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WO2025168022A1 - Compositions et procédés d'inhibition de l'expression de huntingtine (htt) - Google Patents

Compositions et procédés d'inhibition de l'expression de huntingtine (htt)

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Publication number
WO2025168022A1
WO2025168022A1 PCT/CN2025/076099 CN2025076099W WO2025168022A1 WO 2025168022 A1 WO2025168022 A1 WO 2025168022A1 CN 2025076099 W CN2025076099 W CN 2025076099W WO 2025168022 A1 WO2025168022 A1 WO 2025168022A1
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Prior art keywords
htt
dsrna
subject
nucleotide
dsrna agent
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Inventor
Dongxu Shu
Pengcheng Patrick Shao
Shiwei Xia
Yongquan Zhang
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Shanghai Argo Biopharmaceutical Co Ltd
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Shanghai Argo Biopharmaceutical Co Ltd
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Publication of WO2025168022A1 publication Critical patent/WO2025168022A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification

Definitions

  • the invention relates, in part, to compositions and methods that can be used to inhibit a huntingtin (HTT) gene expression.
  • HHT huntingtin
  • HTT Huntingtin
  • IT15 or HD Huntington’s disease
  • HTT gene encodes the huntingtin protein whose expansion of poly glutamine at its N terminus is causally linked to Huntington’s disease.
  • HTT gene spans 169 kb on chromosome 4 and gives rise to large transcripts around 14 kb and consequently HTT proteins about 350 kDa under physiological condition.
  • HTT is widely expressed in various tissues of the body with brain at the highest level. Within the brain HTT is expressed in all neurons as well as in glia cells. Although the exact functions of HTT is still elusive, previous studies have demonstrated its pivotal roles in vesicle traffic, autophagy, transcription and cell division. Consequently, HTT is essential for embryonic development, tissue maintenance and cell survival.
  • Huntington’s disease is a neurodegenerative disease caused by an expansion of the number of CAG repeats in exon 1 of HTT gene. Huntington's disease manifests with a variety of symptoms that include motor, cognitive and psychiatric impairment.
  • the genetic mutation involves a DNA segment of the huntingtin gene known as the CAG trinucleotide repeat. Expansion of the number of CAG repeats to above 40 copies causes adult onset of Huntington’s disease whereas CAG repeats of above 60-70 copies leads to a juvenile and a more progressive form of the disease.
  • polyQ poly glutamine translated from CAG repeats in protein’s N terminus underlies the pathogenesis of HD caused by familial inherited mutation of HTT.
  • Huntington's disease (an autosomal dominant disease) has been shown to be caused by the expansion of the polyglutamine repeat results in a full-length mutant transcript encoding an expanded polyglutamine repeat, as well as a truncated mutant transcript which retains intron 1 and encodes an expanded polyglutamine repeat (WO202376450) .
  • the expansion of the poyglutamine repeat results in a wild-type transcript, a full-length mutant transcript having the expanded polyglutamine repeat, as well as a truncated mutant transcript having the expanded polyglutamine repeat.
  • Huntingtin gene product is expressed at similar levels in patients and controls, it is the expansion of the polyglutamine repeat and the presence of the full-length mutant transcript and the truncated mutant transcript that induces toxicity. Furthermore, HTT-1A mRNA levels are higher in HD patient's motor cortex and hippocampus as compared to controls.
  • the present disclosure features novel HTT gene-specific RNAi agents, compositions that include HTT RNAi agents target full-length of mutant HTT gene, full -length of wild-type HTT gene or a particular portion of intron 1 retained in the truncated mutant HTT gene, thereby inhibiting expression of full-length of mutant HTT gene and/or the truncated mutant HTT transcript encoding an expanded polyglutamine repeat -and methods for inhibiting expression of a HTT gene in vitro and/or in vivo using the HTT RNAi agents and compositions that include HTT RNAi agents described herein.
  • the HTT RNAi agents described herein can selectively and efficiently decrease, inhibit, or silence expression of a HTT gene in a subject, e.g., a human or animal subject for the treatment of Huntington's disease (HD) .
  • HD Huntington's disease
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO: l, 3, 5, or 7 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2, 4, 6, or 8, wherein the sense strand and the antisense strand can be partially, substantially, or fully complementary to each other.
  • the sense strand and the antisense strand can be partially, substantially, or fully complementary to each other.
  • the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein said antisense strand comprises a region of complementarity to a HTT RNA transcript which comprises at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of the antisense sequences listed in any one of Tables 1-3, Tables 1A-3A.
  • the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein said antisense strand comprises a region of complementarity to a HTT RNA transcript which comprises at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides from any one of the antisense sequences listed in any one of Tables 1-3, Tables 1A-3A.
  • the dsRNA agent including a sense nucleotide and an antisense strand, nucleotide positions 2 to 18 in the antisense strand including a region of complementarity to a HTT RNA transcript at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, Tables 1A-3A, and optionally including a targeting ligand.
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from any one of the nucleotide sequences of nucleotides 4394-4524, 6503-6633, 2327-2457, 1892-2022, 3141-3271, 7517-7647, 1503-1633, 2062-2192, 6570-6700, 393-523, 394-524, 395-525, 396-526, 397-527, 398-528, 399-529, 400-530, 401-531, 403-533, 404-534, 410-540, 426-556, 428-558, 432-562, 435-565, 436-566, 438-568, 440-570, 446-576
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from any one of the nucleotide sequences of nucleotides 4399-4419, 6508-6528, 2332-2352, 1897-1917, 3146-3166, 7522-7542, 1508-1528, 2067-2087, 6575-6595, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 404-424, 405-425, 406-426, 408-428, 409-429, 402-429, 415-435, 431-451, 433-453, 437-457, 431-457, 440-460, 441-461, 443-463, 445-465, 451-471, 453-473, 440-473, 486-506,
  • the antisense strand comprises at least 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides which differ by 0, 1, 2, or 3 nucleotides from the complement of nucleotides 4399-4419, 6508-6528, 2332-2352, 1897-1917, 3146-3166, 7522-7542, 1508-1528, 2067-2087, 6575-6595, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 404-424, 405-425, 406-426, 408-428, 409-429, 402-429, 415-435, 431-451, 433-453, 437-457, 431-457, 440-460, 441-461, 443-463, 445-465, 451-471, 453-473, 440-473, 486-506, 495-515, 496-516, 527-547, 595-615, 605-625, 607-627, 610-630, 613-6
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from any one of the nucleotide sequences of nucleotides 28-48, 23-43, 36-56, 4-24, 11-31, 22-42, 28-48, 30-50, 31-51, 32-52, 33-53, 34-54, 35-55, 36-56, 40-60, 57-77, 61-81, 62-82, 73-93, 79-99, 89-109, 91-111, 101-121, 102-122, 106-126, 111-131, 113-133, 120-140, 131-151, 133-153, 136-156, 139-159, 145-165, 150-170, 151-171, 152-172, 153-173, 154-174
  • dsRNA double
  • the antisense strand comprises at least 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides which differ by 0, 1, 2, or 3 nucleotides from the complement of nucleotides 28-48, 23-43, 36-56, 4-24, 11-31, 22-42, 28-48, 30-50, 31-51, 32-52, 33-53, 34-54, 35-55, 36-56, 40-60, 57-77, 61-81, 62-82, 73-93, 79-99, 89-109, 91-111, 101-121, 102-122, 106-126, 111-131, 113-133, 120-140, 131-151, 133-153, 136-156, 139-159, 145-165, 150-170, 151-171, 152-172, 153-173, 154-174, 155-175, 157-177, 158-178, 160-180, 161-181, 163-183, 165-185, 169-189,
  • the HTT RNA transcript is SEQ ID NO: 1 and /or NO: 7.
  • the antisense strand of the dsRNA agent is at least substantially complementary to any one of a target region of SEQ ID NO: 1 and is provided in any one of Tables 1, 2, 3. In some embodiments, the antisense strand of the dsRNA agent is fully complementary to any one of a target region of SEQ ID NO: 1 and is provided in any one of Tables1, 2, 3. In some embodiments, the dsRNA agent includes a sense strand sequence set forth in any one of Tables 1, 2, 3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.
  • the dsRNA agent includes a sense strand sequence set forth in any one of Tables 1, 2, 3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.
  • the dsRNA agent includes an antisense strand sequence set forth in any one of Tables 1, 2, 3.
  • the dsRNA agent includes the sequences set forth as a duplex sequence in any of Tables 1, 2, 3.
  • the antisense strand of the dsRNA agent is at least substantially complementary to any one of a target region of SEQ ID NO: 7 and is provided in any one of Tables 1A, 2A, 3A. In some embodiments, the antisense strand of the dsRNA agent is fully complementary to any one of a target region of SEQ ID NO: 7 and is provided in any one of Tables1A, 2A, 3A. In some embodiments, the dsRNA agent includes a sense strand sequence set forth in any one of Tables 1A, 2A, 3A, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.
  • the dsRNA agent includes a sense strand sequence set forth in any one of Tables 1A, 2A, 3A, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.
  • the dsRNA agent includes an antisense strand sequence set forth in any one of Tables 1A, 2A, 3A.
  • the dsRNA agent includes the sequences set forth as a duplex sequence in any of Tables 1A, 2A, 3A.
  • the dsRNA agent includes at least one phosphorothioate internucleoside linkage.
  • the sense strand includes at least one phosphorothioate internucleoside linkage.
  • the antisense strand includes at least one phosphorothioate internucleoside linkage.
  • the sense strand includes 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.
  • the antisense strand includes 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.
  • At least the terminal two modified or unmodified nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate linkages. In some embodiments, the terminal three modified or unmodified nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate linkages. In some embodiments, at least the terminal two modified or unmodified nucleotides at one end or both ends of the sense strand are linked through phosphorothioate linkages. In some embodiments, the terminal three modified or unmodified nucleotides at one end or both ends of the sense strand are linked through phosphorothioate linkages.
  • the terminal three modified or unmodified nucleotides at 5’ end of the sense strand are linked through phosphorothioate linkages and the terminal two modified or unmodified nucleotides at 3’ end of the sense strand are linked through phosphorothioate linkages.
  • nucleotides of the sense strand and the antisense strand are modified nucleotides.
  • the antisense strand comprises 15 or more modified nucleotides independently selected from a 2’-O-methyl nucleotide, a 2’-fluoro nucleotide and an UNA modified nucleotide, wherein less than 6 modified nucleotides are 2’-fluoro nucleotides.
  • the antisense strand comprises 3 or 5 2’-fluoro nucleotides, preferably, the antisense strand comprises 5 2’-fluoro nucleotides.
  • the sense strand comprises 15 or more modified nucleotides independently selected from a 2’-O-methyl nucleotide and a 2’-fluoro nucleotide, wherein less than 4 modified nucleotides are 2’-fluoro nucleotides. In certain embodiments, the sense strand comprises 3 2’-fluoro nucleotides.
  • the antisense strand comprises 15 or more modified nucleotides independently selected from a 2’-O-methyl nucleotide and a 2’-fluoro nucleotide, wherein at least 14 modified nucleotides are 2’-O-methyl nucleotides and the nucleotides at positions 2, 5, 7, 11, 12, 14, 16 and/or 18 counting from the first matching position of the 5’ end of the antisense strand are independently a 2’-fluoro nucleotide.
  • the antisense strand comprises at least one UNA modified nucleotide and 5 2’-fluoro nucleotides.
  • the antisense strand comprises one UNA modified nucleotide at position 7 and 5 2’-fluoro nucleotides at positions 2, 5, 12, 14 and 16 counting from the first matching position of the 5’ end, and the rest 2’-O-methyl nucleotides. In some embodiments, the antisense strand comprises one UNA modified nucleotide at position 7 and 5 2’-fluoro nucleotides at positions 2, 5, 12, 14 and 18 counting from the first matching position of the 5’ end, and the rest 2’-O-methyl nucleotides.
  • the antisense strand comprises one UNA modified nucleotide at position 7 and 5 2’-fluoro nucleotides at positions 2, 5, 11, 14 and 16 counting from the first matching position of the 5’ end, and the rest 2’-O-methyl nucleotides. In some embodiments, the antisense strand comprises 5 2’-fluoro nucleotides at positions 2, 7, 12, 14 and 16 counting from the first matching position of the 5’ end, and the rest 2’-O-methyl nucleotides. In some embodiments, the antisense strand comprises 5 2’-fluoro nucleotides at positions 2, 7, 11, 14 and 16 counting from the first matching position of the 5’ end, and the rest 2’-O-methyl nucleotides.
  • the sense strand comprises 15 or more modified nucleotides independently selected from a 2’-O-methyl nucleotide and a 2’-fluoro nucleotide, preferably, wherein at least 18 modified nucleotides are 2’-O-methyl nucleotides and the nucleotides at positions 9, 11 and/or 13 counting from the first matching position of the 3’ end of the sense strand are 2’-fluoro nucleotides.
  • the sense strand comprises at least 18 modified nucleotides are 2’-O-methyl nucleotides and the nucleotides at positions 8, 11 and/or 13 counting from the first matching position of the 3’ end of the sense strand are 2’-fluoro nucleotides.
  • the modified sense strand is a modified sense strand sequence set forth in one of Tables 2-3, Tables 2A-3A.
  • the modified antisense strand is a modified antisense strand sequence set forth in one of Tables 2-3, Tables 2A-3A.
  • the dsRNA agent includes at least one modified nucleotide and further includes one or more targeting groups or linking groups.
  • the one or more targeting groups or linking groups are conjugated to the sense strand.
  • the targeting group or linking group includes N-acetyl-galactosamine (GalNAc) .
  • the targeting group has a structure:
  • n are independently selected from 1 or 2.
  • the targeting group has a structure:
  • the dsRNA agent includes a targeting group that is conjugated to the 5’-terminal end of the sense strand. In some embodiments, the dsRNA agent includes a targeting group that is conjugated to the 3'-terminal end of the sense strand.
  • the antisense strand includes one inverted abasic residue at 3’-terminal end.
  • the sense strand includes one or two inverted abasic residues and/or one or two imann residues at 3’ or/and 5’ terminal end. In certain embodiments, each end of the sense strand includes one inverted abasic residue. In certain embodiments, each end of the sense strand includes one imann residue. In certain embodiments, each end of the sense strand includes one inverted abasic residue or imann residues at the 5' terminal end of the sense strand, wherein inverted abasic residue or imann residues is linked to an adjacent nucleotide via a phosphorothioate linkage to the 5' terminal end of the nucleotide sequence of the sense strand.
  • the sense strand further includes targeting group linked to an inverted abasic residue or imann residues at the 5' terminal end of the sense strand, wherein targeting group is linked to an adjacent inverted abasic residue or imann via a phosphorothioate linkage, and optionally targeting group is N-acetyl-galactosamine (GalNAc) .
  • targeting group is linked to an adjacent inverted abasic residue or imann via a phosphorothioate linkage, and optionally targeting group is N-acetyl-galactosamine (GalNAc) .
  • each end of the sense strand includes one inverted abasic residue or imann residues at the 3' terminal end of the sense strand, wherein inverted abasic residue or imann residues is linked to an adjacent nucleotide via a phosphorothioate linkage to the 3' terminal end of the nucleotide sequence of the sense strand.
  • the dsRNA agent has two blunt ends.
  • at least one strand includes a 3’ overhang of at least 1 nucleotide. In some embodiments, at least one strand includes a 3’ overhang of at least 2 nucleotides.
  • the dsRNA comprises a duplex selected from AV04190, AV04183, AV04189, AV04198, AV04181, AV04179, AV04216, AV04187, AV04203, AV04237, AV04211, AV04191, and wherein duplex optionally including a targeting ligand.
  • the dsRNA comprises a duplex selected from AD01638, AD01245, AD01246, AD01247, AD01249, AD01926, AD01927, AD01928, AD01929, AD01930, AD01931, AD01932, AD01933.
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region complementary to part of a HTT RNA transcript, wherein each strand is about 15 to about 30 nucleotides in length, wherein the sense strand comprises sequence may be represented by formula (I) :
  • each N′ F represents a 2'-fluoro-modified nucleotide
  • each N′ N1 and N′ N2 independently represents a modified or unmodified nucleotide
  • each N′ L independently represents a modified or unmodified nucleotide but not a 2'-fluoro-modified nucleotide
  • m′and n′ are each independently an integer of 0 to 7.
  • N′ N1 and N′ N2 include only one 2'-Fluorine modified nucleotides.
  • N′ N1 independently represents a 2'-fluoro-modified nucleotide
  • N′ N2 independently represents a 2’-O-methyl -modified nucleotide
  • N′ N2 independently represents a 2'-fluoro-modified nucleotide
  • N′ N1 independently represents a 2’-O-methyl -modified nucleotide
  • each N′ L independently represents a 2’-O-methyl nucleotide.
  • the sense strand includes one or two inverted abasic residues and/or one or two imann residues at 3’ or/and 5’ terminal end. In certain embodiments, each 3’ and 5’ terminal end of the sense strand independently includes an inverted abasic residue. In certain embodiments, each 3’ and 5’ terminal end of the sense strand independently includes an imann residue. In certain embodiments, the sense strand includes two inverted abasic residues at 3’ and 5’ terminal end and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group, which preferably is aforesaid GLS-15*.
  • the sense strand includes two imann residues at 3’ and 5’ terminal end and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group, which preferably is aforesaid GLS-15*.
  • each end of the sense strand includes one inverted abasic residue or imann residues at the 5' terminal end of the sense strand, wherein inverted abasic residue or imann residues is linked to an adjacent nucleotide via a phosphorothioate linkage to the 5' terminal end of the nucleotide sequence of the sense strand.
  • the sense strand further includes targeting group linked to an inverted abasic residue or imann residues at the 5' terminal end of the sense strand, wherein targeting group is linked to an adjacent inverted abasic residue or imann via a phosphorothioate linkage , and optionally targeting group is N-acetyl-galactosamine (GalNAc) .
  • targeting group is linked to an adjacent inverted abasic residue or imann via a phosphorothioate linkage
  • optionally targeting group is N-acetyl-galactosamine (GalNAc) .
  • each end of the sense strand includes one inverted abasic residue or imann residues at the 3' terminal end of the sense strand, wherein inverted abasic residue or imann residues is linked to an adjacent nucleotide via a phosphorothioate linkage to the 3' terminal end of the nucleotide sequence of the sense strand.
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region complementary to a HTT RNA transcript, wherein each strand is about 18 to about 30 nucleotides in length, wherein the antisense strand comprises sequence may be represented by formula (II) :
  • each N F represents a 2'-fluoro-modified nucleotide
  • each N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , and N M7 independently represents a modified or unmodified nucleotide
  • each N L and /or Nz independently represents a modified or unmodified nucleotide but not a 2'-fluoro-modified nucleotide
  • n is an integer of 0 to 7.
  • the modified nucleotide is a modified nucleotide defined above.
  • N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , and N M7 have only three 2'-fluoro-modified nucleotides.
  • N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , and N M7 independently represents a 2'-fluoro-modified nucleotide, a 2’-O-methyl nucleotide, an UNA modified nucleotide or a nucleotide comprising phosphate mimic.
  • each N L and/or N z independently represents a 2’-O-methyl nucleotide.
  • Nz a nucleotide comprising phosphate mimic, preferably, wherein said nucleotide comprising vinyl phosphonate.
  • Nz is VPu*, which has the structure
  • N M2 , N M3 and N M6 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M4 , N M5 and N M7 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M4 and N M6 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M3 , N M4 and N M7 independently represents a 2’-O-methyl-modified nucleotide.
  • N M1 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M2 , N M4 , N M5 and N M6 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M4 , N M5 and N M6 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M5 and N M6 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M3 , N M4 and N M7 independently represents a 2’-O-methyl-modified nucleotide.
  • N M1 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide and N M6 represents an UNA modified nucleotide, optionally, N M2 , N M4 and N M5 independently represents a 2’-O-methyl -modified nucleotide.
  • N M2 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide and N M6 represents an UNA modified nucleotide, optionally, N M1 , N M4 and N M5 independently represents a 2’-O-methyl -modified nucleotide.
  • N M2 , N M4 and N M7 each independently represents a 2'-fluoro-modified nucleotide and N M6 represents an UNA modified nucleotide, optionally, N M1 , N M3 and N M5 independently represents a 2’-O-methyl -modified nucleotide.
  • n is 1, or n is 2, or n is 3, or n is 5.
  • the antisense strand of the dsRNA agent is at least substantially complementary to any one of a target region of SEQ ID NO: 1 and is provided in any one of Tables 1-3. In some embodiments, the antisense strand of the dsRNA agent is at least substantially complementary to any one of a target region of SEQ ID NO: 7 and is provided in any one of Tables 1A-3A.
  • a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of HTT wherein the dsRNA agent including a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a dsRNA duplex, wherein said sense strand is complementary to the antisense strand, wherein said antisense strand comprises a region of complementarity to a HTT RNA transcript, wherein the region of complementarity comprises at least 15 contiguous nucleotides, wherein the dsRNA duplex comprises represented by formula (III) :
  • each strand is about 18 to about 30 nucleotides in length
  • each N F and N′ F independently represents a 2'-fluoro-modified nucleotide
  • N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 , N′ N1 , and N′ N2 each independently represents a modified or unmodified nucleotide
  • each Nz, N L , and N′ L independently represents a modified or unmodified nucleotide but not a 2'-fluoro-modified nucleotide
  • m′, n′and n are each independently an integer of 0 to 7.
  • N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , and N M7 have only three 2'-fluoro-modified nucleotides
  • N′ N1 and N′ N2 include only one 2'-Fluorine modified nucleotides.
  • Nz, N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , and N M7 independently represents a 2'-fluoro-modified nucleotide, a 2’-O-methyl nucleotide, an UNA modified nucleotide or a nucleotide comprising phosphate mimic.
  • each N L , and/or N′ L independently represents a 2’-O-methyl nucleotide.
  • N′ N1 independently represents a 2'-fluoro-modified nucleotide.
  • N′ N2 independently represents a 2’-O-methyl -modified nucleotide
  • N′ N2 independently represents a 2'-fluoro-modified nucleotide.
  • N′ N1 independently represents a 2’-O-methyl -modified nucleotide
  • N M2 , N M3 and N M6 each independently represents a 2'-fluoro-modified nucleotide.
  • N M1 , N M4 , N M5 and N M7 independently represents a 2’-O-methyl-modified nucleotide
  • N M2 , N M4 and N M6 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M3 , N M5 and N M7 independently represents a 2’-O-methyl-modified nucleotide.
  • N M1 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M2 , N M4 , N M5 and N M6 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M4 , N M5 and N M6 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M4 and N M7 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M3 , N M5 and N M6 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M5 and N M6 each independently represents a 2'-fluoro-modified nucleotide, optionally, N M1 , N M3 , N M4 and N M7 independently represents a 2’-O-methyl-modified nucleotide.
  • N M2 , N M3 and N M7 each independently represents a 2'-fluoro-modified nucleotide and N M6 represents an UNA modified nucleotide, optionally, N M1 , N M4 , and N M5 independently represents a 2’-O-methyl -modified nucleotide.
  • N M2 , N M4 and N M7 each independently represents a 2'-fluoro-modified nucleotide and N M6 represents an UNA modified nucleotide, optionally, N M1 , N M3 , and N M5 independently represents a 2’-O-methyl -modified nucleotide.
  • Nz a nucleotide comprising phosphate mimic, preferably, wherein said nucleotide comprising vinyl phosphonate.
  • Nz is VPu*, which has the structure
  • the dsRNA agent includes a targeting group that is conjugated to the 5’-terminal end of the sense strand, preferably, the targeting group is any one selected from aforesaid GLO-1 through GLO-16 and GLS-1*through GLS-16*, more preferably, the targeting group is aforesaid GLS-15*.
  • the dsRNA agent includes a targeting group that is conjugated to the 5'-terminal end of the sense strand.
  • the antisense strand includes one inverted abasic residue at 3’-terminal end.
  • the sense strand includes one or two inverted abasic residues and/or one or two imann residues at 3’ or/and 5’ terminal end. In certain embodiments, each 3’ and 5’ terminal end of the sense strand independently includes an inverted abasic residue. In certain embodiments, each 3’ and 5’ terminal end of the sense strand independently includes an imann residue. In certain embodiments, the sense strand includes two inverted abasic residues at 3’ and 5’ terminal end and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group, which preferably is aforesaid GLS-15*.
  • the sense strand includes two imann residues at 3’ and 5’ terminal end and either residue at 3’ or 5’ terminal end is further conjugated to a targeting group, which preferably is aforesaid GLS-15*.
  • the dsRNA agent has two blunt ends.
  • at least one strand includes a 3’ overhang of at least 1 nucleotide.
  • at least one strand includes a 3’ overhang of at least 2 nucleotides.
  • the antisense strand of the dsRNA agent is at least substantially complementary to any one of a target region of SEQ ID NO: 1 and is provided in any one of Tables 1-3.
  • the antisense strand of the dsRNA agent is at least substantially complementary to any one of a target region of SEQ ID NO: 7 and is provided in any one of Tables 1A-3A.
  • any one of the sense strands in Table 1 may further be modified in a pattern shown in aforesaid Formula (I) or (III) .
  • any one of the antisense strands in Table 1 may further be modified in a pattern shown in aforesaid Formula (II) or (III) .
  • any one of the duplexes in Table 1 may further be modified in a pattern shown in aforesaid Formula (III) .
  • a composition that includes any embodiment of the aforementioned dsRNA agent aspect of the invention.
  • the composition also includes a pharmaceutically acceptable carrier.
  • the composition also includes one or more additional therapeutic agents.
  • the composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.
  • the composition is formulated for subcutaneous administration, formulated for intrathecally administration, formulated for intracerebroventricular administration, formulated for intrastriatal (IS) administration, or is formulated for intravenous (IV) administration.
  • a cell that includes any embodiment of an aforementioned dsRNA agent aspect of the invention.
  • the cell is a mammalian cell, optionally a human cell, preferentially in CNS (e.g., brain) cells.
  • a method of inhibiting the expression of a HTT gene in a cell including: (i) preparing a cell including an effective amount of any embodiment of the aforementioned dsRNA agent aspect of the invention or any embodiment of an aforementioned composition of the invention. In certain embodiments, the method also includes: (ii) maintaining the prepared cell for a time sufficient to obtain degradation of the mRNA transcript of a HTT gene, thereby inhibiting expression of the HTT gene in the cell.
  • the cell is in a subject and the dsRNA agent is administered to the subject by subcutaneously (SQ) .
  • the cell is in a subject and the dsRNA agent is administered to the subject by IV.
  • the cell is in a subject and the dsRNA agent is administered to the subject by intrathecally. In some embodiments, the cell is in a subject and the dsRNA agent is administered to the subject by intracerebroventricular (ICV) injection. In some embodiments, the cell is in a subject and the dsRNA agent is administered to the subject by intrastriatal (IS) injection.
  • ICV intracerebroventricular
  • IS intrastriatal
  • the method also includes assessing inhibition of the HTT gene, following the administration of the dsRNA agent to the subject, wherein a means for the assessing comprises: (i) determining one or more physiological characteristics of a HTT-associated disease or condition in the subject and (ii) comparing the determined physiological characteristic (s) to a baseline pre-treatment physiological characteristic of the HTT-associated disease or condition and/or to a control physiological characteristic of the HTT-associated disease or condition, wherein the comparison indicates one or more of a presence or absence of inhibition of expression of the HTT gene in the subject.
  • the physiological characteristic is one or more of: the HTT mRNA level and the HTT protein level , the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease. s.
  • a method of inhibiting expression of a HTT gene in a subject including administering to the subject an effective amount of an embodiment of the aforementioned dsRNA agent aspect of the invention or an embodiment of an aforementioned composition of the invention.
  • the dsRNA agent is administered to the subject by subcutaneously.
  • the dsRNA agent is administered to the subject by IV administration.
  • the dsRNA agent is administered to the subject by intrathecally administration.
  • the dsRNA agent is administered to the subject by intracerebroventricular (ICV) injection.
  • the dsRNA agent is administered to the subject by intrastriatal (IS) injection.
  • the method also includes: assessing inhibition of the HTT gene, following the administration of the dsRNA agent, wherein a means for the assessing comprises: (i) determining one or more physiological characteristics of a HTT-associated disease or condition in the subject and (ii) comparing the determined physiological characteristic (s) to a baseline pre-treatment physiological characteristic of the HTT-associated disease or condition and/or to a control physiological characteristic of the HTT-associated disease or condition, wherein the comparison indicates one or more of a presence or absence of inhibition of expression of the HTT gene in the subject.
  • expression of the HTT gene can be assessed based on the level or change in level of any variable associated with HTT gene expression, such as HTT mRNA level, HTT protein level.
  • a reduction in the expression of HTT may also be assessed indirectly by measuring a decrease in biological activity of HTT, the HTT mRNA level and the HTT protein level, e.g., the level of C9orf72 expanded, or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease.
  • HD's one or more of a physical symptom are abnormal body movements called chorea and lack of coordination.
  • a method of treating a disease or condition associated with the presence of HTT protein including: administering to a subject an effective amount of an embodiment of any aforementioned dsRNA agent aspect of the invention or an embodiment of any aforementioned composition of the invention, to inhibit HTT gene expression.
  • the disorder is a HTT associated disorder.
  • the disorder is associated with an abnormality of HTT gene encoded protein.
  • a “HTT-associated disease” is HD (Huntington's disease) .
  • the method also includes: administering an additional therapeutic regimen to the subject.
  • the additional therapeutic regimen includes a treatment for the HTT-associated disease or condition.
  • the additional therapeutic regimen comprises: administering to the subject one or more HTT antisense polynucleotides of the invention, administering to the subject a non-HTT dsRNA therapeutic agent, and a behavioral modification in the subject.
  • the additional therapeutic agent is selected from the group consisting of an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • HTT-associated disease may include a monoamine inhibitor, e.g., tetrabenazine (Xenazine) , deutetrabenazine (Austedo) , and reserpine, an anticonvulsant, e.g., valproic acid (Depakote, Depakene, Depacon) , and clonazepam (Klonopin) , an antipsychotic agent, e.g., risperidone (Risperdal) , and haloperidol (Haldol) , and an antidepressant, e.g., paroxetine (Paxil) .
  • a monoamine inhibitor e.g., tetrabenazine (Xenazine) , deutetrabenazine (Austedo) , and reserpine
  • an anticonvulsant e.g., valproic acid (De
  • the dsRNA agent is administered to the subject by subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject by IV administration. In some embodiments, the dsRNA agent is administered to the subject by intrathecally. In certain embodiments, the dsRNA agent is administered to the subject by intracerebroventricular (ICV) injection administration. In certain embodiments, the dsRNA agent is administered to the subject by intrastriatal (IS) injection administration. In some embodiments, the method also includes determining an efficacy of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject.
  • dsRNA double-stranded ribonucleic acid
  • a means of determining an efficacy of the treatment in the subject comprises: (i) determining one or more physiological characteristics of the HTT-associated disease or condition in the subject and (ii) comparing the determined physiological characteristic (s) to a baseline pre-treatment physiological characteristic of the HTT-associated disease or condition wherein the comparison indicates one or more of a presence, absence, and level of efficacy of the administration of the double-stranded ribonucleic acid (dsRNA) agent to the subject.
  • dsRNA double-stranded ribonucleic acid
  • expression of the HTT gene can be assessed based on the level or change in level of any variable associated with HTT gene expression, such as HTT mRNA level, HTT protein level in the subject, the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease in the subject.
  • any variable associated with HTT gene expression such as HTT mRNA level, HTT protein level in the subject, the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease in the subject.
  • GABA gamma-aminobutyric acid
  • a method of decreasing a level of HTT protein in a subject compared to a baseline pre-treatment level of HTT protein in the subject including administering to the subject an effective amount of an embodiment of any aforementioned dsRNA agent of the invention or an embodiment of any aforementioned composition of the invention, to decrease the level of HTT gene expression.
  • the dsRNA agent is administered to the subject subcutaneously or is administered to the subject by IV administration.
  • the dsRNA agent is administered to the subject by intrathecally.
  • the dsRNA agent is administered to the subject by intracerebroventricular (ICV) injection.
  • ICV intracerebroventricular
  • the dsRNA agent is administered to the subject by intrastriatal (IS) injection. In yet another embodiment, the dsRNA agent is administered to the subject intracisternally.
  • a non-limiting exemplary intracisternal administration comprises an injection into the cisterna magna (cerebellomedullary cistern) by suboccipital puncture.
  • a method of altering a physiological characteristic of a HTT-associated disease or condition in a subject compared to a baseline pre-treatment physiological characteristic of the HTT-associated disease or condition in the subject including administering to the subject an effective amount of an embodiment of any aforementioned dsRNA agent of the invention or an embodiment of any aforementioned composition of the invention, to alter the physiological characteristic of the HTT-associated disease or condition in the subject.
  • the dsRNA agent is administered to the subject subcutaneously or is administered to the subject by IV administration.
  • the dsRNA agent is administered to the subject by intrathecally.
  • the dsRNA agent is administered to the subject by intracerebroventricular (ICV) injection administration. In some embodiments, the dsRNA agent is administered to the subject by intrastriatal (IS) injection. In yet another embodiment, the dsRNA agent is administered to the subject intracisternally.
  • ICV intracerebroventricular
  • IS intrastriatal
  • a non-limiting exemplary intracisternal administration comprises an injection into the cisterna magna (cerebellomedullary cistern) by suboccipital puncture.
  • the method can reduce the expression of an HTT target gene in a brain (e.g., striatum) or spine tissue, for instance, cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine., is formulated for intrathecal administration, is formulated for intracranial administration, is formulated for intraventricular administration, is formulated for intracerebral administration.
  • the cell is a neuron (e.g., primary sensory neuron) .
  • the physiological characteristic and symptoms are one or more of: HTT mRNA level, HTT protein level in the subject, the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease.
  • HD's one or more of a physical symptom are abnormal body movements called chorea and lack of coordination.
  • a composition including an embodiment of any aforementioned antisense polynucleotide agents is provided.
  • the composition also includes a pharmaceutically acceptable carrier.
  • the composition also includes one or more additional therapeutic agents for treatment of a HTT-associated disease or condition.
  • the composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.
  • the composition is formulated for subcutaneous, intracerebroventricular, intrastriatal (IS) , intrathecally or IV administration.
  • a cell that includes an embodiment of any of the aforementioned antisense polynucleotide agents is provided.
  • the cell is a mammalian cell, optionally a human cell.
  • a method of inhibiting expression of a HTT gene in a subject including administering to the subject an effective amount of an embodiment of any of the aforementioned antisense polynucleotide agent.
  • a method of treating a disease or condition associated with the presence of HTT protein including administering to a subject an effective amount of an embodiment of any of the aforementioned antisense polynucleotide agents or an embodiment of any aforementioned composition of the invention, to inhibit HTT gene expression.
  • the disease or condition is HD (Huntington's disease) .
  • a method of decreasing a level of HTT protein in a subject compared to a baseline pre-treatment level of HTT protein in the subject including administering to the subject an effective amount of an embodiment of any of the aforementioned antisense polynucleotide agents or an embodiment of any aforementioned composition of the invention, to decrease the level of HTT gene expression.
  • the antisense polynucleotide agent is administered to the subject subcutaneously or by IV administration.
  • the dsRNA agent is administered to the subject by intracerebroventricular administration.
  • the dsRNA agent is administered to the subject by intrastriatal (IS) administration.
  • the antisense polynucleotide agent is administered to the subject by intrathecally. In yet another embodiment, the antisense polynucleotide agent is administered to the subject intracisternally.
  • a non-limiting exemplary intracisternal administration comprises an injection into the cisterna magna (cerebellomedullary cistern) by suboccipital puncture.
  • an antisense polynucleotide agent for inhibiting expression of HTT gene including from 10 to 30 contiguous nucleotides, wherein at least one of the contiguous nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is about 80%or about 85%complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 7 .
  • a method of altering a physiological characteristic of a HTT-associated disease or condition in a subject compared to a baseline pre-treatment physiological characteristic of the HTT-associated disease or condition in the subject including administering to the subject an effective amount of an embodiment of any of the aforementioned antisense polynucleotide agents or an embodiment of any aforementioned composition of the invention, to alter the physiological characteristic of the HTT disease or condition in the subject.
  • the antisense polynucleotide agent is administered to the subject, intracerebroventricular (ICV) , intrastriatal (IS) , intrathecally or IV administration.
  • the antisense polynucleotide agent is administered to the subject by intrathecally. In yet another embodiment, the antisense polynucleotide agent is administered to the subject intracisternally.
  • a non-limiting exemplary intracisternal administration comprises an injection into the cisterna magna (cerebellomedullary cistern) by suboccipital puncture.
  • the physiological characteristic and symptoms are one or more of: HTT mRNA level, HTT protein level in the subject, or variable progression of motor, cognitive, and behavioral impairment, or symptoms and hallmarks include abnormal body movements called chorea and lack of coordination.
  • SEQ ID NO: 1 and SEQ ID NO: 2 are full -length mutant Homo sapiens HTT mRNA [NCBI Reference Sequence: NM_001388492.1] .
  • SEQ ID NO: 3 and SEQ ID NO: 4 are exon 1 RNA sequence of the HTT gene (a particular portion from nts. 1-408 of full-length mutant HTT transcrip NM_001388492.1) .
  • SEQ ID NO: 5 and SEQ ID NO: 6 are transcript intron 1 (a particular portion from retained in the truncated 5415-12746 of NCBI Reference Sequence: NG_009378.1 (Homo sapiens huntingtin (HTT) , RefSeqGene (LRG_763) on chromosome 4) ) .
  • SEQ ID NO: 7 and SEQ ID NO: 8 are BWS-2a (HTT-1A) transcript comprise exon1 and a particular portion of intron 1 (combination of exon1 and retained in the truncated 1-2710 of SEQ ID NO: 5 (intron 1) ) .
  • SEQ ID Nos: 9-585, 3397-3448 are shown in Table 1 are sense strand sequences. and SEQ ID NOs: 586-1162, 3449-3500 are shown in Table 1 and are antisense strand sequences.
  • SEQ ID Nos: 1163-1625, 3391-3393, 3501-3541 are shown in Table 1A are sense strand, and sequences SEQ ID Nos: 1626-2088 , 3394-3396, 3542-3582 are shown in Table 1A are antisense strand sequences.
  • SEQ ID Nos: 2089-2674 are shown in Table 2 with chemical modifications.
  • SEQ ID NO: 3397 and SEQ ID NO: 3398 are Macaca mulatta (Rhesus monkey) HTT mRNA [NCBI Reference Sequence: XM_028848247.1] .
  • SEQ ID NO: 3399 and SEQ ID NO: 4000 are Mus musculus HTT mRNA [Chromosome 5, NC_000071.7 (NM_010414.3) ] .
  • the invention in part, includes RNAi agents, for example, though not limited to double stranded (ds) RNAi agents, which are capable of inhibiting HTT gene expression.
  • the invention in part also includes compositions comprising HTT RNAi agents and methods of use of the compositions.
  • HTT RNAi agents disclosed herein may be attached to delivery compounds for delivery to cells, including to hepatocytes/CNS (e.g., brain) .
  • Pharmaceutical compositions of the invention may include at least one dsRNA HTT agent and a delivery compound.
  • the delivery compound is a GalNAc-containing delivery, or compound the delivery compound is a phosphohate miminc-containing delivery compound.
  • HTT RNAi agents delivered to cells are capable of inhibiting HTT gene expression, thereby reducing activity in the cell of the HTT protein product of the gene.
  • dsRNAi agents of the invention can be used to treat HTT-associated diseases and conditions.
  • reducing HTT expression in a cell or subject treats a disease or condition associated with HTT expression in the cell or subject, respectively.
  • diseases and conditions that may be treated by reducing HTT activity are: alleviation or amelioration of one or more symptoms associated with unwanted or excessive HTT expression, or the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P.
  • GABA neurotransmitters gamma-aminobutyric acid
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • G, " C, “ “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
  • HAT gene also known as “huntingtin” ; “Huntington Disease Protein” , “IT15” , “HD” , “HD Protein” or “LOMARS” , refers to the gene encoding for a protein called huntingtin protein (HTT) from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise.
  • HTR huntingtin protein
  • wild-type transcript a full-length mutant transcrip, required for normal development and the disease gene linked to Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons caused by an expanded, unstable trinucleotide (CAG) repeat in the huntingtin gene, which translates as a polyglutamine repeat in the protein product.
  • CAG trinucleotide
  • the term also refers to fragments (for example, a particular portion of a HTT gene, exon 1 RNA sequence of the HTT gene is from nts.
  • 1-408 e.g., ) and variants of native HTT that maintain at least one in vivo or in vitro activity of a native HTT, Alternatively, fragments, mutant , non-mutant genes or a combination of native HTT linked to Huntington's disease (e.g. Rodriguez-Lebron et al., 2005, Mol Ther. Vol 12 No. 4: 618-633; Franich et al., 2008, Mol Ther, Vol. 16 No. 5; 947-956; Drouet et al., 2009, Annals of Neurology; Vol. 65 No. 3; 276-285 and McBride et al. Mol Ther.
  • Huntington's disease e.g. Rodriguez-Lebron et al., 2005, Mol Ther. Vol 12 No. 4: 618-633; Franich et al., 2008, Mol Ther, Vol. 16 No. 5; 947-956; Drouet et al., 2009, Annals of Neurology; Vol
  • NM_001388492.1 Homo sapiens huntingtin (HTT) , transcript variant , SEQ ID NO: 1, full-length mutant transcript include exon1, mRNA, reverse complement, SEQ ID NO: 2) ; exon 1 RNA sequence of the HTT gene (SEQ ID NO. 3, a particular portion from nits.
  • HTT Homo sapiens huntingtin
  • transcript intron 1 SEQ ID NO: 5, a particular portion from retained in the truncated 5415-12746 of NCBI Reference Sequence: NG_009378.1 (Homo sapiens huntingtin (HTT) , RefSeqGene (LRG_763) on chromosome 4, reverse complement, SEQ ID NO: 6 )
  • BWS-2a transcript comprise exon1 and a particular portion of intron 1 (SEQ ID NO: 7, combination of exon1 (SEQ ID NO.
  • HTT as used herein, also refers to variations of the HTT gene including variants provided are readily available using publicly available databases, e.g., GenBank, UniProt, Ensembl and OMIM.
  • HTT mRNA sequences U is optionally and independently replaced with T and vice versa
  • the double stranded RNA according to the invention is capable of inducing RNA interference to sequence specifically reduce expression of a RNA transcript comprising BWS-2a (SEQ ID NO: 7) .
  • said induction of RNA interference to reduce expression of an RNA transcript comprising SEQ ID NO. 7 means that it is to reduce human Huntingtin gene expression.
  • a luciferase reporter comprising SEQ ID No.
  • the double stranded RNA according to the invention is capable of sequence specific knock down. Furthermore, as shown in the example section, Huntingtin expression can be determined with specific antibodies to determine the amount of expression in a western blot analysis, as can northern blot analysis detecting the amount of RNA transcript. Hence, the double stranded RNA according to the invention is for use in inducing RNA interference.
  • the double stranded RNA according to the invention is for use in reducing expression of transcripts comprising SEQ ID NO.1166, such as for example SEQ ID NO. 7 or the like with more than 25 varying number of CAG repeats.
  • the iRNAs of the invention have been designed to target a HTT gene, including portions of the gene that are conserved in the HTT orthologs of other mammalian species.
  • the iRNAs of the invention have also been designed to target a particular portion of an HTT gene, for example , exon 1 and/or intron 1 e.g., thereby targeting the full-length wild-type transcript, the full-length mutant transcript, the fragments of transcript, as well as the truncated mutant transcript.
  • HD's most obvious symptoms are abnormal body movements called chorea and lack of coordination, but it also affects a number of mental abilities and some aspects of personality. These physical symptoms commonly become noticeable in a person's forties but can occur at any age. If the age of onset is below 20 years, then it is known as Juvenile HD.
  • Dementia or psychiatric disturbances may precede the movement disorder or develop during its course.
  • Anhedonia or asocial behavior may be the first behavioral manifestation.
  • Motor manifestations include flicking movements of the extremities, a lilting gait, motor impersistence (inability to sustain a motor act, such as tongue protrusion) , facial grimacing, ataxia, and dystonia.
  • HD is caused by a trinucleotide repeat expansion in the Huntingtin (HTT) gene and is one of several polyglutamine expansion (or PolyQ expansion) diseases. This produces an extended form of the mutant Huntingtin protein (mHtt) , which causes cell death in selective areas of the brain.
  • RNAi HTT single-stranded
  • siRNA dsRNA agents
  • RNAi refers to an agent that comprises RNA and mediates targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • an RNAi a target region refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, including messenger RNA (mRNA) that is a product of RNA processing of a primary transcription product.
  • mRNA messenger RNA
  • the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion.
  • a target sequence may be from 8-30 nucleotides long (inclusive) , from 10 -30 nucleotides long (inclusive) , from 12 -25 nucleotides long (inclusive) , from 15 -23 nucleotides long (inclusive) , from 16 -23 nucleotides long (inclusive) , or from 18 –23 nucleotides long (inclusive) , including all shorter lengths within each stated range.
  • a target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long.
  • a target sequence is between 9 and 26 nucleotides long (inclusive) , including all sub-ranges and integers there between.
  • a target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, with the sequence fully or at least substantially complementary to at least part of an RNA transcript of a HTT gene.
  • Some aspects of the invention include pharmaceutical compositions comprising one or more HTT dsRNA agents and a pharmaceutically acceptable carrier.
  • a HTT RNAi as described herein inhibits expression of HTT protein.
  • a “dsRNA agent” means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner.
  • dsRNA agents of the invention 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) .
  • DsRNA agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short interfering RNAs (siRNAs) , RNAi agents, micro RNAs (miRNAs) , short hairpin RNAs (shRNA) , and dicer substrates.
  • the antisense strand of the dsRNA agents described herein is at least partially complementary to the mRNA being targeted. It is understood in the art that different lengths of dsRNA duplex structure can be used to inhibit target gene expression.
  • dsRNAs having a duplex structure of 19, 20, 21, 22, and 23 base pairs are known to be effective to induce RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888) . It is also known in the art that shorter or longer RNA duplex structures are also effective to induce RNA interference.
  • the sense strand and the antisense strand may be the same length or different lengths.
  • each strand is no more than 40 nucleotides in length.
  • each strand is no more than 30 nucleotides in length.
  • each strand is no more than 25 nucleotides in length.
  • each strand is no more than 23 nucleotides in length.
  • each strand is no more than 21 nucleotides in length.
  • the sense and antisense strands of the RNAi agents can each be 15 to 49 nucleotides in length.
  • the antisense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the length of the sense strand is 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.
  • the sense strand and the antisense strand are both 21 nucleotides in length.
  • the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is between 15 and 23 nucleotides in length.
  • the region of complementarity is 19-21 nucleotides in length.
  • the region of complementarity is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • HTT dsRNAs in certain embodiments of the invention can include at least one strand of a length of minimally 21 nt or may have shorter duplexes based on one of the sequences set forth in any one of Tables 1-3, 1A-3A, but minus 1, 2, 3, or 4 nucleotides on one or both ends may also be effective as compared to the dsRNAs set forth in Tables 1-3, 1A-3A, respectively.
  • HTT dsRNA agents may have a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one or more sequences of Tables 1-3, 1A-3A, and differ in their ability to inhibit the expression of a HTT gene by not more than 5%, 10%, 15%, 20%, 25%, or 30%from the level of inhibition resulting from a dsRNA comprising the full sequence.
  • a sense sequence, an antisense sequence and a duplex disclosed in Tables 1-3 may be referred to herein as a “parent” sequence, meaning that the sequences disclosed in Tables 1-3 , 1A-3A, may be modified, shorten, lengthened, include substitutions, etc.
  • Sense and antisense strands included in a dsRNA of the invention are independently selected.
  • independently selected means each of two or more like elements can be selected independent of the selection of the other elements. For example, though not intended to be limiting, in preparing a dsRNA of the invention, one may select the “elements” of the two strands to include in the duplex.
  • the sense sequence may be SEQ ID NO: 2747 (shown in Table 2A) and the other selected element, the antisense sequence, may be SEQ ID NO: 2986, or may be SEQ ID NO: 2986 that is modified, shortened, lengthened, and/or includes 1, 2, or 3 substitutions as compared to its parent sequence SEQ ID NO: 2986.
  • a duplex of the invention need not include both sense and antisense sequences shown as paired in duplexes in Tables 1-3 , 1A-3A. Each sense and antisense strand sequence in the tables is immediately followed by its SEQ ID NO.
  • compositions and methods of the invention comprise a single-strand RNA in a composition and/or administered to a subject.
  • an antisense strand such as one listed in any one of Tables 1-3 , 1A-3A may be a composition or in a composition administered to a subject to reduce HTT polypeptide activity and/or expression of HTT gene in the subject.
  • Tables 1 and Tables 1A shows certain HTT dsRNA agent antisense strand and sense strand core stretch base sequences.
  • a single-strand antisense molecule that may be included in certain compositions and/or administered in certain methods of the invention are referred to herein as a “single-strand antisense agent” or an “antisense polynucleotide agent” .
  • a single-strand sense molecule that may be included in certain compositions and/or administered in certain methods of the invention are referred to herein as a “single-strand sense agent” or a “sense polynucleotide agent” .
  • the term “base sequence” is used herein in reference to a polynucleotide sequence without chemical modifications or delivery compounds.
  • the sense strand UUUUAACUGCGUUGUGAAGAA SEQ ID NO: 1233
  • Table 1A is the base sequence for SEQ ID NO: 2747 in Table 2A and for SEQ ID NO: 3291 in Table 3A, with SEQ ID NO: 2747 and SEQ ID NO: 3291 shown with their chemical modifications, mismacth and a delivery compound.
  • Table 1 and Table 1A includes sense and antisense strands and provides the identification number of duplexes formed from the sense and antisense strand on the same line in Table 1 and Table 1A.
  • an antisense sequence includes nucleobase u or nucleobase a in position 1 of the antisense sequence.
  • an antisense sequence includes nucleobase u in position 1 of the antisense sequence.
  • the term “matching position” in a sense and an antisense strand are the positions in each strand that “pair” when the two strands are duplexed strands.
  • nucleobase in position 1 of the sense strand and position 21 in the antisense strand are in “matching positions” .
  • nucleobase 2 of the sense strand and position 22 of the antisense strand are in matching positions.
  • the first column in Table 1 and Table 1A indicates a Duplex AV#for a duplex that includes the sense and antisense sequences in the same table row.
  • Table 1A discloses the duplex assigned Duplex AV03373. um, which includes sense strand SEQ ID NO: 1233 and antisense strand SEQ ID NO: 1296.
  • each row in Table 1A identifies a duplex of the invention, each comprising the sense and antisense sequences shown in the same row, with the assigned identifier for each duplex shown in the final column in the row.
  • an RNAi agent comprising a polynucleotide sequence shown in any one of Tables 1-3, 1A-3A is attached to a delivery molecule, a non-limiting example of which is a delivery compound comprising a GalNAc compound, or a GLS-15*compound.
  • a dsRNA (also referred to herein as a “duplex” ) is one disclosed in one of Tables 1-3, Tables 1A-3A.
  • Tables 1A-3A discloses a duplex comprising the sequence of the sense strand and the sequence of the antisense strand in that table row.
  • a duplex of the invention may include sense and antisense sequences shown in Tables 1-3, Tables 1A-3A, that differ by zero, one, two, or three nucleotides shown in a sequence shown in Tables 1-3, Tables 1A-3A.
  • an antisense strand in a duplex of the invention may be SEQ ID NO: 3326, 3327, 3350, 3355, or 3356, with zero, one, two, or three different nucleotides than those in SEQ ID: 3326, 3327, 3350, 3355, or 3356.
  • a dsRNA of the invention may comprise a sense strand and an antisense strand of a duplex disclosed in a row in Tables 1-3 and Tables 1A-3A.
  • one or both of the selected sense and antisense strand in the dsRNA may include sequences shown in Tables 1-3 and Tables 1A-3A, but with one or both of the sense and antisense sequences including 1, 2, 3, or more nucleobase substitutions from the parent sequence.
  • the selected sequences may in some embodiments be longer or shorter than their parent sequence.
  • dsRNA agents included in the invention can but need not include exact sequences of the sense and antisense pairs disclosed as duplexes in Tables 1-3 and Tables 1A-3A.
  • a dsRNA agent comprises a sense strand and an antisense strand, nucleotide positions 2 to 18 in the antisense strand comprising a region of complementarity to a HTT RNA transcript, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, Tables 1A-3A, and optionally comprising a targeting ligand.
  • the region of complementarity to the HTT RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, Tables 1A-3A.
  • the antisense strand of the dsRNA is at least substantially complementary to any one of a target region of SEQ ID NO: 1 and is provided in any one of Tables 1-3.
  • the antisense strand of the dsRNA is at least substantially complementary to any one of a target region of SEQ ID NO: 7 and is provided in any one of Tables 1A-3A.
  • an antisense strand of a dsRNA agent of the invention is fully complementary to any one of a target region of SEQ ID NO: 1 and is provided in any one of Tables 1-3.
  • an antisense strand of a dsRNA agent of the invention is fully complementary to any one of a target region of SEQ ID NO: 7 and is provided in any one of Tables 1A-3A.
  • a dsRNA agent includes a sense strand sequence set forth in any one of Tables 1-3, and the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.
  • a dsRNA agent of the invention comprises a sense strand sequence set forth in any one of Tables 1-3, and the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.
  • a dsRNA agent includes a sense strand sequence set forth in any one of Tables 1A-3A, and the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.
  • a dsRNA agent of the invention comprises a sense strand sequence set forth in any one of Tables 1A-3A, and the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.
  • a dsRNA agent of the invention comprises an antisense strand sequence set forth in any one of Tables 1-3, Tables 1A-3A.
  • Some embodiments of a dsRNA agent of the invention comprises the sense and antisense sequences disclosed as duplex in any of Tables 1-3, Tables 1A-3A. As described herein, it will be understood that the sense and antisense strands in a duplex of the invention may be independently selected.
  • mismatches are tolerated for efficacy in dsRNA, especially the mismatches are within terminal region of dsRNA.
  • Certain mismatches tolerate better, for example mismatches with wobble base pairs G: U and A: C are tolerated better for efficacy (Du et el., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.
  • HTT dsRNA agent may contain one or more mismatches to the HTT target sequence.
  • HTT dsRNA agent of the invention includes no mismatches.
  • HTT dsRNA agent of the invention includes no more than 1 mismatch.
  • HTT dsRNA agent of the invention includes no more than 2 mismatches.
  • HTT dsRNA agent of the invention includes no more than 3 mismatches.
  • an antisense strand of a HTT dsRNA agent contains mismatches to a HTT target sequence that are not located in the center of the region of complementarity.
  • the antisense strand of the HTT dsRNA agent includes 1, 2, 3, 4, or more mismatches that are within the last 5, 4, 3, 2, or 1 nucleotide from one or both of the 5' or 3' end of the region of complementarity.
  • the term “complementary, ” when used to describe a first nucleotide sequence (e.g., HTT dsRNA agent sense strand or targeted HTT mRNA) in relation to a second nucleotide sequence (e.g., HTT dsRNA agent antisense strand or a single-stranded antisense polynucleotide) means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize [form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro) ] and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence.
  • Complementary sequences for example, within a HTT dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences.
  • Such sequences can be referred to as “fully complementary” with respect to each other herein. It will be understood that in embodiments when two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs are not regarded herein as mismatches with regard to the determination of complementarity.
  • a HTT dsRNA agent comprising one oligonucleotide 19 nucleotides in length and another oligonucleotide 20 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.
  • “fully complementary” means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • 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 sequences, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not all, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • substantially complementary can be used in reference to a first sequence with respect to a second sequence if the two sequences include one or more, for example at least 1, 2, 3, 4, or 5 mismatched base pairs upon hybridization for a duplex up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs (bp) , while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of HTT gene expression via a RISC pathway.
  • partially complementary may be used herein in reference to a hybridized pair of nucleobase sequences, in which at least 75%, but not all, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • “partially complementary” means at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
  • nucleic acid sequence As used herein, the term “substantially identical” or “substantial identity” used in reference to a nucleic acid sequence means a nucleic acid sequence comprising a sequence with at least about 85%sequence identity or more, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, 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 identical 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 encompasses nucleotide sequences substantially identical to those disclosed herein. e.g., in Tables 1-3, Tables 1A-3A.
  • sequences disclosed herein are exactly identical, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%percent identical to those disclosed herein, e.g., in Tables 1-3, Tables 1A-3A.
  • strand comprising a sequence means an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • double-stranded RNA or “dsRNA, ” as used herein, refers to an RNAi that includes an RNA molecule or complex of molecules having a hybridized duplex region comprising two anti-parallel and substantially or fully complementary nucleic acid strands, which are referred to as having “sense” and “antisense” orientations with respect to a target HTT RNA.
  • the duplex region can be of any length that permits specific degradation of a desired target HTT RNA through a RISC pathway but will typically range from 9 to 30 base pairs in length, e.g., 15-30 base pairs in length.
  • the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base
  • HTT dsRNA agents generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length.
  • One strand of the duplex region of a HTT dsDNA agent comprises a sequence that is substantially complementary to a region of a target HTT RNA.
  • the two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules.
  • the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop” ) between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure.
  • a hairpin look comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more unpaired nucleotides.
  • a HTT dsRNA agent may include a sense and antisense sequence that have no-unpaired nucleotides or nucleotide analogs at one or both terminal ends of the dsRNA agent.
  • An end with no unpaired nucleotides is referred to as a “blunt end” and as having no nucleotide overhang. If both ends of a dsRNA agent are blunt, the dsRNA is referred to as “blunt ended.
  • a first end of a dsRNA agent is blunt, in some embodiments a second end of a dsRNA agent is blunt, and in certain embodiments of the invention, both ends of a HTT dsRNA agent are blunt.
  • the dsRNA does not have one or two blunt ends.
  • a dsRNA can comprise an overhang of at least 1, 2, 3, 4, 5, 6, or more nucleotides.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • nucleotide overhang is on a sense strand of a dsRNA agent, on an antisense strand of a dsRNA agent, or on both ends of a dsRNA agent and nucleotide (s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA.
  • nucleotides in an overhang is replaced with a nucleoside thiophosphate.
  • antisense strand or “guide strand” refers to the strand of a HTT dsRNA agent that includes a region that is substantially complementary to a HTT target sequence.
  • sense strand, ” or “passenger strand” refers to the strand of a HTT dsRNA agent that includes a region that is substantially complementary to a region of the antisense strand of the HTT dsRNA agent.
  • RNA of a HTT RNAi agent is chemically modified to enhance stability and/or one or more other beneficial characteristics.
  • Nucleic acids in certain embodiments of the invention may be synthesized and/or modified by methods well established in the art, for example, those described in “Current protocols in Nucleic Acid Chemistry, "Beaucage, S.L. et al. (Eds. ) , John Wiley & Sons, Inc., New York, N.Y., USA, which is incorporated herein by reference.
  • HTT dsRNA agents of the invention include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, conjugation, inverted linkages, etc. ) 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.
  • RNA compounds useful in certain embodiments of HTT dsRNA agents, HTT antisense polynucleotides, and HTT sense polynucleotides of the invention include, but are not limited to RNAs comprising modified backbones or no natural internucleoside linkages.
  • an RNA having a modified backbone may not have a phosphorus atom in the backbone.
  • RNAs that do not have a phosphorus atom in their internucleoside backbone may be referred to as oligonucleosides.
  • a modified RNA has a phosphorus atom in its internucleoside backbone.
  • RNA molecule or “RNA” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art.
  • ribonucleoside and “ribonucleotide” may be used interchangeably herein.
  • An RNA molecule can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below, and molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex.
  • an RNA molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to the full length of the HTT dsRNA agent molecule’s ribonucleosides that are modified ribonucleosides.
  • the modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule.
  • DsRNA agents, HTT antisense polynucleotides, and/or HTT sense polynucleotides of the invention may, in some embodiments comprise one or more independently selected modified nucleotide and/or one or more independently selected non-phosphodiester linkage.
  • independently selected used in reference to a selected element, such as a modified nucleotide, non-phosphodiester linkage, etc., means that two or more selected elements can but need not be the same as each other.
  • nucleotide base As used herein, a “nucleotide base, ” “nucleotide, ” or “nucleobase” is a heterocyclic pyrimidine or purine compound, which is a standard constituent of all nucleic acids, and includes the bases that form the nucleotides adenine, guanine, cytosine, thymine, and uracil.
  • a nucleobase may further be modified to include, though not intended to be limiting: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases.
  • ribonucleotide or “nucleotide” may be used herein to refer to an unmodified nucleotide, a modified nucleotide, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.
  • PNAs peptide nucleic acids
  • a HTT RNA interference agent includes a single stranded RNA that interacts with a target HTT RNA sequence to direct the cleavage of the target HTT RNA.
  • Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and 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
  • others having mixed N, O, S and CH 2 component parts.
  • Means of preparing modified RNA backbones that do not include a phosphorus atom are routinely practiced in the art and such methods can be used to prepare certain modified HTT dsRNA agents, certain modified HTT antisense polynucleotides, and/or certain modified HTT sense polynucleotides of the invention.
  • RNA mimetics are included in HTT dsRNAs, HTT antisense polynucleotides, and/or HTT sense polynucleotides, such as, but not limited to: replacement of the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units with novel groups.
  • base units are maintained for hybridization with an appropriate HTT nucleic acid target compound.
  • a peptide nucleic acid (PNA) is referred to as a peptide nucleic acid (PNA) .
  • RNA In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Means of preparing RNA mimetics are routinely practiced in the art and such methods can be used to prepare certain modified HTT dsRNA agents of the invention.
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular -CH 2 -NH-CH 2 -, -CH 2 -N (CH 3 ) -O-CH 2 - [known as a methylene (methylimino) or MMI backbone] , -CH 2 -O-N (CH 3 ) -CH 2 -, -CH 2 -N (CH 3 ) -N (CH 3 ) -CH 2 -and -N (CH 3 ) -CH 2 - [wherein the native phosphodiester backbone is represented as -O-P-O-CH 2 -] .
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones are routinely practiced in the art and such methods can be used to prepare certain modified HTT dsRNA agents, certain HTT antisense polynucleotides, and/or certain HTT sense polynucleotides of the invention.
  • Exemplary suitable modifications include O [ (CH 2 ) n O] m CH 3 , O (CH 2 ) n OCH 3 , O (CH 2 ) n NH 2 , O (CH 2 ) n CH 3 , O (CH 2 ) n ONH 2 , and O (CH 2 ) n ON [ (CH 2 ) n CH 3 ) ] 2 , where n and m are from 1 to about 10.
  • the modification includes a 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504) i.e., an alkoxy-alkoxy group.
  • Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O (CH 2 ) 2 ON (CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE) , i.e., 2'-O-CH 2 -O-CH 2 -N (CH 2 ) 2 .
  • Means of preparing modified RNAs such as those described are routinely practiced in the art and such methods can be used to prepare certain modified HTT dsRNA agents of the invention.
  • modifications include 2'-methoxy (2'-OCH 3 ) , 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F) .
  • Similar modifications can also be made at other positions on the RNA of a HTT dsRNA agent, HTT antisense polynucleotide, and/or HTT sense polynucleotide of the invention, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked HTT dsRNAs, HTT antisense polynucleotides, or HTT sense polynucleotides, and the 5' position of 5' terminal nucleotide.
  • HTT dsRNA agent HTT antisense polynucleotide, and/or HTT sense polynucleotide may, in some embodiments, include nucleobase (often referred to in the art simply as "base” ) modifications or substitutions.
  • base as used herein, “unmodified” or “natural” nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil.
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-Me-C) , 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil) , 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl
  • nucleobases that may be included in certain embodiments of HTT dsRNA agents of the invention are known in the art, see for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L, Ed. John Wiley & Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993.
  • Means of preparing dsRNAs, HTT antisense strand polynucleotides and/or HTT sense strand polynucleotides that comprise nucleobase modifications and/or substitutions such as those described herein are routinely practiced in the art and such methods can be used to prepare certain modified HTT dsRNA agents, HTT sense polynucleotides, and/or HTT antisense polynucleotides of the invention.
  • HTT dsRNA agents, HTT antisense polynucleotides, and/or HTT sense polynucleotides of the invention include RNA modified to include one or more locked nucleic acids (LNA) .
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide with a modified ribose moiety comprising an extra bridge connecting the 2' and 4' carbons. This structure effectively “locks” the ribose in the 3'-endo structural conformation.
  • the addition of locked nucleic acids in a HTT dsRNA agent, HTT antisense polynucleotides, and/or HTT sense polynucleotides of the invention may increase stability in serum, and to reduce off-target effects (Elmen, J.
  • HTT dsRNA compounds, sense polynucleotides, and/or antisense polynucleotides of the invention include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: a 2’-O-methyl nucleotide, 2’-Fluoro nucleotide, 2’-deoxy nucleotide, 2’3’-seco nucleotide mimic, locked nucleotide, 2’-F-Arabino nucleotide, 2’-methoyxyethyl nucleotide, 2’-amino-modified nucleotide, 2’-alkyl-modified nucleotide, mopholino nucleotide, and 3’-OMe nucleotide, a nucleotide comprising a 5’-phosphorothioate group, a nucleotide comprising vinyl phosphate, a nucleotide comprising
  • HTT dsRNA compounds 3’ and 5’ end of sense polynucleotides, and/or 3’ end of antisense polynucleotides of the invention, include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2’-OMe nucleotide, inverted 2’-deoxy nucleotide. It is known to skilled in art, including an abasic or inverted abasic nucleotide at the end of oligonucleotide enhances stability (Czauderna et al.
  • a HTT dsRNA compound includes one or more inverted abasic residues (invab) at either 3’-end or 5’-end, or both 3’-end and 5’-end.
  • invab inverted abasic residues
  • Exemplified inverted abasic residues (invab) include, but are not limited to the following:
  • HTT dsRNA compounds include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: isomannide nucleotide or stereoisomer of said isomannide nucleotide.
  • isomannide nucleotides or stereoisomers of said isomannide nucleotides include, but are not limited to: wherein the phrase “Olig” each independently represents a polynucleotide moiety.
  • Exemplified isomannide residues (imann) include, but are not limited to, the following:
  • the isomannide nucleotides may further conjugate to one or more targeting groups or delivery molecules, such as GalNAc moieties.
  • HTT dsRNA compounds, antisense polynucleotides of the invention include at least one modified nucleotide, wherein the at least one modified nucleotide comprises unlocked nucleic acid nucleotide (UNA) or/and glycol nucleic acid nucleotide (GNA) .
  • UNA and GNA are thermally destabilizing chemical modifications, can significantly improves the off-target profile of a siRNA compound (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018; 9 (1) : 723.
  • HTT dsRNA compounds, antisense polynucleotides of the invention further comprise a 5’-phosphate mimic.
  • 5′-phosphate mimic on the antisense strand of a RNAi agent refers to a phosphate analogs corresponding to are bound to the 4'-carbon of the sugar moiety (e.g., a ribose or deoxyribose or analog thereof) of the a nucleotide.
  • the phosphate analog is a vinyl phosphonate, where the carbon atom of the vinyl phosphonate group is bound to the 4'-carbon of the sugar moiety or analog thereof.
  • the phosphorous mimic is resistant to removal in biological systems, relative to unmodified nucleosides and/or the 5′-nucleoside is resistant to cleavage by nucleases.
  • Suitable phosphate mimics are disclosed in, for example WO2011005860, WO2011/139702, WO2013/033230, WO2010/048585, WO2010/048549, WO2011/139699, WO2017214112, WO2018045317, the contents of which are incorporated herein by reference for the methods provided therein.
  • the phosphate mimic is a 5’-vinyl phosphonate (VP) .
  • VP 5’-vinyl phosphonate
  • a vinyl phosphonate of the disclosure has the following structure:
  • a vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure.
  • a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5’ end of the antisense strand of the dsRNA.
  • a vinyl phosphonate modified nucleotide of the disclosure has the structure of formula (IV) :
  • X is O or S
  • R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy) ;
  • B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil.
  • R 5 ' C (H) -P (O) (OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation.
  • Vinyl phosphate modifications are also contemplated for the dsRNAs, the compositions and methods of the instant disclosure.
  • An exemplary vinyl phosphate structure is:
  • a vinyl phosphonate modified nucleotide is VPu*which has the structure of as follows:
  • protecting groups are used during the preparation of the compounds of the invention.
  • the term "protected” means that the indicated moiety has a protecting group appended thereon.
  • compounds contain one or more protecting groups.
  • a wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
  • Protecting groups in general and hydroxyl protecting groups in particular are well known in the art (Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991) .
  • examples of protecting groups include, but are not limited to, methyl, ethyl, benzyl (Bn) , phenyl, isopropyl, tert-butyl, acetyl, chloroacetyl, trichloro acetyl, trifluoroacetyl, pivaloyl, tert-butoxymethyl, methoxymethyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, allyl, cyclohexyl, 9-fluorenylmethoxycarbonyl (Fmoc) , methanesulfonate, toluenesulfonate, triflate, benzoyl, benzoylformate , p-phenylbenzoyl, 4-methoxybenzyl, monomethoxytrityl, dimethoxytrityl, trimethoxy
  • RNA of certain embodiments of HTT dsRNA agents, HTT antisense polynucleotides, and/or HTT sense polynucleotides of the invention comprises chemically linking to the RNA one or more ligands, moieties or conjugates that enhance one or more characteristics of the HTT dsRNA agent, HTT antisense polynucleotide, and/or HTT sense polynucleotide, respectively.
  • a HTT dsRNA agent comprises a targeting compound that is conjugated to the 5'-terminal end of the sense strand.
  • a HTT dsRNA agent comprises a targeting compound that is conjugated to the 3'-terminal end of the sense strand.
  • a HTT dsRNA agent comprises a targeting group that comprises GalNAc.
  • a HTT dsRNA agent does not include a targeting compound conjugated to one or both of the 3'-terminal end and the 5'-terminal end of the sense strand. In certain embodiments of the invention a HTT dsRNA agent does not include a GalNAc containing targeting compound conjugated to one or both of the 5'-terminal end and the 3'-terminal end of the sense strand.
  • targeting and linking agents include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556) , cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060) , a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660: 306-309; Manoharan et al., Biorg.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556) , cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060)
  • Acids Res., 1990, 18: 3777-3783) a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14: 969-973) , or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654) , a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229-237) , or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923-937) .
  • compositions comprising a HTT dsRNA agent, HTT antisense polynucleotide, and/or HTT sense polynucleotide may comprise a ligand that alters distribution, targeting, or etc. of the HTT dsRNA agent.
  • the ligand increases affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • polyamines include: polyethylenimine, polylysine (PLL) , spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • EDTA lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1, 3-Bis-O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1, 3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide) , alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K) , MPEG, [MPEG] 2 , polyamin
  • a ligand included in a composition and/or method of the invention may be a protein, e.g., glycoprotein, or peptide, for example a molecule with a specific affinity for a co-ligand, or an antibody, for example an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, cardiac cell, or bone cell.
  • a ligand useful in an embodiment of a composition and/or method of the invention can be a hormone or hormone receptor.
  • a ligand attached to a HTT dsRNA agent of the invention functions as a pharmacokinetic (PK) modulator.
  • PK modulator that may be used in compositions and methods of the invention includes but is not limited to: a lipophiles, a bile acid, a steroid, a phospholipid analogue, a peptide, a protein binding agent, PEG, a vitamin, cholesterol, a fatty acid, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, a phospholipid, a sphingolipid, naproxen, ibuprofen, vitamin E, biotin, an aptamer that binds a serum protein, etc.
  • a HTT dsRNA agent is in a composition.
  • a composition of the invention may include one or more HTT dsRNA agent and optionally one or more of a pharmaceutically acceptable carrier, a delivery agent, a targeting agent, detectable label, etc.
  • a non-limiting example of a targeting agent that may be useful according to some embodiments of methods of the invention is an agent that directs a HTT dsRNA agent of the invention to and/or into a cell to be treated.
  • a targeting agent of choice will depend upon such elements as: the nature of the HTT-associated disease or condition, and on the cell type being targeted.
  • a therapeutic agent comprises a HTT dsRNA agent with only a delivery agent, such as a delivery agent comprising N-Acetylgalactosamine (GalNAc) , without any additional attached elements.
  • a HTT dsRNA agent may be attached to a delivery compound comprising GalNAc and included in a composition comprising a pharmaceutically acceptable carrier and administered to a cell or subject without any detectable labels, or targeting agents, etc. attached to the HTT dsRNA agent.
  • HTT dsRNA agent of the invention is administered with and/or attached to one or more delivery agents, targeting agents, labeling agents, etc.
  • Labeling agents may be used in certain methods of the invention to determine the location of a HTT dsRNA agent in cells and tissues and may be used to determine a cell, tissue, or organ location of a treatment composition comprising a HTT dsRNA agent that has been administered in methods of the invention.
  • Procedures for attaching and utilizing labeling agents such as enzymatic labels, dyes, radiolabels, etc. are well known in the art.
  • Certain embodiments of methods of the invention includes delivery of a HTT dsRNA agent into a cell.
  • delivery means facilitating or effecting uptake or absorption into the cell. Absorption or uptake of a HTT dsRNA agent can occur through unaided diffusive or active cellular processes, or by use of delivery agents, targeting agents, etc. that may be associated with a HTT dsRNA agent of the invention.
  • Delivery means that are suitable for use in methods of the invention include, but are not limited to: in vivo delivery, in which a HTT dsRNA agent is in injected into a tissue site or administered systemically. In some embodiments of the invention, a HTT dsRNA agent is attached to a delivery agent.
  • a delivery agent that may be used in embodiments of the invention to delivery a HTT dsRNA agent of the invention to a cell, tissue and/or subject is an agent comprising GalNAc that is attached to a HTT dsRNA agent of the invention and delivers the HTT dsRNA agent to a cell, tissue, and/or subject.
  • agents comprising GalNAc that can be used in certain embodiments of methods and composition of the invention are disclosed in PCT Application: WO2020191183A1 (incorporated herein in its entirety) .
  • a non-limiting example of a GalNAc targeting ligand that can be used in compositions and methods of the invention to deliver a HTT dsRNA agent to a cell is a targeting ligand cluster.
  • the aforesaid isomannide nucleotides may further conjugate to one or more GalNAc targeting ligands.
  • Specific examples of isomannide nucleotides conjugated to a GalNAc targeting ligand include, but are not limited to:
  • in vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety.
  • a HTT RNAi agent into a cell may also be done using art-known methods such as electroporation and lipofection.
  • a HTT dsRNA is delivered without a targeting agent. These RNAs may be delivered as “naked” RNA molecules.
  • a HTT dsRNA of the invention may be administered to a subject to treat a HTT-associated disease or condition in the subject, such as a cardiovascular disease, in a pharmaceutical composition comprising the RNAi agent, but not including a targeting agent such as a GalNAc targeting compound.
  • RNAi delivery means such as but not limited to those described herein and those used in the art, can be used in conjunction with embodiments of HTT RNAi agents and treatment methods described herein.
  • Non-limiting examples of non-HTT dsRNA therapeutic agents are a monoamine inhibitor. These and other therapeutic agents and behavior modifications are known in the art and used to treat a HTT-associated disease or condition in a subject and may be administered to a subject in combination with the administration of one or more HTT dsRNA agents of the invention to treat the HTT-associated disease or condition.
  • a HTT dsRNA agent of the invention administered to a cell or subject to treat a HTT-associated disease or condition may act in a synergistic manner with one or more other therapeutic agents or activities and increase the effectiveness of the one or more therapeutic agents or activities and/or to increase the effectiveness of the HTT dsRNA agent at treating the HTT-associated disease or condition.
  • Treatment methods of the invention that include administration of a HTT dsRNA agent can be used prior to the onset of an HTT-associated disease or condition and/or when a HTT-associated disease or condition is present, including at an early stage, mid-stage, and late stage of the disease or condition and all times before and after any of these stages.
  • Methods of the invention may also be to treat subjects who have previously been treated for a HTT-associated disease or condition with one or more other therapeutic agents and/or therapeutic activities that were not successful, were minimally successful, and/or are no longer successful at treating the HTT-associated disease or condition in the subject.
  • a HTT dsRNA agent can be delivered into a cell using a vector.
  • HTT dsRNA agent transcription units can be included in a DNA or RNA vector.
  • Vectors can be used in methods of the invention that result in transient expression of HTT dsRNA, for example for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks.
  • the length of the transient expression can be determined using routine methods based on elements such as, but not limited to the specific vector construct selected and the target cell and/or tissue.
  • transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector.
  • the transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292) .
  • HTT dsRNA agent An individual strand or strands of a HTT dsRNA agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced to a cell using means such as transfection or infection. In certain embodiments each individual strand of a HTT dsRNA agent of the invention can be transcribed by promoters that are both included on the same expression vector. In certain embodiments of the invention a HTT dsRNA agent is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the HTT dsRNA agent has a stem and loop structure.
  • RNA expression vectors are DNA plasmids or viral vectors.
  • Expression vectors useful in embodiments of the invention can be compatible with eukaryotic cells.
  • Eukaryotic cell expression vectors are routinely used in the art and are available from a number of commercial sources. Delivery of HTT dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that allows for introduction into a desired target cell.
  • Certain embodiments of the invention include use of viral vectors for delivery of HTT dsRNA agents into cells.
  • Numerous adenovirus-based delivery systems are routinely used in the art for deliver to, for example, lung, liver, the central nervous system, endothelial cells, and muscle.
  • Non-limiting examples of viral vectors that may be used in methods of the invention are: AAV vectors, a pox virus such as a vaccinia virus, a Modified Virus Ankara (MVA) , NYVAC, an avipox such as fowl pox or canary pox.
  • Certain embodiments of the invention include methods of delivering HTT dsRNA agents into cells using a vector and such vectors may be in a pharmaceutically acceptable carrier that may, but need not, include a slow-release matrix in which the gene delivery vehicle is imbedded.
  • a vector for delivering a HTT dsRNA can be produced from a recombinant cell, and a pharmaceutical composition of the invention may include one or more cells that produced the HTT dsRNA delivery system.
  • compositions Containing HTT dsRNA or ssRNA agents Containing HTT dsRNA or ssRNA agents
  • Certain embodiments of the invention include use of pharmaceutical compositions containing a HTT dsRNA agent or HTT antisense polynucleotide agent and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition containing the HTT dsRNA agent or HTT antisense polynucleotide agent can be used in methods of the invention to reduce HTT gene expression and HTT activity in a cell and is useful to treat a HTT-associated disease or condition.
  • Such pharmaceutical compositions can be formulated based on the mode of delivery.
  • Non-limiting examples of formulations for modes of delivery are: a composition formulated for subcutaneous delivery, a composition formulated for systemic administration via parenteral delivery, a composition formulated for intravenous (IV) delivery, a composition formulated for intrathecally delivery, a composition formulated for intracerebroventricular (ICV) delivery, composition formulated for intrastriatal (IS) injection delivery, a composition formulated for direct delivery into brain, etc, a composition formulated for direct delivery into CNS cell, etc.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
  • a HTT dsRNA agent or HTT antisense polynucleotide agent can also be delivered directly to a target tissue, for example directly into the liver, directly into a Central Nervous System, directly into brain, etc.
  • delivering a HTT dsRNA agent” or “delivering a HTT antisense polynucleotide agent” into a cell encompasses delivering a HTT dsRNA agent or HTT antisense polynucleotide agent, respectively, directly as well as expressing a HTT dsRNA agent in a cell from an encoding vector that is delivered into a cell, or by any suitable means with which the HTT dsRNA or HTT antisense polynucleotide agent becomes present in a cell.
  • Preparation and use of formulations and means for delivering inhibitory RNAs are well known and routinely used in the art.
  • pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc.
  • the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.
  • pharmacologically effective amount refers to that amount of a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention to produce the intended pharmacological, therapeutic or preventive result.
  • a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10%reduction in that parameter.
  • a therapeutically effective amount of a HTT dsRNA agent or HTT antisense polynucleotide agent can reduce HTT polypeptide levels by at least 10%.
  • Methods of the invention in some respects comprise contacting a cell with a HTT dsRNA agent or HTT antisense polynucleotide agent in an effective amount to reduce HTT gene expression in the contacted cell.
  • Certain embodiments of methods of the invention comprise administering a HTT dsRNA agent or a HTT antisense polynucleotide agent to a subject in an amount effective to reduce HTT gene expression and treat a HTT-associated disease or condition in the subject.
  • An “effective amount” used in terms of reducing expression of HTT and/or for treating a HTT-associated disease or condition is an amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount is that amount of a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention that when combined or co-administered with another therapeutic treatment for a HTT-associated disease or condition, results in a therapeutic response that prevents and/or treats the disease or condition.
  • a biologic effect of treating a subject with a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention may be the amelioration and or absolute elimination of symptoms resulting from the HTT-associated disease or condition.
  • a biologic effect is the complete abrogation of the HTT-associated disease or condition, as evidenced for example, by a diagnostic test that indicates the subject is free of the HTT-associated disease or condition.
  • a non-limiting example of a physiological symptom that may be detected includes a reduction in HTT level in liver of a subject following administration of an agent of the invention. Additional art-known means of assessing the status of a HTT-associated disease or condition can be used to determine an effect of an agent and/or methods of the invention on a HTT-associated disease or condition.
  • an effective amount of a HTT dsRNA agent or HTT antisense polynucleotide agent to decrease HTT polypeptide activity to a level to treat a HTT-associated disease or condition will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study. In some embodiments, an effective amount will be that results in a desired response, e.g., an amount that diminishes a HTT-associated disease or condition in cells, tissues, and/or subjects with the disease or condition.
  • an effective amount of a HTT dsRNA agent or HTT antisense polynucleotide agent to treat a HTT-associated disease or condition that can be treated by reducing HTT polypeptide activity may be the amount that when administered decreases the amount of HTT polypeptide activity in the subject to an amount that is less than the amount that would be present in the cell, tissue, and/or subject without the administration of the HTT dsRNA agent or HTT antisense polynucleotide agent.
  • control amount for a subject is a pre-treatment amount for the subject, in other words, a level in a subject before administration of a HTT agent can be a control level for that subject and compared to a level of HTT polypeptide activity and/or HTT gene expression in the subject following siRNA administered to the subject.
  • An effective amount of a compound that decreases HTT polypeptide activity may also be determined by assessing physiological effects of administration of a HTT dsRNA agent or HTT antisense polynucleotide agent on a cell or subject, such as a decrease of a HTT-associated disease or condition following administration.
  • Assays and/or symptomatic monitoring of a subject can be used to determine efficacy of a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention, which may be administered in a pharmaceutical compound of the invention, and to determine the presence or absence of a response to the treatment.
  • a non-limiting example is that one or more art-known tests of a decrease in biological activity of HTT, e.g., or additional pathologies associated with elevated levels of HTT, preferably or the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P.
  • GABA gamma-aminobutyric acid
  • Some embodiments of the invention include methods of determining an efficacy of an dsRNA agent or HTT antisense polynucleotide agent of the invention administered to a subject, to treat a HTT-associated disease or condition by assessing and/or monitoring one or more “physiological characteristics” of the HTT-associated disease or condition in the subject.
  • physiological characteristics of a HTT-associated disease or condition are the HTT mRNA level, the HTT protein level, or the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P.
  • the amount of a HTT dsRNA agent or HTT antisense polynucleotide agent administered to a subject can be modified based, at least in part, on such determinations of disease and/or condition status and/or physiological characteristics determined for a subject.
  • the amount of a treatment may be varied for example by increasing or decreasing the amount of a HTT-dsRNA agent or HTT antisense polynucleotide agent, by changing the composition in which the HTT dsRNA agent or HTT antisense polynucleotide agent, respectively, is administered, by changing the route of administration, by changing the dosage timing and so on.
  • the effective amount of a HTT dsRNA agent or HTT antisense polynucleotide agent will vary with the particular condition being treated, the age and physical condition of the subject being treated; the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any) , the specific route of administration, and additional factors within the knowledge and expertise of the health practitioner.
  • an effective amount may depend upon the desired level of HTT polypeptide activity and or HTT gene expression that is effective to treat the HTT-associated disease or condition.
  • a skilled artisan can empirically determine an effective amount of a particular HTT dsRNA agent or HTT antisense polynucleotide agent of the invention for use in methods of the invention without necessitating undue experimentation.
  • an effective prophylactic or therapeutic treatment regimen can be planned that is effective to treat the particular subject.
  • an effective amount of a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention can be that amount that when contacted with a cell results in a desired biological effect in the cell.
  • HTT gene silencing may be determined in any cell expressing HTT, either constitutively or by genomic engineering, and by any appropriate assay.
  • HTT gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%by administration of a HTT dsRNA agent of the invention.
  • HTT gene expression is reduced by at between 5%and 10%, 5%and 25%, 10%and 50%, 10%and 75%, 25%and 75%, 25%and 100%, or 50%and 100%by administration of a HTT dsRNA agent of the invention.
  • HTT dsRNA agents and HTT antisense polynucleotide agents are delivered in pharmaceutical compositions in dosages sufficient to inhibit expression of HTT genes.
  • a dose of HTT dsRNA agent or HTT antisense polynucleotide agent is in a range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight, 5 to 40 mg/kg body weight, 10 to 30 mg/kg body weight, 1 to 20 mg/kg body weight, 1 to 10 mg/kg body weight, 4 to 15 mg/kg body weight per day, inclusive.
  • HTT dsRNA agent of the invention Various factors may be considered in the determination of dosage and timing of delivery of a HTT dsRNA agent of the invention.
  • the absolute amount of a HTT dsRNA agent or HTT antisense polynucleotide agent delivered will depend upon a variety of factors including a concurrent treatment, the number of doses and the individual subject parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • a maximum dose can be used, that is, the highest safe dose according to sound medical judgment.
  • a pharmaceutical composition of the invention may be administered once daily, or the HTT dsRNA agent or HTT antisense polynucleotide agent may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation.
  • a pharmaceutical composition of the invention is administered to a subject one or more times per day, one or more times per week, one or more times per month, or one or more times per year.
  • Methods of the invention include administration of a pharmaceutical compound alone, in combination with one or more other HTT dsRNA agents or HTT antisense polynucleotide agents, and/or in combination with other drug therapies or treatment activities or regimens that are administered to subjects with a HTT-associated disease or condition.
  • Pharmaceutical compounds may be administered in pharmaceutical compositions.
  • Pharmaceutical compositions used in methods of the invention may be sterile and contain an amount of a HTT dsRNA agent or HTT antisense polynucleotide agent that will reduce activity of a HTT polypeptide to a level sufficient to produce the desired response in a unit of weight or volume suitable for administration to a subject.
  • a dose administered to a subject of a pharmaceutical composition that includes a HTT dsRNA agent or HTT antisense polynucleotide agent to reduce HTT protein activity can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • HTT-associated disease As used herein, “HTT-associated disease” , “HTT-associated diseases and conditions” and “diseases and conditions caused and/or modulated by HTT” is intended to include any disease associated with the HTT gene or protein. Such diseases may be caused, for example, by overproduction of HTT protein, by mutation of the HTT gene, by abnormal cleavage of the HTT protein, by abnormal interaction between HTT and other proteins or other endogenous or exogenous substances. Exemplary HTT -associated diseases is HD (Huntington's disease) .
  • symptom or “hallmark” means any physical feature or test result that indicates the existence or extent of a disease or disorder.
  • a symptom is apparent to a subject or to a medical professional examining or testing the subject.
  • a hallmark is apparent upon invasive diagnostic testing, including, but not limited to, abnormal body movements called chorea and lack of coordination.
  • a subject may be administered a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention at a time that is one or more of before or after diagnosis of a HTT-associated disease or condition.
  • a subject is at risk of having or developing a HTT-associated disease or condition.
  • a subject at risk of developing a HTT-associated disease or condition is one who has an increased probability of developing the HTT-associated disease or condition, compared to a control risk of developing the HTT-associated disease or condition.
  • a level of risk may be statistically significant compared to a control level of risk.
  • a subject at risk may include, for instance, a subject who is, or will be, a subject who has a preexisting disease and/or a genetic abnormality that makes the subject more susceptible to a HTT-associated disease or condition than a control subject without the preexisting disease or genetic abnormality; a subject having a family and/or personal medical history of the HTT-associated disease or condition; and a subject who has previously been treated for a HTT-associated disease or condition.
  • a preexisting disease and/or a genetic abnormality that makes the subject more susceptible to a HTT-associated disease or condition may be a disease or genetic abnormality that when present has been previously identified as having a correlative relation to a higher likelihood of developing a HTT-associated disease or condition.
  • a HTT dsRNA agent or HTT antisense polynucleotide agent may be administered to a subject based on a medical status of the individual subject.
  • a health-care provided for a subject may assess a HTT level measured in a sample obtained from a subject and determine it is desirable to reduce the subject’s HTT level, by administration of a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention.
  • the HTT level may be considered to be a physiological characteristic of a HTT-associated condition, even if the subject is not diagnosed as having a HTT-assoicated disease such as one disclosed herein.
  • a healthcare provider may monitor changes in the subject’s HTT level, as a measure of efficacy of the administered HTT dsRNA agent or HTT antisense polynucleotide agent of the invention.
  • a biological sample such as a blood, serum sample may be obtained from a subject and a HTT level for the subject determined in the sample.
  • a HTT dsRNA agent or HTT antisense polynucleotide agent is administered to the subject and a blood sample is obtained from the subject following the administration and the HTT level determined using the sample and the results compared to the results determined in the subject’s pre-administration (prior) sample.
  • a reduction in the subject’s HTT level in the later sample compared to the pre-administration level indicates the administered HTT dsRNA agent or HTT antisense polynucleotide agent efficacy in reducing the lipid level in the subject.
  • Certain embodiments of methods of the invention include adjusting a treatment that includes administering a dsRNA agent or a HTT antisense polynucleotide agent of the invention to a subject based at least in part on assessment of a change in one or more of the subject’s physiological characteristics of a HTT-associated disease or condition resulting from the treatment.
  • an effect of an administered dsRNA agent or HTT antisense polynucleotide agent of the invention may be determined for a subject and used to assist in adjusting an amount of a dsRNA agent or HTT antisense polynucleotide agent of the invention subsequently administered to the subject.
  • a subject is administered a dsRNA agent or HTT antisense polynucleotide agent of the invention, the subject’s HTT level is determined after the administration, and based at least in part on the determined level, a greater amount of the dsRNA agent or HTT antisense polynucleotide agent is determined to be desirable in order to increase the physiological effect of the administered agent, for example to reduce or further reduce the subject’s HTT level.
  • a subject is administered a dsRNA agent or HTT antisense polynucleotide agent of the invention, the subject’s HTT level is determined after the administration and based at least in part on the determined level, a lower amount of the dsRNA agent or HTT antisense polynucleotide agent is desirable to administer to the subject.
  • some embodiments of the invention include assessing a change in one or more physiological characteristics of resulting from a subject’s previous treatment to adjust an amount of a dsRNA agent or HTT antisense polynucleotide agent of the invention subsequently administered to the subject.
  • Some embodiments of methods of the invention include 1, 2, 3, 4, 5, 6, or more determinations of a physiological characteristic of a HTT-associated disease or condition to assess and/or monitor the efficacy of an administered HTT dsRNA agent or HTT antisense polynucleotide agent of the invention, and optionally using the determinations to adjust one or more of: a dose, administration regimen, and or administration frequency of a dsRNA agent or HTT antisense polynucleotide agent of the invention to treat a HTT-associated disease or condition in a subject.
  • a desired result of administering an effective amount of a dsRNA agent or HTT antisense polynucleotide agent of the invention to a subject is a reduction of the subject’s the HTT mRNA level, the HTT protein level in the subject, or the level of C9orf72 expanded or levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P in the subject.
  • GABA neurotransmitters gamma-aminobutyric acid
  • the terms “treat” , “treated” , or “treating” when used with respect to a HTT-associated disease or condition may refer to a prophylactic treatment that decreases the likelihood of a subject developing the HTT-associated disease or condition, and also may refer to a treatment after the subject has developed a HTT-associated disease or condition in order to eliminate or reduce the level of the HTT-associated disease or condition, prevent the HTT-associated disease or condition from becoming more advanced (e.g., more severe) , and/or slow the progression of the HTT-associated disease or condition in a subject compared to the subject in the absence of the therapy to reduce activity in the subject of HTT polypeptide.
  • inventions can be used to inhibit HTT gene expression.
  • the terms “inhibit, ” “silence, ” “reduce, ” “down-regulate, ” and “knockdown” mean the expression of the HTT gene, as measured by one or more of: a level of RNA transcribed from the gene, a level of activity of HTT expressed, and a level of HTT polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the HTT gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is contacted with (e.g., treated with) a HTT dsRNA agent or HTT antisense polynucleotide agent of the invention, compared to a control level of RNA transcribed from the HTT gene, a level of activity of expressed HTT, or a level of HTT
  • HTT dsRNA agent or HTT antisense polynucleotide agent are available for use in methods of the invention.
  • the particular delivery mode selected will depend at least in part, upon the particular condition being treated and the dosage required for therapeutic efficacy.
  • Methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of treatment of a HTT-associated disease or condition without causing clinically unacceptable adverse effects.
  • a HTT dsRNA agent or HTT antisense polynucleotide agent may be administered via an oral, enteral, mucosal, subcutaneous, and/or parenteral route.
  • a HTT dsRNA agent or HTT antisense polynucleotide agent may be delivered to a subject cell using nanoparticles coated with a delivery agent that targets a specific cell or organelle.
  • a delivery agent that targets a specific cell or organelle.
  • Various delivery means, methods, agents are known in the art. Non-limiting examples of delivery methods and delivery agents are additionally provided elsewhere herein.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated by reference herein) may also be used to deliver HTT dsRNA agents or HTT antisense polynucleotide agents for treatment of a HTT-associated disease or condition.
  • Additional suitable delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of a HTT dsRNA agent or HTT antisense polynucleotide agent, increasing convenience to the subject and the medical care professional.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. (See for example: U.S. Pat. Nos.
  • kits that comprise one or more HTT dsRNA agents and/or HTT antisense polynucleotide agents and instructions for its use in methods of the invention.
  • Kits of the invention may include one or more of a HTT dsRNA agent, HTT sense polynucleotide, and HTT antisense polynucleotide agent that may be used to treat a HTT-associated disease or condition.
  • Kits containing one or more HTT dsRNA agents, HTT sense polynucleotides, and HTT antisense polynucleotide agents can be prepared for use in treatment methods of the invention.
  • Components of kits of the invention may be packaged either in aqueous medium or in lyophilized form.
  • a kit of the invention may also include instructions. Instructions typically will be in written form and will provide guidance for carrying-out a treatment embodied by the kit and for making a determination based upon that treatment.
  • phosphoramidites may be prepared according to procedures described herein and/or prior arts such as, but are not limited to, US426, 220 and WO02/36743.
  • Dichloromethane (19.50kg) was added to the 50 L glass kettle under the protection of nitrogen and started stirring.
  • the temperature was controlled at 20 ⁇ 30 °C, and DMTr imann (1.47 kg) , triethylamine (1.50 kg) , 4-dimethylaminopyridine (0.164 kg) and succinic anhydride (1.34 kg) was added to the glass kettle.
  • the system was kept at 20 ⁇ 30 °C for 18h, samples were taken and the reaction was ended.
  • Saturated sodium bicarbonate solution (22.50 kg) was added into the reaction system, stirred for 10-20 min, and allowed to separate into layers.
  • the organic phase was separated, and the aqueous phase was extracted twice with dichloromethane, and the organic phase was combined and dried over anhydrous sodium sulfate, filtered, and concentrated in vacuum to get the residue forming a gray to off-white solid of 1.83 kg.
  • N, N-dimethylformamide (23.50 kg) was added into a 100L glass kettle and stirred. The temperature was controlled at 20 ⁇ 30 °C. Under the protection of nitrogen, the products of the previous step, O-benzotriazole tetramethylurea hexafluorophosphate (0.33 kg) and N, N-diisopropylethylamine (0.13 kg) were added into the aforesaid 100L glass kettle through the solid feeding funnel and stirred for 10 ⁇ 30 minutes and were discharged into a 50 L zinc barrel for use.
  • Macroporous amine methyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Science and Technology Co., Ltd., batch number HA2X1209, load capacity 0.48 mmol/g) were added into the aforesaid 100 L solid phase synthesis reactor through the solid feeding funnel, the temperature was controlled at 20 ⁇ 30 °C, N, N-dimethylformamide (21.00 kg+21.00 kg) and the reaction solution in the zinc barrel of the previous step were add into the solid phase synthesis reactor. The system was subject to thermal insulation reaction, and the solid load was tracked to ⁇ 250umol/g, and the load detection method was UV.
  • the isomannide residue can be added to the 5'-end or 3'-end of the oligonucleotide chain by a method well known to those skilled in the art, such as the reverse abasic (invab) method, and further added to the target to the group.
  • a method well known to those skilled in the art such as the reverse abasic (invab) method
  • HTT RNAi agent duplexes shown in Table 2-3, Table 2A-3A, above, were synthesized in accordance with the following general procedures:
  • Sense and antisense strand sequences of siRNA were synthesized on oligonucleotide synthesizers using a well-established solid phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain propagation is achieved through 4-step cycles: a deprotection, a condensation, a capping and an oxidation or a sulfurization step for addition of each nucleotide. Syntheses were performed on a solid support made of controlled pore glass (CPG, ) . Monomer phosphoramidites may be purchased from commercial sources or may be the phosporamidite compounds in example 1-2 and/or in WO2023/045995 and/or in WO2016/028649.
  • the phosphoramidite compounds herein may be attached to the 3'-end as a monomeric phosphoramidite, and further be attached to the CPG solid support. In the case of attachment at the 5'-end, the phosphoramidite compounds may be used for the final coupling reaction and can be further conjugated to target ligands if necessary.
  • Phosphoramidites with GalNAc ligand cluster were public in WO2023/045995A1 (incorporated herein in its entirety) .
  • siRNAs used for in vitro screening (Table 2, Table 2A) , syntheses were carried out at 2 ⁇ mol scale, and for siRNAs used for in vivo testing (Table 3, Table 3A) , syntheses were carried out at scale of 5 ⁇ mol or larger.
  • GalNAc ligand GLO-n Phosphoramidites as non-limiting examples were public in WO2023/045995A1 (incorporated herein in its entirety) is attached at 3’-end of sense strand
  • GalNAc ligand attached CPG solid support was used.
  • the GalNAc ligand GLS-5*or GLS-15*as non-limiting example are attached at 5’-end of sense strand
  • GLS-5*or GLS-15*Phosphoramidites with GalNAc ligand cluster are attached at 5’-end of sense strand
  • a GalNAc phosphoramidite was used for the last coupling reaction.
  • the sense strands and the antisense strands were synthesis by solid phase synthesis with 4-step cycles, which detailed as below: Trichloroacetic acid (TCA) 3%in dichloromethane was used for deprotection of 4, 4′-dimethoxytrityl protecting group (DMT) . 5-Ethylthio-1H-tetrazole was used as an activator. I 2 in THF/Py/H 2 O and phenylacetyl disulfide (PADS) in pyridine/MeCN was used for oxidation and sulfurization reactions, respectively. After the final solid phase synthesis step, solid support bound oligomer was cleaved and protecting groups were removed by treating with a 1: 1 volume solution of 40 wt.
  • TCA trifluoride
  • %methylamine in water and 28%ammonium hydroxide solution For the synthesis of siRNAs used for in vitro screening, crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc, and ice cold EtOH was added to precipitate out the single strand product as the sodium salt, which was used for annealing without further purification.
  • crude single strand product was further purified by ion pairing reversed phase HPLC (IP-RP-HPLC) . Purified single strand oligonucleotide product from IP-RP-HPLC was converted to sodium salt by dissolving in 1.0 M NaOAc and precipitation by addition of ice cold EtOH. Annealing of equimolar complementary sense stand and antisense strand oligonucleotide in water was performed to form the double strand siRNA product, which was lyophilized to afford a fluffy white solid.
  • a method used to attach a targeting group comprising GalNAc (also referred to herein as a GalNAc delivery compound) to the 5’-end of a sense strand included use of a GalNAc phosphoramidite (GLS-5*or GLS-15*Phosphoramidites) in the last coupling step in the solid phase synthesis, using a synthetic process such as the process used if oligonucleotide chain propagation of adding a nucleotide to the 5’-end of the sense strand is performed.
  • GalNAc phosphoramidite GLS-5*or GLS-15*Phosphoramidites
  • a method of attaching a targeting group comprising GalNAc to the 3’-end of a sense strand comprised use of a solid support (CPG) that included a GLO-n.
  • a method of attaching a targeting group comprising GalNAc to the 3’-end of a sense strand comprises attaching a GalNAc targeting group to CPG solid support through an ester bond and using the resulting CPG with the attached GalNAc targeting group when synthesizing the sense strand, which results in the GalNAc targeting group attached at the 3’-end of the sense strand.
  • the imann residues can be added to the 5' end or 3' end of the oligonucleotide chain by a method well known to those skilled in the art, such as the inverted abasic residues (invab) method, and/or further added to the target to the GalNAc targeting group.
  • invab inverted abasic residues
  • BE2C cells were trypsinized and adjusted to appropriate density and seeded into 96-well plates.
  • Cells were transfected with test siRNAs or a control siRNA using Lipofectamine RNAiMax (Invitrogen) at the same time of seeding following the protocol according to manufacturer’s recommendation.
  • the siRNAs were tested at three concentrations (10 nM, 3.3nM and 1.1 nM) .
  • duplex sequences used correspond to those shown in Table 2 and Table 2A. Results from these experiments were summarized in Table 5-8.
  • Table 5 provides experimental results of in vitro studies using various HTT RNAi agents to inhibit HTT expression.
  • the duplex sequences used correspond to those shown in Table 2 and Table 2A.
  • Table 8 provides experimental results of in vitro studies using various HTT RNAi agents to inhibit HTT expression.
  • the duplex sequences used correspond to those shown in Table 2.
  • Table 14 provides experimental results of in vivo studies using various HTT RNAi agents to inhibit HTT expression (fused to HTT target sequence comprising nucleotides 2842-5863 Of SEQ ID NO: 1) .
  • the duplex sequences used correspond to those shown in Table 3.

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Abstract

L'invention concerne des compositions et des procédés utiles pour réduire l'expression du gène HTT et pour le traitement de maladies et d'états associés à HTT. L'invention concerne des agents ARNdb HTT, des agents polynucléotidiques antisens HTT, des compositions comprenant des agents ARNdb HTT, et des compositions comprenant des agents polynucléotidiques antisens HTT qui peuvent être utilisés pour réduire l'expression de HTT chez des cellules et des sujets.
PCT/CN2025/076099 2024-02-07 2025-02-07 Compositions et procédés d'inhibition de l'expression de huntingtine (htt) Pending WO2025168022A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007051045A2 (fr) * 2005-10-28 2007-05-03 Alnylam Pharmaceuticals, Inc. Compositions et methodes destinees a inhiber l'expression du gene huntingtine
WO2011032045A1 (fr) * 2009-09-11 2011-03-17 Isis Pharmaceuticals, Inc. Modulation de l'expression de la huntingtine
WO2012109667A1 (fr) * 2011-02-12 2012-08-16 University Of Iowa Research Foundation Composés thérapeutiques
WO2021087036A1 (fr) * 2019-11-01 2021-05-06 Alnylam Pharmaceuticals, Inc. Compositions d'agents à base d'arni ciblant la huntingtine (htt) et leurs procédés d'utilisation
WO2022212231A2 (fr) * 2021-03-29 2022-10-06 Alnylam Pharmaceuticals, Inc. Compositions d'agents d'arni de la huntingtine (htt) et leurs procédés d'utilisation
WO2023045995A1 (fr) * 2021-09-23 2023-03-30 Shanghai Argo Biopharmaceutical Co., Ltd. Agrégats de ligands multivalents avec échafaudage de diamine pour l'administration ciblée d'agents thérapeutiques
WO2023076450A2 (fr) * 2021-10-29 2023-05-04 Alnylam Pharmaceuticals, Inc. Compositions d'agent d'arni de la huntingtine (htt) et leurs procédés d'utilisation

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007051045A2 (fr) * 2005-10-28 2007-05-03 Alnylam Pharmaceuticals, Inc. Compositions et methodes destinees a inhiber l'expression du gene huntingtine
WO2011032045A1 (fr) * 2009-09-11 2011-03-17 Isis Pharmaceuticals, Inc. Modulation de l'expression de la huntingtine
WO2012109667A1 (fr) * 2011-02-12 2012-08-16 University Of Iowa Research Foundation Composés thérapeutiques
WO2021087036A1 (fr) * 2019-11-01 2021-05-06 Alnylam Pharmaceuticals, Inc. Compositions d'agents à base d'arni ciblant la huntingtine (htt) et leurs procédés d'utilisation
WO2022212231A2 (fr) * 2021-03-29 2022-10-06 Alnylam Pharmaceuticals, Inc. Compositions d'agents d'arni de la huntingtine (htt) et leurs procédés d'utilisation
WO2023045995A1 (fr) * 2021-09-23 2023-03-30 Shanghai Argo Biopharmaceutical Co., Ltd. Agrégats de ligands multivalents avec échafaudage de diamine pour l'administration ciblée d'agents thérapeutiques
WO2023076450A2 (fr) * 2021-10-29 2023-05-04 Alnylam Pharmaceuticals, Inc. Compositions d'agent d'arni de la huntingtine (htt) et leurs procédés d'utilisation

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