to the pre-dose (in this case, at day −6 or day −3) cXDH mRNA measurements. The resulting mRNA data are reflected in Table 56:
  • TABLE 56
    Cyno XDH mRNA Levels Normalized to Pre-Dose
    (Day −6 or Day −3) from Example 25 for each Group (n = 3)
    Relative Relative
    cXDH cXDH
    mRNA Low High mRNA Low High
    Expression Error Error Expression Error Error
    Pre-Dose
    (Day −6 or Day −3) Day 29
    Group 1: 1.000 0.107 0.120 0.585 0.098 0.118
    AD10621
    Group 2: 1.000 0.039 0.041 0.695 0.072 0.080
    AD09985
    Group 3: 1.000 0.114 0.128 0.864 0.138 0.165
    AD10621
    Group 4: 1.000 0.121 0.138 0.691 0.131 0.162
    AD09985
    Day 55 Day 99 or Day 100
    Group 1: 0.687 0.052 0.056 0.793 0.082 0.092
    AD10621
    Group 2: 0.708 0.087 0.100 0.678 0.121 0.148
    AD09985
    Group 3: 0.666 0.148 0.190 0.693 0.125 0.153
    AD10621
    Group 4: 0.720 0.112 0.132 0.676 0.149 0.191
    AD09985
  • Additionally. XDH activity was assessed using the XDH Activity Assay method described in Example 13. The resulting XDH activity data are shown in Table 57.
  • TABLE 57
    Cyno XDH Activity Levels Normalized to Pre-Dose
    (Day −6) from Example 25 for each Group (n = 3)
    Pre-Dose (Day Day 99 or
    −6 or Day −3) Day 29 Day 55 Day 100
    Relative Std Relative Std Relative Std Relative Std
    XDH Dev XDH Dev XDH Dev XDH Dev
    Activity (+/−) Activity (+/−) Activity (+/−) Activity (+/−)
    Group 1: 1 0.000 0.153 0.0048 0.399 0.2224 0.855 0.2914
    AD10621
    Group 2: 1 0.000 0.109 0.0139 0.221 0.1523 0.649 0.1959
    AD09985
    Group 3: 1 0.000 0.236 0.0452 0.343 0.3047 0.681 0.0675
    AD10621
    Group 4: 1 0.000 0.506 0.2290 0.517 0.2206 1.215 0.1157
    AD09985
  • As noted above, each of AD10621 (Group 1) and AD09985 (Group 2) obtained ˜85% or greater reductions in XDH activity, indicating these are highly potent XDH RNAi agents capable of reducing XDH protein expression by ˜85% in liver cells (hepatocytes).
    • Example 26. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 58.
  • TABLE 58
    Targeted Positions and Dosing Groups of Example 26
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
     1 N/A Saline (no RNAi agent) Single injection on day 1
     2  488 2.0 mg/kg AD09218 Single injection on day 1
     3  139 2.0 mg/kg AD09725 Single injection on day 1
     4  235 2.0 mg/kg AD09598 Single injection on day 1
     5  239 2.0 mg/kg AD09726 Single injection on day 1
     6  332 2.0 mg/kg AD09727 Single injection on day 1
     7 2320 2.0 mg/kg AD09741 Single injection on day 1
     8 2357 2.0 mg/kg AD09742 Single injection on day 1
     9 2361 2.0 mg/kg AD09743 Single injection on day 1
    10 2696 2.0 mg/kg AD09744 Single injection on day 1
    11 2701 2.0 mg/kg AD09745 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents of Groups 2-11 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at the positions of the gene listed on Table 58. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15 and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 22 are shown in the following Table 59:
  • TABLE 59
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 26
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 (Saline vehicle) 1.000 0.066 1.000 0.104 1.000 0.084
    Group 2 2.0 mg/kg AD09218 0.350 0.043 0.376 0.038 0.400 0.079
    Group 3 2.0 mg/kg AD09725 0.748 0.134 0.853 0.059 0.871 0.129
    Group 4 2.0 mg/kg AD09598 0.729 0.070 0.935 0.235 1.073 0.092
    Group 5 2.0 mg/kg AD09726 0.651 0.104 0.747 0.154 0.806 0.161
    Group 6 2.0 mg/kg AD09727 0.885 0.051 0.927 0.127 0.929 0.140
    Group 7 2.0 mg/kg AD09741 0.616 0.090 0.693 0.064 0.708 0.110
    Group 8 2.0 mg/kg AD09742 0.724 0.101 0.896 0.143 0.863 0.139
    Group 9 2.0 mg/kg AD09743 0.803 0.060 0.907 0.107 0.841 0.130
    Group 10 2.0 mg/kg AD09744 0.477 0.051 0.576 0.170 0.558 0.132
    Group 11 2.0 mg/kg AD09745 0.568 0.045 0.626 0.062 0.719 0.045
    • Example 27. In Vivo Testing of XDH RNAi Agents in XDH-GLuc AAV Mice
  • The XDH-GLUC AAV mouse model described in Example 2, above, using the XDH-GLuc AAV containing the 80-2899 region of the human XDH cDNA sequence was used. At day 1, each mouse was given a single subcutaneous administration of 250 μl/25 g animal weight containing either 2.0 mg/kg (mpk) or 4.0 mg/kg (mpk) of an XDH RNAi agent formulated in isotonic saline, or vehicle control (isotonic saline with no RNAi agent), according to the following Table 60.
  • TABLE 60
    Targeted Positions and Dosing Groups of Example 27
    Targeted Gene
    Position (within
    Group SEQ ID NO: 1) RNAi Agent and Dose Dosing Regimen
    1 N/A Saline (no RNAi agent) Single injection on day 1
    2 2696 2.0 mg/kg AD10621 Single injection on day 1
    3 2696 4.0 mg/kg AD10621 Single injection on day 1
    4 2701 2.0 mg/kg AD09745 Single injection on day 1
    5 2701 4.0 mg/kg AD09745 Single injection on day 1
    6 2701 2.0 mg/kg AD12167 Single injection on day 1
    7 2701 4.0 mg/kg AD12167 Single injection on day 1
    8 2696 2.0 mg/kg AD12168 Single injection on day 1
    9 2696 4.0 mg/kg AD12168 Single injection on day 1
  • Each of the XDH RNAi agents included modified nucleotides that were conjugated at the 5′ terminal end of the sense strand to a targeting ligand that included three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5A, 5B, 5C, and 6 for specific modifications and structure information related to the XDH RNAi agents, including (NAG37)s ligand). The XDH RNAi agents of Groups 2-9 all included nucleotide sequences that were designed to inhibit expression of an XDH gene at positions 2696 and 2701 of the gene. (See, e.g., SEQ ID NO:1 and Table 2 for the XDH gene referenced).
  • The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15 and day 22, and XDH expression levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment through day 22 are shown in the following Table 60.
  • TABLE 60
    Average XDH Normalized to Pre-Treatment &
    Control in XDH-GLUC AAV Mice from Example 27
    Day 8 Day 15 Day 22
    Avg Std Dev Avg Std Dev Avg Std Dev
    Group ID XDH (+/−) XDH (+/−) XDH (+/−)
    Group 1 Saline (no RNAi agent) 1.000 0.064 1.000 0.152 1.000 0.247
    Group 2 2.0 mg/kg AD10621 0.449 0.072 0.317 0.108 0.410 0.095
    Group 3 4.0 mg/kg AD10621 0.317 0.040 0.184 0.038 0.232 0.059
    Group 4 2.0 mg/kg AD09745 0.809 0.214 0.567 0.196 0.690 0.281
    Group 5 4.0 mg/kg AD09745 0.590 0.090 0.347 0.047 0.408 0.026
    Group 6 2.0 mg/kg AD12167 0.712 0.072 0.546 0.124 0.650 0.211
    Group 7 4.0 mg/kg AD12167 0.522 0.087 0.297 0.093 0.385 0.092
    Group 8 2.0 mg/kg AD12168 0.881 0.126 0.497 0.029 0.631 0.120
    Group 9 4.0 mg/kg AD12168 0.500 0.019 0.327 0.028 0.359 0.060
  • As shown in Table 60, the XDH RNAi agent of Group 2 and 3 (AD010621) showed superior XHD inhibition compared to each of the RNAi agents in Groups 4-9 in vivo. For example, a single dose of 2.0 mg/kg of AD10621 reported approximately 59% inhibition of XDH (0.410) and a single 4.0 mg/kg dose reported approximately 77% inhibition (0.232) on day 22.
    • Other Embodiments
  • It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (19)

What is claimed is:
1. A method of treating an XDH-related disease, disorder, or a symptom thereof, in a subject in need thereof, comprising:
providing an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence of ccuccgcaCfAfGfauauugucau (SEQ ID NO: 1664) and the antisense strand comprises a nucleic acid sequence of asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO: 1081), wherein lower case (n)=2′-O-Me modified nucleotide; Nf=2′-F modified nucleotide; and s=phosphorothioate backbone modification; and
administering the subject the RNAi agent, thereby treating the XDH-related disease, disorder, or the symptom thereof.
2. The method of claim 1, wherein the sense strand further comprises an inverted abasic residue at each of the 5′ end and the 3′ end.
3. The method of claim 2, wherein the inverted abasic residue is coupled to an adjacent nucleoside via a phosphorothioate backbone.
4. The method of claim 1, wherein the 5′ end of the sense strand is coupled to a targeting ligand.
5. The method of claim 4, wherein the targeting ligand comprises:
Figure US20230139166A1-20230504-C00024
6. The method of claim 4, wherein the targeting ligand is
Figure US20230139166A1-20230504-C00025
7. The method of claim 1, wherein the RNAi agent is a pharmaceutically acceptable salt.
8. The method of claim 7, wherein the pharmaceutically acceptable salt is a sodium salt.
9. The method of claim 1, wherein the RNAi agent is administered intravenously or subcutaneously.
10. The method of claim 1, wherein the RNAi agent is administered to the subject in an effective amount that reduces XDH gene expression level, XDH mRNA expression level, XDH protein expression level, or XDH activity level in the subject at least about 30% relative to the expression levels of the subject prior to the administration.
11. The method of claim 1, wherein the RNAi agent is administered to the subject in an effective amount that reduces a level of serum uric acid level.
12. The method of claim 1, wherein the RNAi agent is administered at a dose of about 0.05 mg/kg to about 10.0 mg/kg of body weight of the subject.
13. The method of claim 1, wherein the RNAi agent is administered at an amount from about 50 to about 400 mg.
14. The method of claim 1, wherein the RNAi agent is administered in two or more doses.
15. The method of claim 1, wherein the XDH-related disease, disorder, or the symptom thereof comprises hyperuricemia.
16. The method of claim 1, wherein the XDH-related disease, disorder, or the symptom thereof comprises gout.
17. The method of claim 1, wherein the subject is a human subject.
18. A method of inhibiting XDH gene expression, reducing XDH mRNA expression level, or reducing XDH protein expression level in a cell comprising:
contacting an RNAi agent to the cell, thereby inhibiting XDH gene expression, reducing XDH mRNA expression level, or reducing XDH protein expression level in the cell,
wherein the RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence of ccuccgcaCfAfGfauauugucau (SEQ ID NO: 1664) and the antisense strand comprises a nucleic acid sequence of asUfsgsAfcaauaucUfgUfgCfggagsg (SEQ ID NO: 1081), wherein lower case (n)=2′-O-Me modified nucleotide; Nf=2′-F modified nucleotide; and s=phosphorothioate backbone modification.
19. The method of claim 18, wherein the XDH gene expression, the XDH mRNA expression level, or the XDH protein expression level are reduced at least about 30% relative to a cell untreated with the RNAi agent.
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