US20230416737A1 - RNAi Agents for Inhibiting Expression of DUX4, Compositions Thereof, And Methods of Use - Google Patents

RNAi Agents for Inhibiting Expression of DUX4, Compositions Thereof, And Methods of Use Download PDF

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US20230416737A1
US20230416737A1 US18/181,311 US202318181311A US2023416737A1 US 20230416737 A1 US20230416737 A1 US 20230416737A1 US 202318181311 A US202318181311 A US 202318181311A US 2023416737 A1 US2023416737 A1 US 2023416737A1
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seq
rnai agent
dux4
sense strand
nucleotides
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Zhi-Ming Ding
Jonathan Van Dyke
Xiaokai Li
Anthony Nicholas
Casi M. Schienebeck
Tao Pei
Zhao XU
Teng Ai
Susan Phan
Susan Ramos-Hunter
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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: LI, XIAOKAI, SCHIENEBECK, CASI M, VAN DYKE, Jonathan, DING, Zhi-ming, PEI, TAO, PHAN, Susan, RAMOS-HUNTER, Susan, AI, TENG, XU, Zhao, NICHOLAS, ANTHONY
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Definitions

  • RNA interference (RNAi) agents e.g., double stranded RNAi agents, for inhibition of double homeobox 4 (DUX4) gene expression
  • DUX4 RNAi agents for inhibition of double homeobox 4 (DUX4) gene expression
  • compositions that include DUX4 RNAi agents and methods of use thereof.
  • DUX4 is a transcription factor normally expressed during embryogenesis containing two homeobox domains whose partial gene is located within the D4Z4 macrosatellite repeat array on chromosome 4. It is normally heavily epigenetically repressed via methylation in all tissues except testis and has no known physiological function in adult skeletal muscle. Under conditions where DUX4 is hypomethylated and derepressed in skeletal muscle, DUX4 can be expressed and can activate the transcription of germline genes, immunemediators, retrotransposons, endogenous retrovirus elements, and pericentromeric satellite HSATII sequences which can promote the misexpression of non-physiological transcripts, long noncoding RNAs, or antisense transcripts that ultimately cause intracellular and extracellular signaling cascades resulting in muscle degeneration. It is the expression of DUX4 that causes the muscle pathology and weakness responsible for the common symptoms of Facioscapulohuneral Muscular Dystrophy (FSHD), the most common adult myopathy affecting 1 in 15,000 to 1 in 20,000 adults.
  • FSHD onset is relatively late, with typical diagnoses occurring at 20 to 30 years of age, and progression is slow, with muscle weakness severity increasing over years to decades.
  • FSHD patients commonly experience asymmetric muscle weakness and loss of mass in the muscles of the face, back, upper arms, abdominal core, hip girdle, and legs resulting in a significantly reduced quality of life.
  • Both major forms of FSH4D referred to as FSHD1 and FSHD2, are caused by permissive expression of DUX4.
  • FSHD1 occurs when the D4Z4 macrosatellite repeat array is fewer than 11 copies in length.
  • FSHD2 is caused by loss of function mutations in the structural maintenance of the chromosomes hinge domain 1 (SMCHD1) gene responsible, in part, for methylating and repressing the D4Z4 macrosatellite repeat array. Reduced SMCHD1 activity results in epigenetic de-repression and expression of DUX4.
  • DUX4 is not normally expressed in adult skeletal muscle, has no known normal physiological function in skeletal muscle, and, when expressed, results in a gain of function myotoxicity, it is a difficult target for most modalities such as small molecule chemical compounds or antibodies.
  • a therapeutic capable of inhibiting DUX4 expression and preventing, halting, and/or reversing the DUX4 expression-related muscle degeneration, muscle mass loss, and muscle weakness associated with FSHD.
  • RNAi agents also herein referred to as RNAi agent, RNAi trigger, or trigger
  • RNAi agent double stranded RNAi agents
  • DUX4 double homeobox 4
  • FSHD Facioscapulohumeral Muscular Dystrophy
  • the present disclosure features DUX4 RNAi agents, compositions that include such RNAi agents, and methods for inhibiting expression of a DUX4 gene in vitro and/or in vivo using the RNAi agents and compositions that include the RNAi agents described herein.
  • the DUX4 RNAi agents described herein are able to selectively and efficiently decrease, inhibit, or silence expression of a DUX4 gene.
  • the described DUX4 RNAi agents can be used in methods for therapeutic treatment (including preventative, intervention, or prophylactic treatment) of symptoms and diseases such as FSHD, including the most common forms of FSHD1 and FSHD2, which are both caused by permissive expression of DUX4.
  • the methods disclosed herein include the administration of one or more DUX4 RNAi agents to a subject, e.g., a human or animal subject, using any suitable methods known in the art, such as for example, subcutaneous (SQ) injection, intramuscular injection, or intravenous (IV) administration.
  • the disclosure features RNAi agents for inhibiting expression of a DUX4 gene, wherein the RNAi agent includes a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand).
  • the sense strand and the antisense strand can be partially, substantially, or fully complementary to each other.
  • the length of the RNAi agent sense strands described herein each can be 15 to 49 nucleotides in length.
  • the length of the RNAi agent antisense strands described herein each can be 17 to 49 nucleotides in length. In some embodiments, the sense and antisense strands are independently 17 to 26 nucleotides in length.
  • the sense and antisense strands can be either the same length or different lengths. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides in length. In some embodiments, the antisense strands are independently 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the sense strands are independently 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 RNAi agents described herein upon delivery to a cell expressing DUX4 such as a skeletal muscle cell (a skeletal myofiber), inhibit the expression of one or more DUX4 gene transcripts in vivo and/or in vitro.
  • the DUX4 RNAi agents disclosed herein target a double homeobox 4 (DUX4) gene (see, e.g., SEQ ID NO:1 & SEQ ID NO:2, Homo sapiens transcript variant 2).
  • DUX4 double homeobox 4
  • the RNAi agents disclosed herein target a portion of a DUX4 gene having the sequence of any of the sequences disclosed in Table 1.
  • the disclosure features pharmaceutical compositions that include one or more of the disclosed DUX4 RNAi agents that are able to selectively and efficiently decrease expression of a DUX4 gene.
  • the pharmaceutical compositions that include one or more DUX4 RNAi agents described herein can be administered to a subject, such as a human or animal subject, for the treatment (including intervention or prophylactic treatment or inhibition) of symptoms and diseases that can be ameliorated at least in part by a reduction in DUX4 protein levels.
  • the pharmaceutical compositions described herein include an RNAi agent capable of inhibiting the expression of a DUX4 gene and at least one pharmaceutically acceptable excipient.
  • Examples of DUX4 RNAi agent sense strands and antisense strands that can be used in a DUX4 RNAi agent are provided in Tables 3 and Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and Table 5.4.
  • Examples of DUX4 RNAi agent duplexes are provided in Tables 5.1, 5.2, 5.3, and 5.4.
  • Examples of 19-nucleotide core stretch sequences that may consist of or may be included in the sense strands and antisense strands of certain DUX4 RNAi agents disclosed herein, are provided in Table 2.
  • RNAi agent for inhibiting expression of a DUX4 gene comprising:
  • RNAi agent for inhibiting expression of a DUX4 gene comprising:
  • RNAi agent for inhibiting expression of a DUX4 gene comprising:
  • RNAi agent for inhibiting expression of a DUX4 gene comprising:
  • the disclosure features methods for delivering DUX4 RNAi agents to skeletal muscle cells in a subject, such as a mammal, e.g., a human subject, in vivo. Also described herein are compositions for use in such methods.
  • the one or more DUX4 RNAi agents can be delivered to target cells or tissues using any oligonucleotide delivery technology known in the art.
  • a DUX4 RNAi agent is delivered to cells or tissues by covalently linking the RNAi agent to a targeting group.
  • the targeting group can include a cell receptor ligand.
  • a targeting group can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of a DUX4 RNAi agent.
  • a targeting group is linked to the 3′ or 5′ end of the sense strand.
  • a targeting group is linked to the 5′ end of the sense strand.
  • a targeting group is linked internally to a nucleotide on the sense strand and/or the antisense strand of the RNAi agent. In some embodiments, a targeting group is linked to the RNAi agent via a linker.
  • Example targeting ligands that have affinity for skeletal muscle cells and/or receptors present on skeletal muscle cells e.g., integrin alpha-v-beta-6 ( ⁇ v ⁇ 6)
  • Table 6.2 and 6.3 The synthesis and conjugation of certain targeting ligands suitable for use with the DUX4 RNAi agents disclosed herein are shown in Example 1.
  • the DUX4 RNAi agents disclosed herein that are conjugated to targeting groups or targeting ligands that direct the RNAi agent to skeletal muscle cells, whereby the RNAi agents can be selectively internalized either through receptor-mediated endocytosis or by other means.
  • the disclosure features methods for inhibiting DUX4 gene expression in a subject, the methods including administering to the subject an amount of a DUX4 RNAi agent capable of inhibiting the expression of a DUX4 gene, wherein the DUX4 RNAi agent comprises a sense strand and an antisense strand, and wherein the antisense strand includes the sequence of any one of the antisense strand nucleotide sequences in Table 2, Table 3, or Table 5.4.
  • the disclosure features methods of treatment (including prophylactic, intervention, or preventative treatment) of diseases or symptoms that can be ameliorated at least in part by a reduction in DUX4 protein levels, the methods comprising administering to a subject in need thereof a DUX4 RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 5.4.
  • a DUX4 RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 5.4.
  • Pharmaceutical compositions for use in such methods are also described.
  • a DUX4 RNAi agent is linked to one or more linking groups or other non-nucleotide groups or compounds, such as pharmacokinetic/pharmacodynamic (PK/PD) modulators.
  • PK/PD modulators can increase circulation time of the conjugated drug and/or increase the activity of the RNAi agent through improved cell receptor binding, improved cellular uptake, and/or other means. Examples of PK/PD modulators suitable for use with the DUX4 RNAi agents disclosed herein can be found in Table 6.5 and 6.7, herein.
  • a DUX4 RNAi agent is conjugated to a targeting group, a linking group, a PK/PD modulator, and/or another non-nucleotide group. In some embodiments, a DUX4 RNAi agent is conjugated to a targeting group and a PK/PD modulator.
  • DUX4 RNAi agents provides methods for therapeutic (including prophylactic or intervention) treatment of diseases or disorders that can be ameliorated at least in part by a reduction in DUX4 protein levels. Described herein are compositions for delivery of DUX4 RNAi agents to skeletal muscle cells to a subject.
  • the DUX4 RNAi agents disclosed herein are able to reduce DUX4 gene expression in paraspinal, facial, torso, abdominal, and limb muscle tissues of the subject, for example, in the triceps, biceps, quadriceps, pectoralis, gastrocnemius, soleus, masseter, EDL (extensor digitorum longus), TA (Tibialis anterior), trapezius, and/or diaphragm, of the subject.
  • methods for the treatment (including prophylactic or intervention treatment) of a pathological state mediated at least in part by DUX4 expression, such as FSHD are disclosed herein, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 2, Table 4.1, Table 4.2, Table 4.3, Table 4.4, Table 4.5, Table 4.6, or Table 5.4.
  • methods for the treatment (including prophylactic or intervention treatment) of a pathological state mediated at least in part by DUX4 expression are disclosed herein, wherein the methods include administering to a subject a therapeutically effective amount of a DUX4 RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4 herein, and an antisense strand comprising the sequence of any of the sequences in Table 3.
  • methods of inhibiting expression of a DUX4 gene include administering to a subject a DUX4 RNAi agent that includes a sense strand consisting of the nucleobase sequence of any of the sequences in Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4 herein, and the antisense strand consisting of the nucleobase sequence of any of the sequences in Table 3 or Table 5.4.
  • DUX4 RNAi agent that includes a sense strand consisting of the modified sequence of any of the modified sequences in Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or Table 5.4 herein, and the antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 5.4.
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a nucleobase sequence differing by 0 or 1 nucleobases from the nucleotide sequence (5′ ⁇ 3′) selected from the group consisting of:
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence differing by no more than 1 nucleotide from the nucleotide sequence (5′ ⁇ 3′) selected from the group consisting of:
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a nucleobase sequence differing by 0 or 1 nucleobases from the nucleotide sequence (5′ ⁇ 3′) selected from the group consisting of:
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a modified nucleotide sequence differing by no more than 1 nucleotide from the nucleotide sequence (5′ ⁇ 3′) selected from the group consisting of:
  • a DUX4 RNAi agent disclosed herein includes:
  • a DUX4 RNAi agent disclosed herein includes:
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′ ⁇ 3′):
  • the DUX4 RNAi agent further includes a sense strand that is at least partially complementary to the antisense strand; and wherein all or substantially all of the nucleotides on both the antisense strand and the sense strand are modified nucleotides.
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′ ⁇ 3′):
  • the DUX4 RNAi agent further includes a sense strand that is at least partially complementary to the antisense strand; wherein all or substantially all of the nucleotides on both the antisense strand and the sense strand are modified nucleotides; and wherein the sense strand further includes an inverted abasic residue at both the 3′ terminal end of the nucleotide sequence and at the 5′ terminal end of the nucleotide sequence, and the sense strand also includes a targeting ligand at the 5′ terminal end of the sense strand that is covalently linked to the inverted abasic residue, wherein the targeting ligand has affinity for skeletal muscle cells and/or a
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′ ⁇ 3′):
  • the DUX4 RNAi agent further includes a sense strand that is at least partially complementary to the antisense strand; wherein all or substantially all of the nucleotides on both the antisense strand and the sense strand are modified nucleotides; and wherein the sense strand further includes an inverted abasic residue at both the 3′ terminal end of the nucleotide sequence and at the 5′ terminal end of the nucleotide sequence, and the sense strand also includes a targeting ligand that is covalently linked to the 5′ terminal end, wherein the targeting ligand has affinity for skeletal muscle cells and/or a receptor present on skeletal muscle cells, and the sense strand further
  • a DUX4 RNAi agent disclosed herein includes an antisense strand and a sense strand, wherein the antisense strand and the sense strand consist of, consist essentially of, or comprise nucleotide sequences that differ by 0 or 1 nucleotides from one of the following nucleotide sequence (5′ ⁇ 3′) pairs:
  • a DUX4 RNAi agent disclosed herein includes an antisense strand and a sense strand, wherein the antisense strand and the sense strand consist of, consist essentially of, or comprise nucleotide sequences that differ by 0 or 1 nucleotides from one of the following nucleotide sequences (5′ ⁇ 3′) pairs:
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′ ⁇ 3′):
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that consists of, consists essentially of, or comprises a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′->3′):
  • a DUX4 RNAi agent disclosed herein includes an antisense strand and a sense strand that consists of, consists essentially of, or comprises modified nucleotide sequences that differs by 0 or 1 nucleotides from one of the following nucleotide sequence pairs (5′ ⁇ 3′):
  • a DUX4 RNAi agent disclosed herein includes an antisense strand and a sense strand that consists of, consists essentially of, or comprises one of the following nucleotide sequence pairs (5′ ⁇ 3′):
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that includes a nucleobase sequence that differs by 0 or 1 nucleobases from the nucleotide sequences selected from the group consisting of (5′ ⁇ 3′):
  • a DUX4 RNAi agent disclosed herein includes an antisense strand that includes a nucleobase sequence that differs by 0 or 1 nucleobases from the nucleotide sequences selected from the group consisting of (5′ ⁇ 3′):
  • a DUX4 RNAi agent disclosed herein includes an antisense strand and a sense strand that each include a nucleobase sequences that differs by 0 or 1 nucleobases from the nucleotide sequence pairs selected from the group consisting of (5′ ⁇ 3′):
  • oligonucleotide and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodified.
  • RNAi agent also referred to as an “RNAi trigger” 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 mRNA 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. DUX4 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 “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, and thus 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 or modified nucleotides 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 a DUX4 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 inventions 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 or intervention 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.
  • a typical pharmaceutically acceptable salt of the disclosed DUX4 RNAi agents is in the form of a sodium salt.
  • 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.
  • FIG. 1 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 3.
  • FIG. 2 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 4.
  • FIG. 3 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 4.
  • FIG. 4 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 5.
  • FIG. 5 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 6.
  • FIG. 6 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 6.
  • FIG. 7 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 7.
  • FIG. 8 Graph depicting time on Rotarod apparatus of FSHD-like model mice, as more fully described in Example 7.
  • FIG. 9 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 8.
  • FIG. 10 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 9.
  • FIG. 11 Graph depicting mean bodyweights of FSHD-like model mice, as more fully described in Example 10.
  • FIG. 12 Graph depicting time on Rotarod apparatus of FSHD-like model mice, as more fully described in Example 10.
  • FIG. 13 Graph depicting DUX4 expression in patient-derived myotubules, as more fully described in Example 11.
  • FIG. 14 Graph depicting relative gene expression of several biomarker genes known to be related to FSHD in patient-derived myotubules, as more fully described in Example 11.
  • FIG. 15 A Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000232 (see, e.g., Table 5.4), having an avb6-SM45b targeting ligand linked via an L4 linker to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and having a PK/PD modulator with the structure of LP1b linked via the C6-SS—C6 linker at the 3′ end of the sense strand. (See, e.g., Examples 1 and 3 herein).
  • FIGS. 15 A to 15 I The following abbreviations are used in FIGS. 15 A to 15 I : a, c, g, and u are 2′-O-methyl modified nucleotides; Af, Cf, Gf, and Uf are 2′-fluoro modified nucleotides; o is a phosphodiester linkage; s is a phosphorothioate linkage; invAb is an inverted abasic residue (see, e.g., Table 6.1); cPrpu is a 5′-cyclopropyl phosphonate-2′-O-methyluridine modified nucleotide (see, e.g., Table 6.1); avb6-SM45b is the small molecule targeting ligand of SM45b (see, e.g., Table 6.3); -L4- is the linker having the structure as described in Example 3; avb6-pep1 is the avb6 peptide 1 targeting
  • FIG. 15 B Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000247 (see, e.g., Table 5.4), shown having an avb6-peptide 1 targeting ligand linked to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and with the PK/PD modulator having the structure of LP38b linked to the C6-SS—C6 linker at the 3′ end of the sense strand (See, e.g., Examples 1 and 3 herein).
  • FIG. 15 C Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000278 (see, e.g., Table 5.4), having an avb6-SM45b targeting ligand linked via an L4 linker to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and having a PK/PD modulator with the structure of LP1b linked via the C6-SS—C6 linker at the 3′ end of the sense strand. (See, e.g., Examples 1 and 3 herein).
  • FIG. 15 D Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC0000280 (see, e.g., Table 5.4), having an avb6-SM45b targeting ligand linked via an L4 linker to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and having a PK/PD modulator with the structure of LP1b linked via the C6-SS—C6 linker at the 3′ end of the sense strand. (See, e.g., Examples 1 and 3 herein)
  • FIG. 15 E Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC0000281 (see, e.g., Table 5.4), having an avb6-SM45b targeting ligand linked via an L4 linker to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and having a PK/PD modulator with the structure of LP1b linked via the C6-SS—C6 linker at the 3′ end of the sense strand. (See, e.g., Examples 1 and 3 herein)
  • FIG. 15 F Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000446 (see, e.g., Table 5.4), shown having an avb6-peptide 1 targeting ligand linked to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and with the PK/PD modulator having the structure of LP29b linked to the C6-SS—C6 linker at the 3′ end of the sense strand (See, e.g., Examples 1 and 3 herein).
  • FIG. 15 G Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000447 (see, e.g., Table 5.4), shown having an avb6-peptide 1 targeting ligand linked to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and with the PK/PD modulator having the structure of LP29b linked to the C6-SS—C6 linker at the 3′ end of the sense strand (See, e.g., Examples 1 and 3 herein).
  • FIG. 15 H Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000448 (see, e.g., Table 5.4), shown having an avb6-peptide 1 targeting ligand linked to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and with the PK/PD modulator having the structure of LP29b linked to the C6-SS—C6 linker at the 3′ end of the sense strand (See, e.g., Examples 1 and 3 herein).
  • FIG. 15 I Schematic diagram of the modified sense and antisense strands of the DUX4 RNAi agent conjugate having the structure of AC000449 (see, e.g., Table 5.4), shown having an avb6-peptide 1 targeting ligand linked to the (NH 2 —C 6 ) linking group at the 5′ end of the sense strand, and with the PK/PD modulator having the structure of LP28b linked to the C6-SS—C6 linker at the 3′ end of the sense strand (See, e.g., Examples 1 and 3 herein).
  • FIG. 16 A through FIG. 16 E Chemical structure representation of DUX4 RNAi agent conjugate of AC000446 (see, e.g., Table 5.4), shown in a free acid form.
  • FIG. 17 A through FIG. 17 E Chemical structure representation of DUX4 RNAi agent conjugate having the structure of AC0000446 (see, e.g., Table 5.4), shown in a sodium salt form.
  • FIG. 18 A through FIG. 18 E Chemical structure representation of DUX4 RNAi agent conjugate of AC000448 (see, e.g., Table 5.4), shown in a free acid form.
  • FIG. 19 A through FIG. 19 E Chemical structure representation of DUX4 RNAi agent conjugate having the structure of AC0000448 (see, e.g., Table 5.4), shown in a sodium salt form.
  • FIG. 20 A through FIG. 20 E Chemical structure representation of DUX4 RNAi agent conjugate of AC000449 (see, e.g., Table 5.4), shown in a free acid form.
  • FIG. 21 A through FIG. 21 E Chemical structure representation of DUX4 RNAi agent conjugate having the structure of AC0000449 (see, e.g., Table 5.4), shown in a sodium salt form.
  • RNAi agents for inhibiting expression of a DUX4 gene referred to herein as DUX4 RNAi agents or DUX4 RNAi triggers.
  • Each DUX4 RNAi agent comprises a sense strand and an antisense strand.
  • the sense strand can be 15 to 49 nucleotides in length.
  • the antisense strand each can be 17 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 17 to 27 nucleotides in length.
  • the sense and antisense strands are each independently 19-21 nucleotides in length.
  • both the sense and antisense strands are each 21-26 nucleotides in length. In some embodiments, the sense and antisense strands are each 21-24 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some embodiments, a sense strand is 23 nucleotides in length and an antisense strand is 21 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21 nucleotides in length.
  • the RNAi agent sense strands are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length.
  • the RNAi agent antisense strands are 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • a double-stranded RNAi agent has a duplex length of about 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
  • nucleotide sequences used in forming DUX4 RNAi agents are provided in Tables 2, 3, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and 5.4.
  • Examples of RNAi agent duplexes, that include the sense strand and antisense strand sequences in Tables 2, 3, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, are shown in Tables 5.1, 5.2, 5.3, and 5.4.
  • the region of perfect, substantial, or partial complementarity between the sense strand and the antisense strand (sometimes referred to the “duplex region”) is 12-26 (e.g., 12, 13, 14, 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 DUX4 RNAi agents described herein includes at least 12 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 a DUX4 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 DUX4 mRNA target.
  • this sense strand core stretch is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, this sense strand core stretch is 17 nucleotides in length. In some embodiments, this sense strand core stretch is 19 nucleotides in length. In some embodiments, this sense strand core stretch is 21 nucleotides in length.
  • An antisense strand of a DUX4 RNAi agent described herein includes at least 17 consecutive nucleotides that have at least 85% complementarity to a core stretch of the same number of nucleotides in a DUX4 mRNA, and in some embodiments, 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 DUX4 mRNA target.
  • this antisense strand core stretch is 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • this antisense strand core stretch is 19 nucleotides in length. In some embodiments, this antisense strand core stretch is 17 nucleotides in length. In some embodiments, this antisense strand core stretch is 21 nucleotides in length. In some embodiments, this antisense strand core stretch is 23 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 DUX4 RNAi agent sense and antisense strands anneal to form a duplex.
  • a sense strand and an antisense strand of a DUX4 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 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% or 100% complementary to a corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of a DUX4 RNAi agent have a region of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% base paired or 100% base paired.)
  • the antisense strand of a DUX4 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 5.4.
  • the sense strand of a DUX4 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2 or Table 4.1, or Table 4.2, or Table 4.3, or Table 4.4, or Table 4.5, Table 4.6, or Table 5.4.
  • 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 DUX4 mRNA.
  • the sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in the DUX4 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 embodiments, 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.
  • a DUX4 RNAi agent has an antisense strand having a 3′ extension and a sense strand having a 5′ extension. In some embodiments, 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.
  • a DUX4 RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, a DUX4 RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding DUX4 mRNA sequence. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding DUX4 mRNA sequence.
  • a DUX4 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 DUX4 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 TTTT (each listed 5′ to 3′).
  • a sense strand can have a 3′ extension and/or a 5′ extension.
  • a DUX4 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 DUX4 mRNA sequence.
  • the sense strand 5′ extension is one of the following sequences, but is not limited to: CA, AUAGGC, AUAGG, AUAG, AUA, A, AA, AC, GCA, GGCA, GGC, UAUCA, UAUC, UCA, UAU, U, UU (each listed 5′ to 3′).
  • DUX4 RNAi agent antisense strand includes a sequence of any of the sequences in Tables 2 or 3.
  • a DUX4 RNAi agent antisense strand comprises or consists of any one of the modified sequences in Table 3 or Table 5.4.
  • a DUX4 RNAi agent antisense strand includes the sequence of nucleotides (from 5′ end ⁇ 3′ end) 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 5.4.
  • a DUX4 RNAi agent sense strand includes the sequence of any of the sequences in Tables 2 or 4.
  • a DUX4 RNAi agent sense strand includes the sequence of nucleotides (from 5′ end ⁇ 3′ end) 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, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • a DUX4 RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4.1, Table 4.2, Table 4.3, Table 4.4, Table 4.5, Table 4.6, or Table 5.4.
  • the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form blunt ends. In some embodiments, 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 DUX4 RNAi agents disclosed herein may also be comprised of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the DUX4 RNAi agent are modified nucleotides.
  • the DUX4 RNAi agents disclosed herein may further be comprised of one or more modified internucleoside linkages, e.g., one or more phosphorothioate or phosphorodithioates linkages.
  • a DUX4 RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2′-modified nucleotide is combined with modified internucleoside linkage.
  • a DUX4 RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a DUX4 RNAi agent is prepared as a 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.
  • a DUX4 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%
  • 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′-Methyl, 2′-fluoro nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides.
  • PNAs peptide nucleic acids
  • 2′-modified nucleotides include, but are not limited to, 2′-O-methyl nucleotides (also referred to as 2′-methoxy 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.
  • 2′-O-methyl nucleotides also referred to as 2′-methoxy nucleotides
  • 2′-fluoro nucleotides also referred to herein as 2′-deoxy-2′-fluoro nucleotides
  • 2′-deoxy nucleotides 2′-methoxyethyl (2′-O-
  • DUX4 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. Various modified nucleotides are well known and described in the art.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aninopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, inosine (hypoxanthine), xanthine, 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 6.1 herein.
  • one or more nucleotides of a DUX4 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, diphosphorothioates, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkyl-phospho
  • 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 a DUX4 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • an antisense strand of a DUX4 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 a DUX4 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • an antisense strand of a DUX4 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.
  • a DUX4 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 linkage 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.
  • a DUX4 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.
  • a DUX4 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 (also referred to in the art as “inverted abasic sites”) are added as capping residues (see Table 6.1).
  • 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 embodiments, one or more inverted abasic residues (invAb) are added to the 5′ end of the sense strand. In some embodiments, 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 embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between the PK/PD modulator and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, 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 internucleoside 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. Chemical structures for inverted abasic deoxyribose residues are shown in Table 6.1 below.
  • DUX4 RNAi agent embodiments disclosed herein were designed to target specific positions on a DUX4 gene (i.e., specific positions on a DUX4 gene transcript).
  • an antisense strand sequence is designed to target a DUX4 gene at a specific 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 a DUX4 gene at position 408 requires that when base pairing to the gene, the 5′ terminal nucleobase of the antisense strand is aligned with position 428 of the DUX4 gene.
  • a DUX4 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 DUX4 RNAi agent must be aligned with position 428 of the gene; however, the 5′ terminal nucleobase of the antisense strand may be, but is not required to be, complementary to position 428 of a DUX4 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 DUX4 RNAi agent is important to the level of inhibition achieved by the DUX4 RNAi agent.
  • the DUX4 RNAi agents disclosed herein target a DUX4 gene at or near the positions of the DUX4 sequence shown in Table 1.
  • the antisense strand of a DUX4 RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to a target DUX4 19-mer sequence disclosed in Table 1.
  • a DUX4 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 embodiments, a DUX4 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 a 19-mer target sequence disclosed in Table 1.
  • a DUX4 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 a 19-mer target sequence disclosed in Table 1. In some embodiments, a DUX4 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 DUX4 gene, or can be non-complementary to the DUX4 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.
  • a DUX4 RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2, Table 3, or Table 5.4.
  • a DUX4 RNAi sense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) 1-17, 1-18, or 2-18 of any of the sense strand sequences in Table 2 or Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6 or 5.4.
  • a DUX4 RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5′ end ⁇ 3′ end) 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) 1-17 or 1-18 of any of the sense strand sequences in Table 2 or Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6 or 5.4.
  • the DUX4 RNAi agents include core 19-mer nucleotide sequences shown in the following Table 2.
  • the DUX4 RNAi agent sense strands and antisense strands that comprise or consist of the nucleotide sequences in Table 2 can be modified nucleotides or unmodified nucleotides.
  • the DUX4 RNAi agents having the sense and antisense strand sequences that comprise or consist of any of the nucleotide sequences in Table 2 are all or substantially all modified nucleotides.
  • the antisense strand of a DUX4 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2. In some embodiments, the sense strand of a DUX4 RNAi 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 embodiments, 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 DUX4 RNAi agent sense and antisense strands are provided in Table 3 and Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and 5.4.
  • Modified DUX4 RNAi agent antisense strands, as well as their underlying unmodified nucleobase sequences are provided in Table 3.
  • Modified DUX4 RNAi agent sense strands, as well as their underlying unmodified nucleobase sequences are provided in Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6 and 5.4.
  • each of the nucleotides in each of the underlying base sequences listed in Tables 3 and Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and 5.4, as well as in Table 2, above, can be a modified nucleotide.
  • the DUX4 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 Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4 can be hybridized to any antisense strand containing a sequence listed in Table 2, Table 3, or Table 5.4 provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.
  • a DUX4 RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2, Table 3, or Table 5.4.
  • a DUX4 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 Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • antisense strands containing modified nucleotides are provided in Table 3.
  • Examples of sense strands containing modified nucleotides are provided in Tables 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and 5.4.
  • 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.
  • 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.
  • the phosphorothioate chemical structures depicted herein typically show the anion on the sulfur atom
  • the inventions disclosed herein encompass all phosphorothioate tautomers (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 DUX4 RNAi agents and compositions of DUX4 RNAi agents disclosed herein.
  • targeting groups and linking groups used with the DUX4 RNAi agents disclosed herein are included in the chemical structures provided below in Table 6.1.
  • Each sense strand and/or antisense strand can have any targeting groups or linking groups listed herein, as well as other targeting or linking groups, conjugated to the 5′ and/or 3′ end of the sequence.
  • the example DUX4 RNAi agent sense strand nucleotide sequences are shown to further include reactive linking groups at both the 5′ terminal end and the 3′ terminal end of the sense strand.
  • the DUX4 RNAi agent sense strand sequences shown in Table 4.1 above have an (NH2-C6) linking group at the 5′ end of the nucleotide sequence.
  • the DUX4 RNAi agent nucleotide sequences shown in Table 4.1 above have a (C6-SS—C6) linking group near the 3′ end of the nucleotide sequence.
  • Such reactive linking groups are positioned to facilitate the linking of targeting ligands, targeting groups, and/or PK/PD modulators to the DUX4 RNAi agents disclosed herein.
  • Linking or conjugation reactions are well known in the art and provide for formation of covalent linkages between two molecules or reactants. Suitable conjugation reactions for use in the scope of the inventions herein include, but are not limited to, amide coupling reaction, Michael addition reaction, hydrazone formation reaction, and click chemistry cycloaddition reaction.
  • targeting ligands can be synthesized as a tetrafluorophenyl (TFP) ester, which react with an amino group (e.g., NH2-C6) to attach the targeting ligand to the DUX4 RNAi agents disclosed herein.
  • TFP tetrafluorophenyl
  • targeting ligands are synthesized as azides, which can be conjugated to a propargyl or DBCO group, for example, via click chemistry cycloaddition reaction.
  • nucleotide sequences shown in Table 4.1 were synthesized with a dT nucleotide at the 3′ terminal end of the sense strand, followed by (3′ ⁇ 5′) a linker (e.g., C6-SS—C6).
  • a linker e.g., C6-SS—C6
  • a suitable and commercially available dT-loaded resin can be used to initiate the synthesis of the oligonucleotide strand.
  • the (C6-SS—C6) linker can, in some embodiments, then be used facilitate the linkage to additional components, such as, for example, a PK/PD modulator or one or more targeting ligands.
  • the C6-SS—C6 is first reduced cleaving among other things the dT residue off the molecule, which can then facilitate the conjugation of the desired PK/PD modulator.
  • Table 4.2 below shows the nucleotide sequences identified in Table 4.1, above, but without the inclusion of the 3′ terminal dT nucleotide, as these properly reflect the sequence of the DUX4 RNAi agents disclosed herein when delivered in vivo.
  • Table 4.3 shows the nucleotide sequences identified in Table 4.1, above, but without the terminal linking groups present (i.e., the nucleotide sequences with only capping groups).
  • AM09965-SS (NH2-C6)s(invAb)scaggauucAfGfAfucug 117 CAGGAUUCAGAUCUGGUUUCA 181 guuucas(invAb)(C6-SS-C6) AM09966-SS (NH2-C6)s(invAb)saggauucaGfAfUfcugg 118 AGGAUUCAGAUCUGGUUUCAA 182 uuucaas(invAb)(C6-SS-C6) AM09967-SS (NH2-C6)s(invAb)scuguucuuCfCfGfugaa 119 CUGUUCUUCCGUGAAAUUCUA 183 auucuas(invAb)(C6-SS-C6) AM09968-SS (NH2-C6)s(invAb)succuggauGfAfUfuagu 120 UCCUGGAUGAUUAGUUCAGAA 184 ucagaas(invAb)(C6
  • AM09965-SS (invAb)scaggauucAfGfAfucugguuucas(invAb) 132 CAGGAUUCAGAUCUGGUUUCA 181 AM09966-SS (invAb)saggauucaGfAfUfcugguuucaas(invAb) 133 AGGAUUCAGAUCUGGUUUCAA 182 AM09967-SS (invAb)scuguucuuCfCfGfugaaauucuas(invAb) 134 CUGUUCUUCCGUGAAAUUCUA 183 AM09968-SS (invAb)succuggauGfAfUfuaguucagaas(invAb) 135 UCCUGGAUGAUUAGUUCAGAA 184 AM10194-SS (invAb)sgcccuuguUfCfUfuccgugaaaus(invAb) 136 GCCCUUGUUCUUCCGUGAAAU 185 AM10196-
  • AM09965-SS caggauucAfGfAfucugguuuca 147 CAGGAUUCAGAUCUGGUUUCA 181 AM09966-SS aggauucaGfAfUfcugguuucaa 148 AGGAUUCAGAUCUGGUUUCAA 182 AM09967-SS cuguucuuCfCfGfugaaauucua 149 CUGUUCUUCCGUGAAAUUCUA 183 AM09968-SS uccuggauGfAfUfuaguucagaa 150 UCCUGGAUGAUUAGUUCAGAA 184 AM10194-SS gcccuuguUfCfUfuccgugaaau 151 GCCCUUGUUCUUCCGUGAAAU 185 AM10196-SS a_2NaaccuggAfUfUfagaguuacau 152 (A 2N )AACCUGGAUUAGAGUUACAU 186 AM10198-SS ggaugauuAfGfU
  • one or more targeting ligands and/or PK/PD modulators are linked or conjugated to the RNAi agent.
  • a targeting ligand (or targeting group) and/or a PK/PD modulator is linked to the 5′ end of the sense strand, the 3′ end of the sense strand, and/or to one or more internal nucleotides.
  • the synthesis of the sense strand and/or the antisense strand can be designed such that reactive groups are readily available to facilitate linkage to additional components, such as a targeting ligand or PK/PD modulator.
  • Table 4.5 depicts the sense strand of the DUX4 RNAi agents disclosed above in Table 4.1 after linking to one or more targeting ligands and/or PK/PD modulators (collectively, shown below, as Z).
  • Pharmacological moieties are linked to the DUX4 RNAi agents using reactions described in Example 1, below.
  • the linking groups may have the structure (NH—C6), (NH—C6)s, or (C6-S), the structure of each of which is shown in Table 6.1, below.
  • RNAi Agent Sense Strand Sequences Showing Targeting Ligand and/or PK/PD modulator Positions (Z pharmacological moiety (e.g., targeting ligand, targeting group, and/or PK/PD modulator))
  • the DUX4 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 4.1, 4.2, 4.3, 4.4, 4.5, 4.6 or 5.4 can be hybridized to any antisense strand containing a sequence listed in Table 2, Table 3, or Table 5.4, 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 antisense strand of a DUX4 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3.
  • the sense strand of a DUX4 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • a DUX4 RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3. In some embodiments, a DUX4 RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or 2-24 of any of the sequences in Table 2, Table 3, or Table 5.4. In certain embodiments, a DUX4 RNAi agent antisense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 3.
  • a DUX4 RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2 or Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • a DUX4 RNAi agent sense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) 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, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23, 1-24, 2-24, 3-24, or 4-24 of any of the sequences in Table 2 or Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • a DUX4 RNAi agent sense strand comprises or consists of a modified sequence of any one
  • the nucleotide at position 1 of the antisense strand can be perfectly complementary to a DUX4 gene, or can be non-complementary to a DUX4 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 DUX4 RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3.
  • a DUX4 RNAi sense strand comprises the sequence of nucleotides (from 5′ end ⁇ 3′ end) 1-17 or 1-18 of any of the sense strand sequences in Table 2 or Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • a DUX4 RNAi agent includes (i) an antisense strand comprising the sequence of nucleotides (from 5′ end ⁇ 3′ end) 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) 1-17 or 1-18 of any of the sense strand sequences in Table 2 or Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4.
  • 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.
  • the DUX4 RNAi agent has a sense strand consisting of the modified sequence of any of the modified sequences in Table 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 5.4 and an antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 5.4.
  • Certain representative sequence pairings are exemplified by the Duplex ID Nos. shown in Table 5.1, 5.2, 5.3, and 5.4.
  • a DUX4 RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the Duplex ID Nos. presented herein.
  • a DUX4 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.
  • a DUX4 RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the duplexes represented by any of the Duplex ID Nos.
  • a DUX4 RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein.
  • a DUX4 RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting ligand, targeting group, and/or linking group, wherein the targeting ligand, targeting group, and/or linking group is covalently linked to the sense strand or the antisense strand.
  • a DUX4 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 Table 5.1 (or Table 5.2, Table 5.3, or Table 5.4), and further comprises a targeting group.
  • a DUX4 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 5.1 (or Table 5.2, or 5.3, or Table 5.4), and further comprises an integrin receptor ligand targeting group.
  • a DUX4 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 5.1, 5.2, 5.3, or 5.4, and comprises one or more linking groups selected from the group consisting of (NH2-C6), (C6-NH2), (C6-SS—C6), or (6-SS-6), each as defined in Table 6.1.
  • a DUX4 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 4.1, 4.2, 4.3, 4.4, 4.5, 4.6 or 5.4.
  • a DUX4 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 Table 5.1 (or Table 5.2, 5.3 or 5.4), and further comprises an integrin targeting group.
  • a DUX4 RNAi agent comprises, consists of, or consists essentially of any of the duplexes of Table 5.1 (or Table 5.2, 5.3, or 5.4).
  • a DUX4 RNAi agent is prepared or provided as a salt, mixed salt, a free-acid, or a free base.
  • a XDH RNAi agent is prepared as a pharmaceutically acceptable salt.
  • a XDH RNAi agent is prepared as a pharmaceutically acceptable sodium salt.
  • compositions that include a combination or cocktail of at least two DUX4 RNAi agents having different sequences.
  • the two or more DUX4 RNAi agents are each separately and independently linked to targeting groups.
  • the two or more DUX4 RNAi agents are each linked to targeting groups that include or consist of targeting ligands.
  • the two or more DUX4 RNAi agents are each linked to targeting groups.
  • a DUX4 RNAi agent contains or is conjugated to one or more non-nucleotide groups including, but not limited to, a targeting group, a linking group, a pharmacokinetic/pharmacodynamic (PK/PD) modulator, a delivery polymer, or a delivery vehicle.
  • the non-nucleotide group can enhance targeting, delivery, or attachment of the RNAi agent.
  • linking groups are provided in Table 6.1
  • examples of targeting groups or targeting ligands are provided in Tables 6.2 and 6.3.
  • 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.
  • a DUX4 RNAi agent contains a non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5′ end of a DUX4 RNAi agent sense strand.
  • a non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker/linking group. In some embodiments, 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 conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.
  • Targeting groups or targeting ligands 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 molecule, cell receptor ligands, hapten, 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 in some instances can serve as linkers.
  • a linker such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) residues, which in some instances can serve as linkers.
  • the DUX4 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.
  • the DUX4 RNAi agents disclosed herein are synthesized having an NH 2 —C 6 group (represented as (NH2-C6) in the modified sequences herein) at the 5′-terminus of the sense strand of the RNAi agent.
  • the terminal amino group subsequently can be reacted to form a conjugate with, for example, a group that includes a targeting ligand.
  • the DUX4 RNAi agents disclosed herein are synthesized having one or more alkyne groups at the 5′-terminus of the sense strand of the RNAi agent.
  • the terminal alkyne group(s) can subsequently be reacted to form a conjugate with, for example, a group that includes a targeting ligand.
  • RNAi agents comprise a targeting group, which includes 2 or more targeting ligands.
  • a targeting group may be conjugated at the 5′ or 3′ end of the sense strand of an RNAi agent.
  • a targeting group may be conjugated to an internal nucleotide on an RNAi agent.
  • a targeting group may consist of two targeting ligands linked together, referred to as a “bidentate” targeting group.
  • a targeting group may consist of three targeting ligands linked together, referred to as a “tridentate” targeting group.
  • a targeting group may consist of four targeting ligands linked together, referred to as a “tetradentate” targeting group.
  • the use of a targeting ligand facilitates cell-specific targeting to cells having desired receptors on its respective surface, and binding of the targeting ligand can facilitate entry of the therapeutic agent, such as an RNAi agent, to which it is linked, into cells such as skeletal muscle cells.
  • Targeting ligands can be monomeric or monovalent (e.g., having a single targeting moiety) or multimeric or multivalent (e.g., having multiple targeting moieties).
  • the targeting group can be attached to the 3′ and/or 5′ end of the RNAi oligonucleotide using methods known in the art.
  • Embodiments of the present disclosure include pharmaceutical compositions for delivering a DUX4 RNAi agent to a skeletal muscle cell in vivo.
  • Such pharmaceutical compositions can include, for example, a DUX4 RNAi agent conjugated to a targeting group that comprises a targeting ligand.
  • the DUX4 RNAi agents disclosed herein can reduce DUX4 gene expression in one or more of the following tissues: paraspinal, facial, torso, abdominal, and limb muscle tissues, including for example, in the triceps, biceps, quadriceps, pectoralis, gastrocnemius, soleus, masseter, EDL (extensor digitorum longus), TA (Tibialis anterior), trapezius, and/or diaphragm.
  • tissues paraspinal, facial, torso, abdominal, and limb muscle tissues, including for example, in the triceps, biceps, quadriceps, pectoralis, gastrocnemius, soleus, masseter, EDL (extensor digitorum longus), TA (Tibialis anterior), trapezius, and/or diaphragm.
  • a linking group is conjugated to the RNAi agent.
  • the linking group facilitates covalent linkage of the agent to a targeting group, pharmacokinetic modulator, 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 include, but are not limited to: C6-SS—C6, 6-SS-6, reactive groups such as primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues/nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and/or PEG groups.
  • reactive groups such as primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues/nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and/or PEG groups.
  • 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, pharmacokinetic modulator, 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 may 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.
  • targeting groups are linked to the DUX4 RNAi agents without the use of an additional linker.
  • the targeting group is designed having a linker readily present to facilitate the linkage to a DUX4 RNAi agent.
  • the two or more RNAi agents can be linked to their respective targeting groups using the same linkers.
  • the two or more RNAi agents are linked to their respective targeting groups using different linkers.
  • Any of the DUX4 RNAi agent nucleotide sequences listed in Tables 2, 3, and 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and 5.4, whether modified or unmodified, can contain 3′ and/or 5′ targeting group(s), linking group(s), and/or pharmacokinetic modulator(s).
  • any of the DUX4 RNAi agent sequences listed in Tables 3 and 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, and 5.4, or are otherwise described herein, which contain a 3′ or 5′ targeting group, linking group, or pharmacokinetic modulator can alternatively contain no 3′ or 5′ targeting group, linking group, or PK/PD modulator, or can contain a different 3′ or 5′ targeting group, linking group, or PK/PD modulator including, but not limited to, those depicted in Tables 6.1, 6.2, 6.3, 6.4, 6.5, 6.6 or 6.7.
  • any of the DUX4 RNAi agent duplexes listed in Table 5.1 (or Table 5.2, 5.3 or 5.4), whether modified or unmodified, can further comprise a targeting group, linking group, or PK/PD modulator, including, but not limited to, those depicted in Tables 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, or 6.7, and in some embodiments the targeting group, linking group and/or PK/PD modulator can be attached to the 3′ or 5′ terminus of either the sense strand or the antisense strand of the DUX4 RNAi agent duplex.
  • linking groups can be commercially acquired or alternatively, are incorporated into commercially available nucleotide phosphoramidites.
  • a targeting ligand is linked to the DUX4 RNAi agents disclosed herein. Examples of certain targeting ligands are provided in Table 6.2:
  • the targeting groups in Table 6.2 are synthesized with reactive groups allowing for efficient coupling of a targeting ligand that includes one or more targeting groups to the RNAi agents disclosed herein.
  • the targeting groups identified in Table 6.2 are synthesized as azides to facilitate linkage to the RNAi agent.
  • the DUX4 RNAi agents are linked to a targeting ligand having a structure disclosed in Table 6.3:
  • Example targeting ligands for combination with DUX4 RNAi agents Compound Number Formula 40b 41b 42b 43b 44b 45b 46b 47b 48b 49b 50b 51b 52b 53b 54b 55b 56b 57b 58b 59b 60b ⁇ 6 peptide 1 or a pharmaceutically acceptable salt thereof, wherein indicates the point of connection to the DUX4 RNAi agents.
  • a delivery vehicle may 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 for nucleic acid delivery.
  • the RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesteryl and cholesteryl derivatives), encapsulating in nanoparticles, liposomes, micelles, conjugating to polymers or DPCs (see, for example WO 2000/053722, WO 2008/022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), by iontophoresis, or by incorporation into other delivery vehicles or systems available in the art such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors.
  • the RNAi agents can be conjug
  • the DUX4 RNAi agents disclosed herein are further or alternatively linked to one or more PK/PD modulators.
  • PK/PD modulators examples of certain pharmacodynamic/pharmacokinetic (PK/PD) modulators suitable for use with the RNAi agents disclosed herein are provided in Table 6.4.
  • Table 6.4 PK/PD modulators were acquired from commercial suppliers where indicated and were otherwise synthesized using commercially available materials:
  • the PK/PD modulators of Table 6.4 have the following structures following conjugation to the DUX4 RNAi agents as shown in Table 6.5:
  • the PK/PD modulator that may be conjugated to the DUX4 RNAi agents described herein may be selected from the group consisting of the PK/PD modulators in Table 6.6:
  • the PK/PD modulators of Table 6.6 have the following structures following conjugation to the DUX4 RNAi agents as shown in Table 6.7:
  • DUX4 RNAi agents may comprise one or more PK/PD modulators. In some embodiments, the DUX4 RNAi agents disclosed herein comprise one, two, three, four, five, six, seven or more PK/PD modulators.
  • PK/PD modulators may be conjugated to a DUX4 RNAi agent using any known method in the art. Many PK/PD modulators, including several of those above, are commercially available. In some embodiments, such as several of the compounds shown in Table 6.4, PK/PD modulators can include a maleimide moiety and be reacted with an RNAi agent comprising a disulfide linkage to form an RNAi agent comprising a PK/PD modulator. The disulfide may be reduced, and added to a maleimide by way of a Michael-Addition reaction. An example reaction scheme is shown below:
  • R ZZ comprises an RNAi agent, and indicates a point of connection to any suitable group known in the art.
  • alkyl group such as hexyl (C 6 H 13 ).
  • PK/PD modulator precursors may comprise a sulfone moiety and may react with a disulfide.
  • An example reaction scheme is shown below:
  • R ZZ comprises an RNAi agent, and indicates a point of connection to any suitable group known in the art.
  • alkyl group such as hexyl (C 6 H 13 ).
  • PK/PD modulator precursors may comprise an azide moiety and be reacted with an RNAi agent comprising an alkyne to form a compound comprising a PK/PD modulator conjugated to an RNAi agent according to the general reaction scheme below:
  • R ZZ comprises an RNAi agent.
  • PK/PD modulator precursors may comprise an alkyne moiety and be reacted with an RNAi agent comprising a disulfide to form a compound comprising a PK/PD modulator conjugated to an RNAi agent according to the general reaction scheme below:
  • R ZZ comprises an RNAi agent, and indicates a point of connection to any suitable group known in the art.
  • alkyl group such as hexyl (C 6 H 13 ).
  • PK/PD modulators may be conjugated to the 5′ end of the sense or antisense strand, the 3′ end of the sense or antisense strand, or to an internal nucleotide of a DUX4 RNAi agent.
  • a DUX4 RNAi agent is synthesized with a disulfide-containing moiety at the 3′ end of the sense strand, and a PK/PD modulator may be conjugated to the 3′ end of the sense strand using the general synthetic scheme shown above.
  • the DUX4 RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “medicaments”).
  • pharmaceutical compositions include at least one DUX4 RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of DUX4 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 mRNA, or inhibition in expression of the target gene.
  • the diseases to be treated is FSHD, including FSHD1 and FSHD2.
  • the pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene.
  • the method includes administering a DUX4 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 a DUX4 RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.
  • one or more of the described DUX4 RNAi agents are administered to a mammal in a pharmaceutically acceptable carrier or diluent.
  • the mammal is a human.
  • the pharmaceutical compositions including one or more DUX4 RNAi agents can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, for example, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device), and intraparenchymal administration.
  • compositions that include a DUX4 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 DUX4 RNAi agent, thereby inhibiting the expression of DUX4 mRNA in the subject.
  • the subject has been previously identified or diagnosed as having a disease or disorder that is mediated at least in part by DUX4 expression.
  • the subject has been previously identified or diagnosed as having a condition, disease, or disorder that would benefit from a reduction of DUX4 protein levels in one or more cells or tissues.
  • the subject has been previously diagnosed with having one or more skeletal muscular diseases such as FSHD, such as FSHD1 or FSHD2.
  • the subject has been suffering from symptoms associated with one or more skeletal muscle diseases.
  • the described pharmaceutical compositions that include a DUX4 RNAi agent are used for treating or managing clinical presentations in a subject that would benefit from the inhibition of expression of DUX4.
  • a therapeutically or 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 DUX4 RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
  • compositions that include a DUX4 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 DUX4 mRNA.
  • the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions that include a DUX4 RNAi agent thereby treating the symptom.
  • the route of administration is the path by which a DUX4 RNAi agent is brought into contact with the body.
  • methods of administering drugs, 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 DUX4 RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route.
  • the pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally, or topically.
  • compositions including a DUX4 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 via subcutaneous injection, intramuscular injection, or intravenous administration.
  • the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions described herein are formulated for administration to a subject.
  • pharmaceutical formulations that include the DUX4 RNAi agents disclosed herein suitable for SQ or IV administration can be prepared in an aqueous sodium phosphate buffer (e.g., the DUX4 RNAi agent formulated in 0.5 mM sodium phosphate monobasic, 0.5 mM sodium phosphate dibasic, in water)
  • an aqueous sodium phosphate buffer e.g., the DUX4 RNAi agent formulated in 0.5 mM sodium phosphate monobasic, 0.5 mM sodium phosphate dibasic, in water
  • 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., DUX4 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, NJ) or phosphate buffered saline (PBS). It 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.
  • 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.
  • 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 DUX4 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 DUX4 RNAi agent (e.g., a DUX4 RNAi agent that targets a different sequence within the DUX4 target).
  • the second therapeutic can be a small molecule drug, an antibody, an antibody fragment, and/or an aptamer.
  • an effective amount of a DUX4 RNAi agent disclosed herein will be in the range of from about 0.0001 to about 20 mg/kg of body weight/dose, e.g., from about 0.5 to about 10 mg/kg of body weight/dose.
  • the amount administered and dosing frequency e.g., daily, bi-weekly, weekly, monthly, quarterly, or semi-annually
  • 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 a DUX4 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 DUX4 RNAi agents when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.
  • the pharmaceutical compositions described herein can be packaged, for example, in pre-filled syringes or vials.
  • the DUX4 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 a reduction and/or inhibition in expression of DUX4 mRNA.
  • the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) having a disease or disorder for which the subject would benefit from reduction in DUX4 protein levels, including but not limited to, for example, FSHD, including FSHD1 and FSHD2.
  • Treatment of a subject can include therapeutic and/or prophylactic treatment.
  • the subject is administered a therapeutically effective amount of any one or more DUX4 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 described DUX4 RNAi agents are used to treat at least one symptom mediated at least in part by DUX4 protein levels, in a subject.
  • the subject is administered a therapeutically effective amount of any one or more of the described DUX4 RNAi agents.
  • the subject is administered a prophylactically effective amount of any one or more of the described 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 DUX4 gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the DUX4 RNAi agents described herein.
  • the DUX4 RNAi agents are used to treat or manage a clinical presentation or pathological state in a subject, wherein the clinical presentation or pathological state is mediated at least in part by DUX4 expression.
  • the subject is administered a therapeutically effective amount of one or more of the DUX4 RNAi agents or DUX4 RNAi agent-containing compositions described herein.
  • the method comprises administering a composition comprising a DUX4 RNAi agent described herein to a subject to be treated.
  • the gene expression level or mRNA level of a DUX4 gene in certain skeletal muscle cells of subject to whom a described DUX4 RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 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 DUX4 RNAi agent or to a subject not receiving the DUX4 RNAi agent.
  • the DUX4 protein levels of a subject to whom a described DUX4 RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 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 DUX4 RNAi agent or to a subject not receiving the DUX4 RNAi agent.
  • the gene expression level, protein level, and/or mRNA level in the subject may be reduced in a cell, group of cells, tissue, and/or other fluid of the subject.
  • the DUX4 mRNA levels in certain skeletal muscle cells or skeletal muscle tissues in a subject to whom a described DUX4 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%, or 98% relative to the subject prior to being administered the DUX4 RNAi agent or to a subject not receiving the DUX4 RNAi agent.
  • the level of DUX4 protein in the skeletal muscle cells and/or skeletal muscle tissue of a subject to whom a described DUX4 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%, or 98% relative to the subject prior to being administered the DUX4 RNAi agent or to a subject not receiving the DUX4 RNAi agent.
  • the DUX4 protein level and/or DUX4 mRNA level in the subject may be reduced in a cell, group of cells, tissue, blood, and/or other fluid (e.g., serum) of the subject, as would be understood by the person of ordinary skill in the art.
  • fluid e.g., serum
  • the level of DUX4 mRNA of a subject to whom a described DUX4 RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 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 DUX4 RNAi agent or to a subject not receiving the DUX4 RNAi agent in one or more skeletal muscle cells or skeletal muscle tissues.
  • the level of DUX4 mRNA and/or DUX4 protein in a subset of skeletal muscle cells, of a subject to whom a described DUX4 RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 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 DUX4 RNAi agent or to a subject not receiving the DUX4 RNAi agent.
  • the DUX4 RNAi agents can reduce DUX4 gene expression in one or more of the following muscle tissues: triceps, biceps, quadriceps, gastrocnemius, soleus, masseter EDL (extensor digitorum longus), TA (Tibialis anterior), trapezius, and/or diaphragm.
  • a reduction in gene expression, mRNA, and protein levels can be assessed by any methods known in the art.
  • the Examples set forth herein provide appropriate ways for measuring DUX4 protein levels and DUX4 mRNA levels in a subject. Reduction or decrease in DUX4 mRNA level and/or DUX4 protein levels, are collectively referred to herein as a reduction or decrease in DUX4 or inhibiting or reducing the expression of a DUX4 gene.
  • the Examples set forth herein illustrate known methods for assessing inhibition of DUX4 gene expression.
  • Cells, tissues, organs, and non-human organisms that include at least one of the DUX4 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.
  • DUX4 RNAi agents disclosed herein were synthesized in accordance with the following:
  • RNA and 2′-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA).
  • the 2′-O-methyl phosphoramidites that were used included the following: (5′-O-dimethoxytrityl-N 6 -(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 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-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5′-O-dimethoxytrityl-2′-O-methyl
  • the 2′-deoxy-2′-fluoro-phosphoramidites carried the same protecting groups as the 2′-O-methyl RNA amidites.
  • 5′-dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia).
  • the inverted abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA).
  • UNA phosphoramidites include 5′-(4,4′-Dimethoxytrityl)-N6-(benzoyl)-2′,3′-seco-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5′
  • cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017/214112 and Erich F. Altenhafer et al., Synthesis of a novel cyclopropyl phosphonate nucleotide as a phosphate mimic , Chemical Communications (June 2021) (DOI:10.1039/d1cc02328d). TFA aminolink phosphoramidites were also commercially purchased (ThermoFisher).
  • 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 ⁇ PBS. 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 experimentally determined.
  • Peptide 1 was prepared by modification of Arg-Gly-Asp(tBu)-Leu-Ala-Abu-Leu-Cit-Aib-Leu-Peg 5 -CO 2-2 -Cl-Trt resin 1 that was obtained using general Fmoc peptide chemistry on CS Bio peptide synthesizer utilizing Fmoc-Peg 5 -CO 2 H preloaded 2-Cl-Trt resin on (0.79 mmol/g) at 4.1 mmol scale as described above. Following cleavage from resin the peptide 6-2 was converted into tetrafluorophenyl ester 6-3, and the crude product was used in the next step without purification.
  • Targeting Ligands Either prior to or after annealing, the 5′ or 3′ amine functionalized sense strand is conjugated to a targeting ligand, either directly or via the use of a linker such as an alkyne functionalized linker (for example, DBCO or Linkers 1-10 as shown in Table 6.1), which can then be used to facilitate the conjugation to the targeting ligand(s).
  • a linker such as an alkyne functionalized linker (for example, DBCO or Linkers 1-10 as shown in Table 6.1), which can then be used to facilitate the conjugation to the targeting ligand(s).
  • the 5′ or 3′ tridentate alkyne functionalized sense strand is conjugated to the ⁇ v ⁇ 6 Integrin Ligands.
  • the following example describes the conjugation of ⁇ v ⁇ 6 integrin ligands to the annealed duplex: Stock solutions of 0.5M Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5M of Cu(II) sulfate pentahydrate (Cu(II)SO 4 ⁇ 5H 2 O) and 2M solution of sodium ascorbate were prepared in deionized water. A 75 mg/mL solution in DMSO of ⁇ v ⁇ 6 integrin ligand was made.
  • RNAi agent comprising an amine, such as C6-NH2, NH2-C6, or (NH2-C6)s, as shown in Table 6.1, above.
  • RNAi agent was dissolved in DMSO and 10% water (v/v %) at 25 mg/mL. Then 50-100 equivalents TEA and three equivalents of activated ester targeting ligand were added to the mixture. The reaction was allowed to stir for 1-2 hours while monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: Acetonitrile; column: XBridge C18). After the reaction was complete, 12 mL of acetonitrile was added followed by 0.4 mL of PBS and then the mixture was centrifuged. The solid pellet was collected and dissolved in 0.4 mL of 1 ⁇ PBS and then 12 mL of acetonitrile was added. The resulting pellet was collected and dried on high vacuum for 1 hour.
  • one or more lipid PK/PD modulator precursors can be linked to the RNAi agents disclosed herein.
  • the following describes the general process used to link a maleimide-containing lipid PK/PD modulator precursor to the (C6-SS—C6) or (6-SS-6) functionalized sense strand of an RNAi agent by undertaking a dithiothreitol reduction of disulfide followed by a thiol-Michael Addition of the respective maleimide-containing lipid PK/PD modulator precursor: In a vial, functionalized sense strand was dissolved at 50 mg/mL in sterilized water. Then 20 equivalents of each of 0.1M Hepes pH 8.5 buffer and dithiothreitol were added. The mixture was allowed to react for one hour, then the conjugate was precipitated in acetonitrile and PBS, and the solids were centrifuged into a pellet.
  • the pellet was brought up in a 70/30 mixture of DMSO/water at a solids concentration of 30 mg/mL. Then, the maleimide-containing lipid PK/PD modulator precursor was added at 1.5 equivalents. The mixture was allowed to react for 30 minutes.
  • the solvent was removed by rotary evaporator, and desalted with a 3K spin column using 2 ⁇ 10 mL exchanges with sterilized water.
  • the solid product was dried using lyophilization and stored for later use.
  • the pellet was brought up in a 70/30 mixture of DMSO/water at a solids concentration of 30 mg/mL. Then, the sulfone-containing lipid PK/PD modulator precursor was added at 1.5 equivalents. The vial was purged with N2, and heated to 40° C. while stirring. The mixture was allowed to react for one hour.
  • the solvent was removed by rotary evaporator, and desalted with a 3K spin column using 2 ⁇ 10 mL exchanges with sterilized water.
  • the solid product was dried using lyophilization and stored for later use.
  • TG-TBTA resin loaded with Cu(I) was weighed into a glass vial.
  • the vial was purged with N 2 for 15 minutes.
  • functionalized sense strand was dissolved in a separate vial in sterilized water at a concentration of 100 mg/mL.
  • two equivalents of the azide-containing lipid PK/PD modulator precursor 50 mg/mL in DMF is added to the vial.
  • TEA, DMF and water are added until the final reaction conditions are 33 mM TEA, 60% DMF, and 20 mg/mL of the conjugated product.
  • the solution was then transferred to the vial with resin via a syringe.
  • the N 2 purge was removed and the vial was sealed and moved to a stir plate at 40° C.
  • the mixture was allowed to react for 16 hours.
  • the resin was filtered off using a 0.45 ⁇ m filter.
  • the acetonitrile was removed using a rotary evaporator, and desalted with a 3K spin column using 2 ⁇ 10 mL exchanges with sterilized water.
  • the solid product was dried using lyophilization and stored for later use.
  • the mixture was allowed to react for one hour, then purified on XBridge BEH C4 Column using a mobile phase A of 100 mM HFIP, 14 mM, and TEA, and a mobile phase B of Acetonitrile using the following formula, wherein % B indicates the amount of mobile phase B while the remainder is mobile phase A.
  • the product was precipitated once by adding 12 mL of acetonitrile and 0.4 mL 1 ⁇ PBS, and the resulting solid was centrifuged into a pellet. The pellet was re-dissolved in 0.4 mL 1 ⁇ PBS and 12 mL of acetonitrile. The pellet was dried on high vacuum for one hour.
  • the pellet was brought up in a vial a 70/30 mixture of DMSO/water at a solids concentration of 30 mg/mL. Then, the alkyne-containing lipid PK/PD modulator precursor was added at 2 equivalents relative to siRNA. Then 10 equivalents of TEA was added. The vial was purged using N2, and the reaction mixture was heated to 40° C. while stirring. The mixture was allowed to react for one hour.
  • the fractions containing the product were collected, and acetonitrile was removed using a rotary evaporator.
  • the product was desalted with a 3K spin column, using 2 ⁇ 10 mL exchanges with sterilized water. The product was then dried using lyophilization and stored for later use.
  • FLExDUX4 mice B6(Cg)-Gt(ROSA)26Sortm1.1(DUX4*)Plj/J
  • HSA-MCM mice Tg(ACTA1-cre/Esr1*)2Kesr/J) by Jackson Laboratories (JAX) to produce homozygous offspring that express human DUX4 in skeletal muscle upon administration of tamoxifen.
  • FLExDUX4 Mouse Background The FLExDUX4 mice were created using a cre-dependent one-way genetic switch (FLEx) system. Homozygote mice carrying this DUX4 conditional allele are viable and fertile. Two sets of incompatible outward facing recombination sites (loxP and lox511) flank an inverted human DUX4 sequence, including exons 1-3 and both introns. The DUX4 gene encodes several alternative mRNA splicing variants. The hereditary muscle disorder, facioscapulohumeral muscular dystrophy (FSHD) is caused by the expression of DUX4 encoded by the DUX4-full-length (DUX4-fl) mRNA isoform.
  • FLEx cre-dependent one-way genetic switch
  • DUX4-fl mRNA which encodes a paired homeobox domain transcription factor
  • FSHD the rare expression of DUX4-fl (in less than 1% of muscle fibers) initiates a pathogenic cascade of events including apoptosis, differentiation defects, muscle atrophy, and susceptibility to oxidative stress.
  • FSHD is characterized by a slowly progressing muscular dystrophy that predominantly affects the skeletal muscles of the face, scapula, and upper arms but can affect muscles of the abdomen, hip girdle, and lower legs with ⁇ 20% of patients ultimately losing ambulation.
  • the DUX4 promoter drives expression of a short non-pathogenic isoform (DUX4-s) and a longer cytotoxic isoform (DUX4-fl).
  • This strain contains 4 point mutations in the 5′ splicing donor sites for the two DUX4-s mRNAs, abolishing expression of the short isoforms and only generating the pathogenic DUX4-fl mRNA isoform.
  • DUX4-fl expression is determined by which tissue(s) express Cre recombinase.
  • tissue(s) express Cre recombinase.
  • Cre recombinase When bred to mice that express Cre recombinase, the resulting offspring will have the loxP or lox511 sites recombined, resulting in the inversion of the human DUX4-fl sequence, ending in a sense orientation.
  • mice have low level DUX4-fl expression in the absence of Cre Recombinase. These mice exhibit alopecia, and, with age, soft stool, inflammation, and muscle weakness. Homozygous are more affected, as are males compared to females.
  • HSA-MCM Mouse Background HSA-MCM mice express MerCreMer double fusion protein under the control of the human ACTA1 (actin, alpha 1, skeletal muscle) promoter. Heterozygous mice are viable and fertile. Homozygotes are also viable but exhibit significantly reduced fertility. Of note, the MerCreMer double fusion protein has substantially greater Cre recombinase activity with less promiscuity compared with the CreMer single fusion protein.
  • mice When HSA-MCM mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination results in deletion of the floxed sequences in skeletal muscles of the limbs, face/tongue, and diaphragm of the offspring.
  • the MerCreMer double fusion protein consists of Cre recombinase flanked on each end with a mutated murine estrogen receptor (mer) ligand binding domain (amino acids 281-599, G525R); which does not bind its natural ligand (17 ⁇ -estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, MerCreMer can only gain access to the nuclear compartment after exposure to tamoxifen.
  • a mutated murine estrogen receptor (mer) ligand binding domain amino acids 281-599, G525R
  • Tamoxifen induction of DUX4 expression Tamoxifen dissolved in corn oil (1 mg/mL) was administered via oral gavage 2 or 3 times weekly to induce increased DUX4 expression in skeletal muscle for the duration of the study (generally 18 to 31 days).
  • Bodyweight assessments As increased DUX4 expression is known to result in muscle wasting and bodyweight loss in this animal model of FSHD, for the Examples disclosed herein, bodyweights were recorded throughout the duration of various studies, including on days of tamoxifen or RNAi agent administration and on the day of tissue harvest. Bodyweights were normalized to the first day of tamoxifen administration and average bodyweight of the “baseline” control group which was administered corn oil (containing no tamoxifen) and saline (containing no RNAi agent).
  • Tissue collection Mice were anesthetized with 3-4% isoflurane and euthanized via exsanguination. Tissues of interest intended for gene expression analysis were harvested and snap frozen in liquid nitrogen and then later stored at ⁇ 80° C. Tissues of interest intended for histology were fixed in formalin then embedded in paraffin wax and stained via histochemical or immunohistochemical protocols.
  • RNA expression analysis Whole frozen tissues were homogenized using a Precellys Tissue Homogenization System (Bertin) and RNA was isolated via acid guanidinium thiocyanate-phenol-chloroform extraction. Extracted RNA was used to synthesize complimentary DNA using a SuperScriptTM VILOTM cDNA Synthesis Kit (Thermo) and DUX4 expression was measured using a QX200 droplet digital PCR (Bio-Rad). Wfdc3 and Myo1 g expression was measured using a QuantFlex7 qRT-PCR (Applied Biosystems) systems employing Taqman primer/probe sets (Thermo-Fisher) designed to detect genes of interest. Gene expression was normalized to a reference gene (e.g. Arl1) and the average of the “baseline” control group which was administered corn oil (containing no tamoxifen) and saline (containing no RNAi agent).
  • a reference gene e.g. Arl1
  • baseline containing
  • WAP-type four-disulfide core domain 3 (Wfdc3) expression as biomarker of DUX4 activity in mouse muscle: WAP-type four-disulfide core domain 3 is a well-documented direct murine target of overexpressed DUX4-fl protein. Gene expression of Wfdc3 is measured (using qRT-PCR as described above) and used as a biomarker of DUX4 activity in collected muscle tissue.
  • Myosin 1G (Myo1 g) expression as biomarker of DUX4 activity in mouse muscle Myo1 g is a well-documented direct murine target of overexpressed DUX4-fl protein. Gene expression of Myo1 g is measured (using qRT-PCR as described above) and used as a biomarker of DUX4 activity in collected muscle tissue
  • DUX4 RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • mice were injected between the skin and muscle (i.e. subcutaneous injections) into the loose skin region over the neck and shoulder area with either isotonic saline (vehicle control) or a DUX4 RNAi agent formulated in isotonic saline.
  • isotonic saline vehicle control
  • DUX4 RNAi agent formulated in isotonic saline.
  • an oral gavage of 100 ⁇ L/20 g mouse of either corn oil (negative control) or tamoxifen dissolved in corn oil (1 mg/mL) was administered three times per week (days 4, 6, 8, 10, 12, 15, 17, and 19) to induce increased expression of DUX4.
  • the dosing regimen and details are set forth in the following Table:
  • RNAi agent Dosing Induction Agent Dosing Group RNAi agent and Dose Regimen Administration Regimen 1 Baseline N/A Corn oil 3 times per week (no RNAi agent, saline injection) (negative control) starting on day 4 2 Positive Control N/A Tamoxifen 3 times per week (no RNAi agent, saline injection) starting on day 4 3 SM45b-L4-AD07218-Bis 5 mg/kg administered Tamoxifen 3 times per week (PEG47 + C22) on days 1 and 7 starting on day 4 4 SM45b-L4-AD07219-Bis 5 mg/kg administered Tamoxifen 3 times per week (PEG47 + C22) on days 1 and 7 starting on day 4 5 SM45b-L4-AD07275-Bis 5 mg/kg administered Tamoxifen 3 times per week (PEG47 + C22) on days 1 and 7 starting on day 4 6 SM45b-L4-AD07
  • RNAi agents in Example 2 were synthesized having nucleotide sequences directed to target the DUX4 gene (i.e., mRNA transcript), and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the linker L4, which may be conjugated to the targeting ligand, a small molecule having affinity for a receptor present on skeletal muscle cells (referred to herein as a “skeletal muscle cell receptor small molecule”).
  • a functionalized amine reactive group NH 2 —C 6
  • a small molecule having affinity for a receptor present on skeletal muscle cells referred to herein as a “skeletal muscle cell receptor small molecule”.
  • the DUX4 RNAi agents were linked to a small molecule targeting ligand SM45b having affinity for skeletal muscle cells.
  • DUX4 RNAi agents were linked to a compound having the following chemical structure:
  • the targeting ligand SM45-p was synthesized as an azide, which allowed for convenient coupling to Linker L4.
  • Linker L4 was originally synthesized as a tetrafluorophenyl (TFP) ester functionalized compound having the following structure:
  • the TFP ester reactive group was first linked to the terminal amine (NH 2 —C 6 ) on the 5′ end of the sense strand.
  • the azide of SM45 was then coupled to the alkyne of linker (L4).
  • DUX4 RNAi agents in Example 2 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to a PK/PD modulator.
  • C6-SS—C6 disulfide functional group
  • a Bis(PEG47+C22) moiety was attached to the 3′ terminal end of the sense strand to serve as a pharmacokinetic/pharmacodynamic (PK/PD) modulator having the following structure:
  • the maleimide was linked to the 3′ end of the sense strand by reducing the terminal 3′ disulfide bond and performing Michael addition to the terminal 3′ thiol.
  • a PK/PD modulator can increase circulation time of the conjugated drug and/or increase the activity of the RNAi agent through improved cell receptor binding, improved cellular uptake, and/or other means.
  • the DUX4 RNAi agent sense strands had the general structure as shown in Table 4.5.
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • Wfdc3 transcript levels serve as a biomarker for DUX4 protein activity levels. Average relative Wfdc3 transcript levels in harvested tissue were similarly determined as shown in the following Tables for various muscle types:
  • Body weight measurements were taken on days 4, 6, 8, 10, 12, 14, 18, 20, and 21. Preservation of body weight can be indicative of a preventative effect. Body weights as normalized to Day 4 (pre-tamoxifen administration) and baseline are shown in FIG. 1 .
  • AD07218 included nucleotide sequences designed to inhibit a DUX4 gene (i.e., a DUX4 mRNA transcript) at position 408 of the gene;
  • AD07219 and AD07275 included nucleotide sequences designed to inhibit a DUX4 gene at position 409 of the gene;
  • AD07220 and AD07276 included nucleotide sequences designed to inhibit a DUX4 gene at position 1437 of the gene;
  • AD07221 and AD07277 included nucleotide sequences designed to inhibit a DUX4 gene at position 1518 of the gene;
  • AD07396 (Group 10) included nucleotide sequences designed to inhibit a DUX4 gene at position 1496 of the gene.
  • the DUX4 RNAi agents provide for a reduction in DUX4 gene expression in the FSHD-like mouse model, with the DUX4 RNAi agents targeting positions 408, 409, and 1437 in particular evidencing substantial inhibition of DUX4 gene expression.
  • Tables 8.1-8.9 the relative expression of DUX4 in Groups 3, 5, and 7 in which a DUX4 RNAi agent was administered remained well below the tamoxifen group and at or below the baseline group in all muscles indicating a preventative effect. This effect was confirmed by the prevention of dramatic increase in Wfdc3 expression in Groups 5 and 7 as shown in Tables 9.1-9.9 and in the prevention of bodyweight loss in Groups 5 and 7 ( FIG. 1 ).
  • DUX4 RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • mice were injected between the skin and muscle (i.e. subcutaneous injections) into the loose skin region over the neck and shoulder area with either isotonic saline (vehicle control) or a DUX4 RNAi agent formulated in isotonic saline.
  • isotonic saline vehicle control
  • DUX4 RNAi agent formulated in isotonic saline.
  • an oral gavage of 100 ⁇ L/20 g mouse of either corn oil (negative control) or tamoxifen dissolved in corn oil (1 mg/mL) was administered three times per week (i.e., days 4, 6, 8, 10, 12, 15, 17, and 19) to induce increased expression of DUX4.
  • the dosing regimen and details are set forth in the following Table:
  • RNAi agent Dosing Induction Agent Dosing Group RNAi agent and Dose Regimen Administration Regimen 1 Baseline (no RNAi agent, saline injection) N/A Corn oil 3 times per week (negative control) starting on day 4 2 Positive Control (no RNAi agent, saline injection) N/A Tamoxifen 3 times per week starting on day 4 3 SM45b-L4-AD07276-Bis(PEG47 + C22) 5 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 3 SM45b-L4-AD07510-Bis(PEG47 + C22) 5 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 5 SM45b-L4-AD07511-Bis(PEG47 + C22) 5 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 6 SM45b-L4-AD07511-Bis(PEG47 + C22)
  • RNAi agents in Example 4 were synthesized having nucleotide sequences directed to target the DUX4 gene (i.e., DUX4 mRNA transcript), and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the skeletal muscle cell receptor small molecule targeting ligand SM45.
  • the targeting ligand SM45 was synthesized as an azide, which allowed for convenient coupling to Linker L4. (See, e.g., Example 3, above, for structural and related information for SM45 and L4).
  • Example 2 The DUX4 RNAi agents in Example 2 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to the PK/PD modulator Bis(PEG47+C22). (See, e.g., Example 3, above, for structural information and related information).
  • C6-SS—C6 disulfide functional group
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • Body weight measurements were taken on days 1, 4, 7, 8, 10, 12, 14, 18, 21, and 22, and as noted above preservation of body weight can be indicative of a preventative effect on muscle wasting.
  • the RNAi agents of Group 10 (AD07394), Group 11 (AD07395), Group 12 (AD07398), and Group 13 (AD07399) did not show an acceptable preservation of bodyweight compared to the positive control (tamoxifen administration only), and thus further assessments were not made for these Groups. Additionally, while Group 8 (AD07514) and Group 13 (AD07399) both showed some preventative effect of maintaining body weight, bodyweights declined more than several other RNAi agents that targeted the same position of the DUX4 gene, and thus further assessments were not made for these Groups either. Body weights as normalized to Day 4 (pre-tamoxifen administration) and baseline are shown in FIGS. 2 and 3 .
  • AD07276, AD07510, AD07511, AD07512, AD07513, AD07514, AD07515 included nucleotide sequences designed to inhibit a DUX4 gene (i.e., DUX4 mRNA transcript) at position 1437 of the gene;
  • AD07394 and AD07395 included nucleotide sequences designed to inhibit a DUX4 gene at position 1433 of the gene;
  • AD07398 and AD07399 Groups 12 and 13 included nucleotide sequences designed to inhibit a DUX4 gene at position 1522 of the gene.
  • the DUX4 RNAi agents targeting position 1437 of the gene provide for a reduction in DUX4 gene expression in the FSHD-like mouse model.
  • DUX4 gene expression levels were observed to be below baseline and Wfdc3 gene expression levels were observed to be far below baseline in 7 of 7 muscles assayed from mice administered AD07511 (see Tables 11.1-11.7 and 12.1-12.7).
  • DUX4 RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • mice were injected between the skin and muscle (i.e. subcutaneous injections) into the loose skin region over the neck and shoulder area with either isotonic saline (vehicle control) or a DUX4 RNAi agent formulated in isotonic saline.
  • isotonic saline vehicle control
  • DUX4 RNAi agent formulated in isotonic saline.
  • an oral gavage of 100 ⁇ L/20 g mouse of either corn oil (negative control) or tamoxifen dissolved in corn oil (1 mg/mL) was administered three times per week (days 4, 6, 8, 10, 12, 15, 17, and 19) to induce increased expression of DUX4.
  • the dosing regimen and details are set forth in the following Table:
  • RNAi Induction Induction agent Agent RNAi agent Dosing Admin- Dosing Group and Dose Regimen istration Regimen 1 Baseline N/A Corn oil 3 times per week (no RNAi agent, (negative starting on day 4 saline injection) control) 2 Positive Control N/A Tamoxifen 3 times per week (no RNAi agent, starting on day 4 saline injection) 3 SM45b-L4- 5 mg/kg Tamoxifen 3 times per week AD07218- administered on starting on day 4 Bis(PEG47 + C22) days 1 and 7 4 SM45b-L4- 5 mg/kg Tamoxifen 3 times per week AD07274- administered on starting on day 4 Bis(PEG47 + C22) days 1 and 7 5 SM45b-L4- 5 mg/kg Tamoxifen 3 times per week AD07775- administered on starting on day 4 Bis(PEG47 + C22) days 1 and 7 6 SM45b-L
  • RNAi agents in Example 5 were synthesized having nucleotide sequences directed to target the DUX4 gene, and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the skeletal muscle cell receptor small molecule targeting ligand SM45.
  • the targeting ligand SM45 was synthesized as an azide, which allowed for convenient coupling to Linker L4. (See, e.g., Example 3, above, for structural and related information for SM45 and L4).
  • Example 2 The DUX4 RNAi agents in Example 2 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to the PK/PD modulator Bis(PEG47+C22). (See, e.g., Example 3, above, for structural information and related information).
  • C6-SS—C6 disulfide functional group
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • Body weight measurements were taken on days 1, 4, 7, 9, 11, 14, 16, 18, and 21, and as noted above preservation of body weight can be indicative of preventative effect on muscle wasting.
  • the DUX4 RNAi agents of AD07274, AD07776, and AD07778 performed the best of the RNAi agents tested with respect to retaining animal body weight after administration of tamoxifen, and were subject to additional assessments. Body weights as normalized to day 4 (pre-tamoxifen administration) and baseline are shown in FIG. 4 .
  • the RNAi agents included nucleotide sequences designed to inhibit a DUX4 gene at position 408 of the gene. As shown herein, the DUX4 RNAi agents showed substantial reductions in relevant parameters, with AD07776 and AD07778 having particular potency in reducing DUX4 and Wfdc3 gene expression.
  • DUX4 RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • mice were injected between the skin and muscle (i.e. subcutaneous injections) into the loose skin region over the neck and shoulder area with either isotonic saline (vehicle control) or a DUX4 RNAi agent formulated in isotonic saline.
  • isotonic saline vehicle control
  • DUX4 RNAi agent formulated in isotonic saline.
  • an oral gavage of 100 ⁇ L/20 g mouse of either corn oil (negative control) or tamoxifen dissolved in corn oil (1 mg/mL) was administered three times per week (days 4, 6, 8, 10, 12, 15, 17, and 19) to induce increased expression of DUX4.
  • the dosing regimen and details are set forth in the following Table:
  • RNAi agent agent Dosing Admin- Dosing Group and Dose Regimen istration Regimen 1 Baseline (no N/A Corn oil 3 times per week RNAi agent, (negative starting on day 4 saline injection) control) 2 Positive Control N/A Tamoxifen 3 times per week (no RNAi agent, starting on day 4 saline injection) 3 SM45b-L4- 1 mg/kg a Tamoxifen 3 times per week AD07511- dministered on starting on day 4 Bis(PEG47 + C22) days 1 and 7 4 SM45b-L4- 5 mg/kg Tamoxifen 3 times per week AD07511- administered on starting on day 4 Bis(PEG47 + C22) days 1 and 7 5 SM45b-L4- 1 mg/kg Tamoxifen 3 times per week AD07843- administered on starting on day 4 Bis(PEG47 + C22) days 1 and 7 6
  • RNAi agents in Example 6 were synthesized having nucleotide sequences directed to target the DUX4 gene, and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the respective targeting ligand.
  • a functionalized amine reactive group NH 2 —C 6
  • the targeting ligand SM45 was synthesized as an azide, which allowed for convenient coupling to Linker L4. (See, e.g., Example 3, above, for structural and related information for SM45 and L4).
  • a peptide having affinity for a receptor present on skeletal muscle cells was conjugated to the sense strand of the DUX4 RNAi agent.
  • the skeletal muscle cell receptor peptide (Peptide 1) was linked to the RNAi agent via an amide coupling reaction as described in Example 1, above at the 5′ end of the sense strand.
  • ⁇ v ⁇ 6 Peptide 1 is represented by the following structure:
  • the DUX4 RNAi agents in Example 6 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to a PK/PD modulator.
  • C6-SS—C6 disulfide functional group
  • a Bis(PEG47+C22) moiety was attached to the 3′ terminal end of the sense strand to serve as a pharmacokinetic/pharmacodynamic (PK/PD) modulator (See, e.g., Example 3, above, for structural information and related information).
  • PK/PD pharmacokinetic/pharmacodynamic
  • PK/PD pharmacokinetic/pharmacodynamic
  • R is the remainder of the RNAi agent.
  • the maleimide was linked to the 3′ end of the sense strand by reducing the terminal 3′ disulfide bond and performing Michael addition to the terminal 3′ thiol.
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • Body weight measurements were taken on days 1, 4, 6, 7, 8, 10, 12, 15, 17, 19, and 22, and were normalized to Day 4 (pre-tamoxifen administration) and baseline, as shown in FIG. 5 (1 mg/kg) and FIG. 6 (5 mg/kg). Bodyweight was preserved above positive control levels in all groups treated with 1 or 5 mg/kg RNAi agent. Of particular note, animals treated with AD07511, AD07776, and AD07778 maintained bodyweight at levels equivalent to baseline. at both 1 and 5 mg/kg.
  • AD07511 included nucleotide sequences designed to inhibit a DUX4 gene at position 1437 of the gene; and AD077778 included nucleotide sequences designed to inhibit a DUX4 gene at position 408 of the gene.
  • DUX4 RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • mice were injected between the skin and muscle (i.e. subcutaneous injections) into the loose skin region over the neck and shoulder area with either isotonic saline (vehicle control) or a DUX4 RNAi agent formulated in isotonic saline.
  • isotonic saline vehicle control
  • DUX4 RNAi agent formulated in isotonic saline.
  • an oral gavage of 100 ⁇ L/20 g mouse of either corn oil (negative control) or tamoxifen dissolved in corn oil (1 mg/mL) was administered three times per week (days 4, 6, 8, 10, 12, 15, 17, and 19) to induce increased expression of DUX4.
  • the dosing regimen and details are set forth in the following Table:
  • RNAi agent Dosing Induction Agent Dosing Group RNAi agent and Dose Regimen Administration Regimen 1 Baseline (no RNAi agent, saline N/A Corn oil 3 times per week injection) (negative control) starting on day 4 2 Positive Control (no RNAi agent, N/A Tamoxifen 3 times per week saline injection) starting on day 4 3 ⁇ v ⁇ 6 Peptide 1-AD07511-LP38b 1 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 4 ⁇ v ⁇ 6Peptide 1-AD07511-LP38b 5 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 5 ⁇ v ⁇ 6Peptide 1-AD07776-LP38b 1 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 6 ⁇ v ⁇ 6 Peptide 1-AD07776-LP38b 5 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 6
  • RNAi agents in Example 7 were synthesized having nucleotide sequences directed to target the DUX4 gene, and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the skeletal muscle cell receptor peptide referred to as Peptide 1 (See, e.g., Example 6, above, for structural information and related information).
  • Example 7 The DUX4 RNAi agents in Example 7 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to the PK/PD modulator (LP38b).
  • C6-SS—C6 disulfide functional group
  • LP38b PK/PD modulator
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • Body weight measurements were taken on days 1, 3, 4, 5, 8, 10, 11, 12, 15, 17, 18, and 19, and were normalized to Day 1 (pre-tamoxifen administration) and baseline, as shown in FIG. 7 . Bodyweight was preserved above positive control levels in all groups treated with 1 or 5 mg/kg RNAi agent.
  • mice in Example 7 were further subjected to the Rotarod apparatus to conduct a gross motor coordination assessment, as describe in Example 2 above.
  • the animals dosed with the DUX4 RNAi agents (Groups 3-6) were able to maintain their balance and gross motor function on the Rotarod apparatus similar to the negative control saline group that was not administered tamoxifen.
  • the animals dosed with tamoxifen but no DUX4 RNAi agent began falling off the Rotarod apparatus much faster starting around day 11, indicating a loss of muscle function.
  • both of the DUX4 RNAi agents show substantial inhibition of DUX4 gene expression, and preservation of gross motor function and bodyweight in the model mice dosed with the DUX4 RNAi agents.
  • DUX4 RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • mice were injected between the skin and muscle (i.e. subcutaneous injections) into the loose skin region over the neck and shoulder area with either isotonic saline (vehicle control) or a DUX4 RNAi agent formulated in isotonic saline.
  • isotonic saline vehicle control
  • DUX4 RNAi agent formulated in isotonic saline.
  • an oral gavage of 100 ⁇ L/20 g mouse of either corn oil (negative control) or tamoxifen dissolved in corn oil (1 mg/mL) was administered three times per week (days 4, 6, 8, 10, 12, 15, 17, and 19) to induce increased expression of DUX4.
  • the dosing regimen and details are set forth in the following Table:
  • RNAi agent Dosing Induction Agent Dosing Group RNAi agent and Dose Regimen Administration Regimen 1 Baseline (no RNAi agent, saline injection) N/A Corn oil 3 times per week (negative control) starting on day 4 2 Positive Control (no RNAi agent, saline injection) N/A Tamoxifen 3 times per week starting on day 4 3 SM45b-L4-AD07511-Bis(PEG47 + C22) 1 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 4 SM45b-L4-AD07778-Bis(PEG47 + C22) 1 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 5 ⁇ v ⁇ 6 Peptide 1-AD07511-LP29b 1 mg/kg administered on Tamoxifen 3 times per week days 1 and 7 starting on day 4 6 ⁇ v ⁇ 6 Peptide 1-AD07778
  • RNAi agents in Example 8 were synthesized having nucleotide sequences directed to target the DUX4 gene, and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the respective targeting ligand or linker.
  • a functionalized amine reactive group NH 2 —C 6
  • the targeting ligand selected was the small molecule skeletal muscle receptor SM45b, which was synthesized as an azide, which allowed for convenient coupling to Linker L4. (See, e.g., Example 3, above, for structural and related information for SM45-p and L4).
  • Peptide 1 was conjugated to the sense strand of the DUX4 RNAi agent.
  • Peptide 1 was linked to the (NH2-C6) functionalized RNAi agent via an amide coupling reaction at the 5′ terminal end of the sense strand (See Example 6 for structural information.)
  • the DUX4 RNAi agents in Example 6 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to a PK/PD modulator.
  • C6-SS—C6 disulfide functional group
  • a Bis(PEG47+C22) moiety was attached to the 3′ terminal end of the sense strand to serve as a pharmacokinetic/pharmacodynamic (PK/PD) modulator (See, e.g., Example 3, above, for structural information and related information).
  • PK/PD pharmacokinetic/pharmacodynamic
  • PK/PD pharmacokinetic/pharmacodynamic
  • R comprises the DUX4 RNAi agent.
  • LP29-p was linked to the 3′ end of the sense strand by reducing the terminal 3′ disulfide bond of the (C6-SS—C6) functional group and coupling the maleimide of LP29-p to the terminal 3′ thiol via Michael addition.
  • an LP38b moiety was attached to the 3′ terminal end of the sense strand to serve as a pharmacokinetic/pharmacodynamic (PK/PD) modulator. (See, e.g., Example 6, above, for structural information and related information).
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • animals were sacrificed and muscles were harvested, processed, and analyzed in accordance with the procedures described in Example 2.
  • Body weight measurements were taken on days 1, 4, 6, 7, 8, 10, 12, 15, 17, 19 and 22, and were normalized to day 4 (pre-tamoxifen administration) and baseline, as shown in FIG. 9 .
  • the FSHD-like transgenic mouse model as described in Example 2 were used.
  • the DUX4 RNAi agent assessed was DUX4 RNAi agent AD07778 linked to the targeting ligand of peptide 1 and the PK/PD modulator LP29b (see AC000448 in Table 5.4 for fully modified and conjugated sense and antisense strand structure), which was synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • the objective of this study was to assess dose response and timing effect of this DUX4 RNAi agent on the knockdown of DUX4 mRNA expression, the reduction of biomarkers of DUX4 protein activity, and pharmacodynamic effect after a twice weekly subcutaneous dose followed by weekly subcutaneous doses in FLExDUX4/HSA-MCM mice.
  • the DUX4 RNAi agent was administered within 2 days of initiation of tamoxifen administration. In this way, the DUX4 RNAi agent was believed to be delivered to skeletal muscle cells (myofibers) as DUX4 expression was induced and increasing.
  • the DUX4 RNAi agent was administered after manifestation of the FSHD-like phenotype (by Day 10 after initiation of tamoxifen administration). In this way, the DUX4 RNAi agent was delivered to myofibers after DUX4 expression had already begun to take myotoxic effect.
  • RNAi agent Dosing Induction Agent Induction Agent Group
  • RNAi agent and Dose Regimen Administration Dosing Regimen A Baseline (no RNAi agent, N/A Corn oil Day 1, and then 2 saline injection) (negative control) times per week for the first week and 3 times per week beginning at week 2
  • Positive Control no RNAi N/A Tamoxifen Day 1, and then 2 agent, saline injection
  • C Prevention study
  • D Prevention study
  • Each mouse was administered corn oil control or 1 mg/mL tamoxifen solution via oral gavage at a dose volume of 100 ⁇ L per 20 g body weight (5 mg/kg) twice weekly during Week 1 and three times weekly during Weeks 2 through 4.
  • RNAi agent in Example 9 (Groups C, D, and E) were synthesized having nucleotide sequences directed to target the DUX4 gene, and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the respective targeting ligand or linker.
  • Peptide 1 was conjugated to the sense strand of the DUX4 RNAi agent.
  • Peptide 1 was linked to the (NH2-C6) functionalized RNAi agent via an amide coupling reaction at the 5′ terminal end of the sense strand (See Example 6 for structural information.)
  • DUX4 RNAi agents in Example 6 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to a PK/PD modulator.
  • C6-SS—C6 disulfide functional group
  • An LP29b moiety was attached to the 3′ terminal end of the sense strand to serve as a pharmacokinetic/pharmacodynamic (PK/PD) modulator, having the following structure:
  • R comprises the DUX4 RNAi agent.
  • the maleimide LP29-p was linked to the 3′ end of the sense strand by reducing the terminal 3′ disulfide bond and performing Michael addition to the terminal 3′ thiol to synthesize the RNAi agent.
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • Body weight measurements were taken on days of tamoxifen and RNAi agent administration (Days 1, 3, 4, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, and 31). To control for individual variance, each individual animal's bodyweight was normalized to Day 1 and then to the mean of the baseline group's bodyweight at each time point. A two-way ANOVA followed by a Dunnett's multiple comparison test was used to determine significant differences between group body weights at each time point.
  • animals administered tamoxifen only (Group B) and the group with tamoxifen and 5 mg/kg RNAi agent administered for the first time on Day 10 (Group E) had significantly lower bodyweight compared to baseline animals (Group A) and those administered 1 or 5 mg/kg on Day 3 (Groups C and D).
  • the DUX4 RNAi agent Peptide 1-AD07778-LP29 administered at 1 or 5 mg/kg beginning on Day 3 (Groups C and D) and beginning on Day 10 (Group E) prevented Wfdc3 and Myo1 g expression increase or reduced relative Wfdc3 and Myo1 g expression to or below baseline.
  • Administration of the DUX4 RNAi agent resulted in Wfdc3 and Myo1 g mean relative expression levels that were significantly lower than tamoxifen only (Group B) regardless of dose level or administration timing (Groups C, D and E; p ⁇ 0.0001 for all respective comparisons).
  • Muscle sections were cut from formalin-fixed gastrocnemius, TA, and triceps collected on days of harvest and stained using H&E and PSR.
  • gastrocnemius, TA, and triceps administration of tamoxifen induced a dramatic increase in centrally located nuclei, indicating active muscle repair, and fibrosis in FLExDUX4/HSA-MCM animals (Group B).
  • TA TA
  • triceps administration of tamoxifen induced a dramatic increase in centrally located nuclei, indicating active muscle repair, and fibrosis in FLExDUX4/HSA-MCM animals (Group B).
  • fewer centralized nuclei and less fibrosis was observed in muscle sections from animals administered tamoxifen and DUX4 RNAi agent-treated animals (Groups C, D, and E) when compared to those administered tamoxifen only (Group B).
  • the DUX4 RNAi agent administered was sufficient to return DUX4 expression levels to baseline, prevent or reduce increased expression of DUX4 target genes and markers of DUX4 activity (Wfdc3 and Myo1 g), prevent bodyweight loss and return bodyweight to baseline levels, and reduce signs of myotoxicity (fibrosis, increased central nuclei, elevated serum creatinine kinase, muscle weight loss—4 of 9 muscles) thereby alleviating the FSHD-like phenotype observed in the FLExDUX4/HSA-MCM transgenic mouse model when administered tamoxifen via oral gavage.
  • the FSHD-like transgenic mouse model as described in Example 2 were used.
  • the DUX4 RNAi agent assessed was DUX4 RNAi agent AD07778 linked to the targeting ligand of peptide 1 and the PK/PD modulator LP29b (see AC000448 in Table 5.4 for fully modified and conjugated sense and antisense strand structure), which was synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • RNAi agent Dosing Induction Agent Induction Agent Group
  • RNAi agent and Dose Regimen Administration Dosing Regimen 1 Baseline (no RNAi agent, N/A Corn oil Day 1, and then 2 saline injection) (negative control) times per week for the first week and 3 times per week beginning at week 2
  • Positive Control no RNAi N/A Tamoxifen Day 1, and then 2 agent, saline injection
  • Prevention study 5 mg/kg administered on Tamoxifen Day 1, and then 2 ⁇ v ⁇ 6 Peptide 1-AD07778-LP29b days 1 and 4 times per week for the first week and 3 times per week beginning at week 2 5
  • Each mouse was administered corn oil control or 1 mg/mL tamoxifen solution via oral gavage at a dose volume of 100 ⁇ L per 20 g body weight (5 mg/kg) twice weekly during Week 1 and three times weekly during Weeks 2 through 4.
  • RNAi agent in Example 10 (Groups 2 and 4-6) was synthesized having nucleotide sequences directed to target the DUX4 gene, and included a functionalized amine reactive group (NH 2 —C 6 ) at the 5′ terminal end of the sense strand to facilitate conjugation to the respective targeting ligand or linker.
  • Peptide 1 was conjugated to the sense strand of the DUX4 RNAi agent.
  • Peptide 1 was linked to the (NH2-C6) functionalized RNAi agent via an amide coupling reaction at the 5′ terminal end of the sense strand (See Example 6 for structural information.)
  • DUX4 RNAi agents in Example 6 were further synthesized with a disulfide functional group (C6-SS—C6) at the 3′ terminal end of the sense strand to facilitate conjugation to a PK/PD modulator.
  • C6-SS—C6 disulfide functional group
  • An LP29b moiety was attached to the 3′ terminal end of the sense strand to serve as a pharmacokinetic/pharmacodynamic (PK/PD) modulator, having the following structure:
  • R comprises the DUX4 RNAi agent.
  • the maleimide LP29-p was linked to the 3′ end of the sense strand by reducing the terminal 3′ disulfide bond and performing Michael addition to the terminal 3′ thiol to synthesize the RNAi agent.
  • RNAi agent nucleotide sequences were synthetized as shown herein in Table 3, Table 4.1, Table 4.6, and Tables 5.1, Table 5.2, Table 5.3, and Table 5.4 (showing the fully modified conjugate).
  • mice in Example 10 were further subjected to the Rotarod apparatus to conduct a gross motor coordination assessment, as describe in Example 2 above.
  • the animals dosed with the DUX4 RNAi agents (Groups 3-5) were able to maintain their balance and gross motor function on the Rotarod apparatus more similar to the negative control saline group that was not administered tamoxifen (Group 1).
  • the animals dosed with tamoxifen but no DUX4 RNAi agent (Group 2) were unable to maintain balance and motor function for long and began falling off the Rotarod apparatus much sooner by day 11 (as compared to Groups 1, 3 and 4) indicating a loss of muscle function in the animals of Group 2.
  • the DUX4 RNAi agent showed substantial inhibition of DUX4 gene expression and preservation gross motor function or reversed gross motor function loss (as shown by Group 5 beginning around day 15) in the model mice.
  • Frozen untransformed FSHD patient-derived myoblasts were acquired from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, NJ). Upon differentiation into myotubes in vitro, these cells have been shown to express relatively high levels of DUX4 and target genes of DUX4 protein. The FSHD patient-derived myoblasts were then expanded and differentiated into myotubes in vitro.
  • the objective of this study was to assess dose response of this DUX4 RNAi agent on the knockdown of DUX4 mRNA expression and the reduction of biomarkers of DUX4 protein activity in FSHD patient-derived myotubes following transfection.
  • the FSHD patient-derived myoblasts were expanded and differentiated into myotubes in vitro.
  • the DUX4 RNAi agent was transfected into differentiating myotubes using a commercially available lipofectamine transfection reagent (RNAiMAX; Thermo).
  • RNAiMAX lipofectamine transfection reagent
  • Myotube cultures were harvested once mature myotube morphology was observed and DUX4 and DUX4 target gene relative expression examined.
  • DUX4 RNAi agent assessed in patient-derived myotubes was DUX4 RNAi agent AD07778 linked to the targeting ligand of peptide 1 and the PK/PD modulator LP29b (see AC000448 in Table 5.4 for fully modified and conjugated sense and antisense strand structure), which was synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis, as set forth in Example 1 herein.
  • the DUX4 RNAi agent was tested at 1.0, 10, and 100 nM concentrations.
  • a “scrambled control” was also evaluated, which included the same targeting ligands and PK/PD modifier as the DUX4 RNAi agent AD07778 linked to the targeting ligand of peptide 1 and the PK/PD modulator LP29b, but the scrambled control was modified in a manner such that it was expected to have no activity and would not inhibit DUX4 gene expression.
  • FIG. 11 shows a dose-dependent inhibition of the patient-derived myotubes with the DUX4 RNAi agent, suggesting that the DUX4 RNAi agent is effective to reduce DUX4 protein expression by elimination of DUX4 mRNA in human muscle cells.
  • the data were normalized against “scrambled control.”.
  • biomarkers of DUX4 expression were evaluated to determine how they were impacted by the DUX4 RNAi agent. These include CCNA1, KHDC1L, LEUTX MDB3L2, PRAMEF2, PRAMEF6, SLC2A3, SLC34A2, TRIM43, and ZSCAN4. These genes are known gene targets of the DUX4 transcription factor and whose increased expression has been characterized in FSHD patient muscle biopsies as markers of increased DUX4 expression. As shown in FIG. 12 , cells in which the DUX4 RNAi agent was administered also showed reductions in expression levels for these FSHD biomarker genes.

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US20230265430A1 (en) 2023-08-24
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WO2022056266A3 (fr) 2022-04-28
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