RNAi agents for inhibiting expression of DUX4, compositions thereof, and methods of use
Described are RNAi agents, compositions that include RNAi agents, and methods for inhibition of a double homeobox 4 (DUX4) gene. The DUX4 RNAi agents and RNAi agent conjugates disclosed herein inhibit the expression of a DUX4 gene. Pharmaceutical compositions that include one or more DUX4 RNAi agents, optionally with one or more additional therapeutics, are also described. Delivery of the described DUX4 RNAi agents to skeletal muscle cells in vivo, provides for inhibition of DUX4 gene expression and a reduction in DUX4 levels, which can provide a therapeutic benefit to subjects, including human subjects, suffering from certain skeletal muscle-related diseases or disorders including Facioscapulohumeral Muscular Dystrophy (FSHD).
1. An RNAi agent for inhibiting expression of a double homeobox 4 (DUX4) gene, comprising:
i. an antisense strand consisting of the nucleotide sequence cPrpusGfsasAfaccagauCfuGfaAfuccusg (SEQ ID NO: 99); and
ii. a sense strand comprising the nucleotide sequence caggauucAfGfAfucugguuuca (SEQ ID NO: 147);
wherein a represents 2′-O-methyl adenosine, c represents 2′-O-methyl cytidine, g represents 2′-O-methyl guanosine, and u represents 2′-O-methyl uridine; Af represents 2′-fluoro adenosine, Cf represents 2′-fluoro cytidine, Gf represents 2′-fluoro guanosine, and Uf represents 2′-fluoro uridine; cPrpu represents a 5′-cyclopropyl phosphonate-2′-O-methyl uridine; and s represents a phosphorothioate linkage.
2. The RNAi agent of claim 1 , wherein the sense strand comprises a targeting ligand having the structure:
or a pharmaceutically acceptable salt thereof, wherein indicates the point of connection to the RNAi agent.
3. The RNAi agent of claim 2 , wherein the targeting ligand is linked to the 5′ terminal end of the sense strand.
4. The RNAi agent of claim 1 , wherein the RNAi agent is further linked to a pharmacokinetic/pharmacodynamic (PK/PD) modulator.
5. The RNAi agent of claim 4 , wherein the PK/PD modulator is linked to the sense strand.
6. The RNAi agent of claim 5 , wherein the PK/PD modulator is linked to the 3′ terminal end of the sense strand.
7. The RNAi agent of claim 4 , wherein the PK/PD modulator is:
wherein R z comprises the RNAi agent.
8. An RNAi agent for inhibiting expression of a double homeobox 4 (DUX4) gene, comprising:
i. an antisense strand consisting of the nucleotide sequence cPrpusGfsasAfaccagauCfuGfaAfuccusg (SEQ ID NO: 99); and
ii. a sense strand comprising the nucleotide sequence αvβ6-peptide 1-(NH-C6)s(invAb)scaggauucAfGfAfucugguuucas(invAb)(C6-S)-LP29b (SEQ ID NO:236);
wherein a represents 2′-O-methyl adenosine, c represents 2′-O-methyl cytidine, g represents 2′-O-methyl guanosine, and u represents 2′-O-methyl uridine; Af represents 2′-fluoro adenosine, Cf represents 2′-fluoro cytidine, Gf represents 2′-fluoro guanosine, and Uf represents 2′-fluoro uridine; cPrpu represents a 5′-cyclopropyl phosphonate-2′-O-methyl uridine; s represents a phosphorothioate linkage, and (invAb)s represents
(C6-S) represents
LP29b represents
wherein R z is the remainder of the RNAi agent,
(NH-C6)s represents
αvβ6-peptide 1 represents
9. The RNAi agent of claim 8 , wherein the RNAi agent is a pharmaceutically acceptable salt.
10. The RNAi agent of claim 9 , wherein the RNAi agent is in the sodium salt form.
11. A pharmaceutical composition comprising the RNAi agent of claim 1 , wherein the composition further comprises a pharmaceutically acceptable excipient.
12. A method for inhibiting expression of a DUX4 gene in a cell, the method comprising introducing into a cell an effective amount of the RNAi agent of claim 1 .
13. The method of claim 12 , wherein the cell is within a subject.
14. The method of claim 13 , wherein the subject is a human subject.
15. The method of claim 14 , wherein the DUX4 gene expression is inhibited by at least about 40% in the skeletal muscle cells of the subject.
16. The method of claim 12 , wherein the subject's DUX4 protein levels are reduced by at least about 40%.
17. The method of claim 12 , wherein the DUX4 gene expression is reduced in one or more of paraspinal, facial, torso, abdominal, and limb muscle tissues of the subject.
18. The method of claim 12 , wherein the DUX4 gene expression is reduced in one or more of the triceps, biceps, quadriceps, gastrocnemius, soleus, EDL (extensor digitorum longus), TA (Tibialis anterior), or diaphragm of the subject.
19. The method of claim 12 , wherein the RNAi agent is administered at a dose of about 0.5 mg/kg to about 20.0 mg/kg of body weight.
20. A method of treating one or more symptoms or diseases that can be ameliorated at least in part by a reduction in DUX4 protein levels or a reduction in DUX4 mRNA levels, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 11 .
21. The method of claim 20 , wherein the disease is Facioscapulohumeral Muscular Dystrophy (FSHD).
22. The method of claim 21 , wherein the RNAi agent is administered by subcutaneous (SQ) injection.