WO2024073604A2 - Compositions et procédés pour le traitement de maladies neurodégénératives - Google Patents

Compositions et procédés pour le traitement de maladies neurodégénératives Download PDF

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WO2024073604A2
WO2024073604A2 PCT/US2023/075425 US2023075425W WO2024073604A2 WO 2024073604 A2 WO2024073604 A2 WO 2024073604A2 US 2023075425 W US2023075425 W US 2023075425W WO 2024073604 A2 WO2024073604 A2 WO 2024073604A2
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formula
nucleotides
sirna molecule
length
antisense strand
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PCT/US2023/075425
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Corrie Gallant-Behm
Wimalanathan KOKULAPALAN
Matthew Hassler
Daniel Curtis
Bruno Miguel DA CRUZ GODINHO
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Atalanta Therapeutics, Inc.
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Publication of WO2024073604A2 publication Critical patent/WO2024073604A2/fr

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    • 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 N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/52—Physical structure branched
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

  • This disclosure relates to small interfering RNA (siRNA) molecules, and compositions containing the same, that target RNA transcripts (e.g., mRNA) of a transactive response DNA binding protein 43 (TARDBP) gene.
  • target RNA transcripts e.g., mRNA
  • TARDBP transactive response DNA binding protein 43
  • the disclosure further describes methods for silencing of TARDBP and the treatment of diseases that may benefit from the silencing of TARDBP (e.g., neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia) by delivering TARDBP- targeting siRNA molecules to a target tissue of a subject in need.
  • diseases e.g., neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia
  • TARDBP transactive response DNA binding protein 43 encodes a protein that is implicated in the pathology of diseases including neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia). Accordingly, there is a need for therapeutics capable of selectively diminishing TARDBP activity in a manner that provides effective treatment for neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia) or other TARDBP-re ⁇ ated diseases or disorders.
  • neurodegenerative diseases e.g., amyotrophic lateral sclerosis and frontotemporal dementia
  • compositions and methods for reduction of transactive response DNA binding protein 43 (TARDBP) expression by way of small interfering RNA (siRNA)- mediated silencing of TARDBP transcripts The compositions and methods provide the benefit of exhibiting high selectivity toward TARDBP over other genes.
  • the siRNA molecules of the disclosure can be used to silence the TARDBP gene, thereby preventing the translation of the corresponding mRNA transcript and reducing transactive response DNA binding protein 43 (TDP-43) protein expression.
  • TDP-43 transactive response DNA binding protein 43
  • This reduction of TDP-43 levels thus prevents disease onset or progression, as mislocalization and aggregation of TDP-43 is a cause of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • siRNA molecules of the disclosure can be delivered directly to a subject in need of TARDBP silencing by way of, for example, injection intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, intra-cisterna magna injection, such as by catheterization, intravenous injection, subcutaneous injection, or intramuscular injection.
  • the disclosure provides an siRNA molecule containing an antisense strand and sense strand having complementarity to the antisense strand.
  • the antisense strand has complementarity sufficient to hybridize to a region within a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand may be, for example, from 10 to 50 nucleotides in length (e.g., from 10 to 45 nucleotides in length, from 10 to 40 nucleotides in length, from 10 to 35 nucleotides in length, from 10 to 30 nucleotides in length, from 10 to 29 nucleotides in length, from 10 to 28 nucleotides in length, from 10 to 27 nucleotides in length, from 10 to 26 nucleotides in length, from 10 to 25 nucleotides in length, from 10 to 24 nucleotides in length, from 10 to 23 nucleotides in length, from 10 to 22 nucleotides in length, from 10 to 21 nucleotides in length, or from 10 to 20 nucleotides in length).
  • 10 to 50 nucleotides in length e.g., from 10 to 45 nucleotides in length, from 10 to 40 nucleotides in length, from 10 to 35 nucleotides in length, from 10
  • the antisense strand is 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length, 14 nucleotides in length, 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, or more.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 15 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 16 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 17 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 18 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 19 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 20 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to a region of 21 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 70% (e.g., at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100%) complementarity to the region within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 75% complementarity to the region within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 - 192.
  • the antisense strand may have 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%, 99%, or 100% complementarity to the region within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has from 10 to 30 contiguous nucleotides (e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • contiguous nucleotides e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides
  • the antisense strand has from 12 to 30 contiguous nucleotides (e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • contiguous nucleotides e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides
  • the antisense strand has from 15 to 30 contiguous nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • contiguous nucleotides e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides
  • the antisense strand has from 18 to 30 contiguous nucleotides (e.g., 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • contiguous nucleotides e.g., 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides
  • the antisense strand has from 18 to 25 contiguous nucleotides (e.g., 18, 19, 20, 21 , 22, 23, 24, or 25 contiguous nucleotides) that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID Nos: 1-192.
  • the antisense strand has from 18 to 21 contiguous nucleotides (e.g., 18, 19, 20, or 21 contiguous nucleotides) that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has 9 or fewer nucleotide mismatches relative to a region of 21 contiguous nucleobases of the TARDBP RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192, optionally wherein the antisense strand contains 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch relative to the region of the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-576.
  • the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-576.
  • the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-576, optionally wherein the antisense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 385-576.
  • the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 385-576.
  • the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 193-384.
  • the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 193-384.
  • the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 193-384, optionally wherein the sense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 193-384.
  • the siRNA molecule has a sense strand having the nucleic acid sequence of any one of SEQ ID NOs: 193-384.
  • the antisense strand has a structure represented by Formula I, wherein Formula I is, in the 5’-to-3’ direction:
  • the antisense strand has a structure represented by Formula A1 , wherein Formula A1 is, in the 5’-to-3’ direction:
  • A represents a 2’-O-Me ribonucleoside
  • B represents a 2’-F ribonucleoside
  • O represents a phosphodiester internucleoside linkage
  • S represents a phosphorothioate internucleoside linkage
  • the antisense strand has a structure represented by Formula II, wherein Formula II is, in the 5’-to-3’ direction:
  • antisense strand has a structure represented by Formula A2, wherein Formula A2 is, in the 5’-to-3’ direction:
  • A represents a 2’-O-Me ribonucleoside
  • B represents a 2’-F ribonucleoside
  • O represents a phosphodiester internucleoside linkage
  • S represents a phosphorothioate internucleoside linkage
  • the sense strand has a structure represented by Formula III, wherein Formula III is, in the 5’-to-3’ direction:
  • F is represented by the formula (C-P 2 ) 3 -D-P 1 -C-P 1 -C, (C-P 2 ) 3 -D-P 2 -C-P 2 -C, (C-P 2 ) 3 -D-P 1 -C-P 1 -D, or (C-P 2 ) 3 -D-P 2 -C-P 2 -D;
  • A’, C, D, P 1 , and P 2 are as defined in Formula II; and m is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7).
  • the sense strand has a structure represented by Formula S1 , wherein
  • Formula S1 is, in the 5’-to-3’ direction:
  • Formula S1 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand has a structure represented by Formula S2, wherein Formula S2 is, in the 5’-to-3’ direction:
  • Formula S2 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand has a structure represented by Formula S3, wherein Formula S3 is, in the 5’-to-3’ direction:
  • Formula S3 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand has a structure represented by Formula S4, wherein Formula S4 is, in the 5’-to-3’ direction:
  • Formula S4 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the antisense strand has a structure represented by Formula IV, wherein Formula IV is, in the 5’-to-3’ direction: A-(A’)j-C-P 2 -B-(C-P 1 )k-C’
  • the antisense strand has a structure represented by Formula A3, wherein Formula A3 is, in the 5’-to-3’ direction:
  • A represents a 2’-O-Me ribonucleoside
  • B represents a 2’-F ribonucleoside
  • O represents a phosphodiester internucleoside linkage
  • S represents a phosphorothioate internucleoside linkage
  • the sense strand has a structure represented by Formula V, wherein Formula V is, in the 5’-to-3’ direction:
  • F is represented by the formula D-P 1 -C-P 1 -C, D-P 2 -C-P 2 -C, D-P 1 -C-P 1 -D, or D-P 2 -C-P 2 -D;
  • A’, C, D, P 1 and P 2 are as defined in Formula IV; and m is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7).
  • the sense strand has a structure represented by Formula S5, wherein Formula S5 is, in the 5’-to-3’ direction:
  • the sense strand has a structure represented by Formula S6, wherein Formula S6 is, in the 5’-to-3’ direction:
  • Formula S6 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand has a structure represented by Formula S7, wherein Formula S7 is, in the 5’-to-3’ direction:
  • Formula S7 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand has a structure represented by Formula S8, wherein Formula S8 is, in the 5’-to-3’ direction:
  • Formula S8 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the antisense strand has a structure represented by Formula VI, wherein Formula VI is, in the 5’-to-3’ direction:
  • I is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7).
  • the antisense strand has a structure represented by Formula A4, wherein Formula A4 is, in the 5’-to-3’ direction:
  • Formula A4 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand has a structure represented by Formula VII, wherein Formula VII is, in the 5’-to-3’ direction:
  • Formula VII wherein A’ is represented by the formula C-P 2 -D-P 2 ; each H is represented by the formula (C-P 1 )2; each I is represented by the formula (D-P 2 );
  • B, C, D, P 1 and P 2 are as defined in Formula VI; m is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7).
  • the sense strand has a structure represented by Formula S9, wherein Formula S9 is, in the 5’-to-3’ direction:
  • Formula S9 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the antisense strand also has a 5’ phosphorus stabilizing moiety at the 5’ end of the antisense strand.
  • the sense strand also has a 5’ phosphorus stabilizing moiety at the 5’ end of the sense strand.
  • each 5’ phosphorus stabilizing moiety is, independently, represented by any one of Formulas IX, XX, XI, XII, XIII, XIV, XV, or XVI:
  • Nuc represents a nucleobase, optionally wherein the nucleobase is selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, a cation (e.g., a monovalent cation), or hydrogen.
  • Nuc represents a nucleobase, optionally wherein the nucleobase is selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine
  • R represents an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, a cation (e.g., a monovalent cation), or hydrogen.
  • the nucleobase is an adenine, uracil, guanine, thymine, or cytosine.
  • the 5’ phosphorus stabilizing moiety is (E)-vinylphosphonate represented by Formula XI.
  • the siRNA molecule also has a hydrophobic moiety at the 5’ or the 3’ end of the siRNA molecule.
  • the hydrophobic moiety is selected from a group consisting of cholesterol, vitamin D, or tocopherol.
  • the siRNA molecule is a branched siRNA molecule.
  • the branched siRNA molecule is di-branched, tri-branched, or tetrabranched.
  • the siRNA molecule is di-branched, optionally wherein the di-branched siRNA molecule is represented by any one of Formulas XVII, XVIII, or XIX:
  • the di-branched siRNA molecule is represented by Formula XVII.
  • the di-branched siRNA molecule is represented by Formula XVIII.
  • the di-branched siRNA molecule is represented by Formula XIX.
  • the siRNA molecule is tri-branched, optionally wherein the tribranched siRNA molecule is represented by any one of Formulas XX, XXI, XXII, or XXIII: Formula XX; Formula XXI; Formula XXII; Formula XXIII; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.
  • the tri-branched siRNA molecule is represented by Formula XX. In some embodiments, the tri-branched siRNA molecule is represented by Formula XXI. In some embodiments, the tri-branched siRNA molecule is represented by Formula XXII. In some embodiments, the tri-branched siRNA molecule is represented by Formula XXIII.
  • the siRNA molecule is tetra-branched, optionally wherein the tetrabranched siRNA molecule is represented by any one of Formulas XXIV, XXV, XXVI, XXVII, or XXVIII:
  • the tetra-branched siRNA molecule is represented by Formula XXIV. In some embodiments, the tetra-branched siRNA molecule is represented by Formula XXV. In some embodiments, the tetra-branched siRNA molecule is represented by Formula XXVI. In some embodiments, the tetra-branched siRNA molecule is represented by Formula XXVII. In some embodiments, the tetra-branched siRNA molecule is represented by Formula XXVIII.
  • the linker is selected from a group consisting of one or more contiguous subunits of an ethylene glycol (e.g., polyethylene glycol (PEG), such as, e.g., triethylene glycol (TrEG) or tetraethylene glycol (TEG)), alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.
  • PEG polyethylene glycol
  • TrEG triethylene glycol
  • TEG tetraethylene glycol
  • the linker is an ethylene glycol oligomer. In some embodiments, the linker is an alkyl oligomer. In some embodiments, the linker is a carbohydrate oligomer. In some embodiments, the linker is a block copolymer. In some embodiments, the linker is a peptide oligomer. In some embodiments, the linker is an RNA oligomer. In some embodiments, the linker is a DNA oligomer.
  • the ethylene glycol oligomer is a PEG. In some embodiments, the PEG is a TrEG. In some embodiments, the PEG is a TEG.
  • the oligomer or copolymer contains 2 to 20 contiguous subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous subunits).
  • the linker attaches one or more (e.g., 1 , 2, 3, 4, or more) siRNA molecules by way of a covalent bond-forming moiety.
  • the covalent bond-forming moiety is selected from the group consisting of an alkyl, ester, amide, carbamate, phosphonate, phosphate, phosphorothioate, phosphoroamidate, triazole, urea, and formacetal.
  • the linker includes a structure of Formula L1 :
  • the linker includes a structure of Formula L2:
  • the linker includes a structure of Formula L3:
  • the linker includes a structure of Formula L4:
  • the linker includes a structure of Formula L5:
  • the linker includes a structure of Formula L6:
  • the linker includes a structure of Formula L7:
  • the linker includes a structure of Formula L8:
  • the linker includes a structure of Formula L9:
  • 50% or more of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (e.g., 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %,
  • ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).
  • 60% or more of the ribonucleotides in the antisense strand are 2'-O- Me ribonucleotides (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).
  • 70% or more of the ribonucleotides in the antisense strand are 2'-O- Me ribonucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).
  • 80% or more of the ribonucleotides in the antisense strand are 2'-O- Me ribonucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).
  • 90% or more of the ribonucleotides in the antisense strand are 2'-O- Me ribonucleotides (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).
  • 10% or less of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, 100% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.
  • 9 internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.
  • the length of the antisense strand is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides,
  • the length of the antisense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides.
  • the length of the antisense strand is 28 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides.
  • the siRNA molecules of the branched compound are joined to one another by way of a linker (e.g., an ethylene glycol oligomer, such as tetraethylene glycol). In some embodiments, the siRNA molecules of the branched compound are joined to one another by way of a linker between the sense strand of one siRNA molecule and the sense strand of the other siRNA molecule.
  • a linker e.g., an ethylene glycol oligomer, such as tetraethylene glycol
  • the siRNA molecules are joined by way of linkers between the antisense strand of one siRNA molecule and the antisense strand of the other siRNA molecule. In some embodiments, the siRNA molecules of the branched compound are joined to one another by way of a linker between the sense strand of one siRNA molecule and the antisense strand of the other siRNA molecule.
  • the length of the sense strand is between 12 and 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides).
  • 14 and 18 nucleotides e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18
  • the length of the sense strand is 15 nucleotides. In some embodiments, the length of the sense strand is 16 nucleotides. In some embodiments, the length of the sense strand is 17 nucleotides. In some embodiments, the length of the sense strand is 18 nucleotides. In some embodiments, the length of the sense strand is 19 nucleotides. In some embodiments, the length of the sense strand is 20 nucleotides. In some embodiments, the length of the sense strand is 21 nucleotides. In some embodiments, the length of the sense strand is 22 nucleotides. In some embodiments, the length of the sense strand is 23 nucleotides.
  • the length of the sense strand is 24 nucleotides. In some embodiments, the length of the sense strand is 25 nucleotides. In some embodiments, the length of the sense strand is 26 nucleotides. In some embodiments, the length of the sense strand is 27 nucleotides. In some embodiments, the length of the sense strand is 28 nucleotides. In some embodiments, the length of the sense strand is 29 nucleotides. In some embodiments, the length of the sense strand is 30 nucleotides.
  • four internucleoside linkages are phosphorothioate linkages.
  • the antisense strand is 18 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 18 nucleotides in length.
  • the antisense strand is 19 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 18 nucleotides in length.
  • the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 17 nucleotides in length.
  • the antisense strand is 20 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 15 nucleotides in length.
  • the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 20 nucleotides in length.
  • the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 17 nucleotides in length.
  • the antisense strand is 22 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 22 nucleotides in length.
  • the antisense strand is 23 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 18 nucleotides in length.
  • the antisense strand is 23 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 23 nucleotides in length.
  • the antisense strand is 24 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 18 nucleotides in length.
  • the antisense strand is 24 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 23 nucleotides in length.
  • the antisense strand is 24 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 17 nucleotides in length.
  • the antisense strand is 25 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 22 nucleotides in length.
  • the antisense strand is 25 nucleotides in length and the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 15 nucleotides in length.
  • the antisense strand is 26 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 20 nucleotides in length.
  • the antisense strand is 26 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 25 nucleotides in length.
  • the antisense strand is 26 nucleotides in length and the sense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 17 nucleotides in length.
  • the antisense strand is 27 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 22 nucleotides in length.
  • the antisense strand is 27 nucleotides in length and the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 27 nucleotides in length.
  • the antisense strand is 28 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 18 nucleotides in length.
  • the antisense strand is 28 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 23 nucleotides in length.
  • the antisense strand is 28 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 27 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 28 nucleotides in length.
  • the antisense strand is 29 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 18 nucleotides in length.
  • the antisense strand is 29 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 23 nucleotides in length.
  • the antisense strand is 29 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 27 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 28 nucleotides in length.
  • the antisense strand is 29 nucleotides in length and the sense strand is 29 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 17 nucleotides in length.
  • the antisense strand is 30 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 22 nucleotides in length.
  • the antisense strand is 30 nucleotides in length and the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 27 nucleotides in length.
  • the antisense strand is 30 nucleotides in length and the sense strand is 28 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 29 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 30 nucleotides in length.
  • the disclosure provides a pharmaceutical composition containing an siRNA molecule of any of the preceding aspects or embodiments of the disclosure, and a pharmaceutically acceptable excipient, carrier, or diluent.
  • the disclosure provides a method of delivering an siRNA molecule to a subject diagnosed as having a neurodegenerative disease by administering a therapeutically effective amount of the siRNA molecule or a pharmaceutical composition of any of the preceding aspects or embodiments of the disclosure to the subject.
  • the disclosure provides a method of treating a neurodegenerative disease in a subject in need thereof by administering a therapeutically effective amount of an siRNA molecule or a pharmaceutical composition of any of the preceding aspects or embodiments of the disclosure to the subject.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In some embodiments of the methods described herein, the neurodegenerative disease is frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the disclosure provides a method of reducing TARDBP expression in a subject in need thereof by administering a therapeutically effective amount of an siRNA or pharmaceutical composition of any of the preceding aspects or embodiments of the disclosure.
  • the siRNA molecule or the pharmaceutical composition is administered to the subject by way of intracerebroventricular, intrastriatal, intraparenchymal or intrathecal injection. In some embodiments, the siRNA molecule or the pharmaceutical composition is administered to the subject by way of intravenous, intramuscular, or subcutaneous injection.
  • the subject is a human.
  • the disclosure provides a kit containing an siRNA molecule or pharmaceutical composition of any of the preceding aspects or embodiments of the disclosure, and a package insert that instructs a user of the kit to perform the method of any of the preceding aspects or embodiments of the disclosure.
  • nucleic acids refers to RNA or DNA molecules consisting of a chain of ribonucleotides or deoxyribonucleotides, respectively.
  • therapeutic nucleic acid refers to a nucleic acid molecule (e.g., ribonucleic acid) that has partial or complete complementarity to, and interacts with, a disease- associated target mRNA and mediates silencing of expression of the mRNA.
  • carrier nucleic acid refers to a nucleic acid molecule (e.g., ribonucleic acid) that has sequence complementarity with, and hybridizes with, a therapeutic nucleic acid.
  • 3' end refers to the end of the nucleic acid that contains an unmodified hydroxyl group at the 3' carbon of the ribose ring.
  • nucleoside refers to a molecule made up of a heterocyclic base and its sugar.
  • nucleotide refers to a nucleoside having a phosphate group, or a variant thereof, on its 3' or 5' sugar hydroxyl group.
  • phosphate group variants include, but are not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, phosphorothioates, and phosphoramidites.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring (e.g., modified) portions that function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • siRNA refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway.
  • siRNA molecules may vary in length (generally, between 10 and 30 base pairs) and may contain varying degrees of complementarity to their target mRNA.
  • siRNA includes duplexes of two separate strands, as well as single strands that optionally form hairpin structures including a duplex region.
  • antisense strand refers to the strand of the siRNA duplex that contains some degree of complementarity to the target gene.
  • sense strand refers to the strand of the siRNA duplex that contains complementarity to the antisense strand.
  • interfering RNA molecule refers to an RNA molecule, such as a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO) that suppresses the endogenous function of a target RNA transcript.
  • siRNA small interfering RNA
  • miRNA microRNA
  • shRNA short hairpin RNA
  • ASO antisense oligonucleotide
  • the terms “express” and “expression” refer to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); and (3) translation of an RNA into a polypeptide or protein.
  • expression and the like are used interchangeably with the terms “protein expression” and the like.
  • Expression of a gene or protein of interest in a patient can manifest, for example, by detecting: an increase in the quantity or concentration of mRNA encoding corresponding protein (as assessed, e.g., using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the quantity or concentration of the corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), among others), and/or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay described herein or known in the art) in a sample obtained from the patient.
  • RNA detection procedures described herein or known in the art such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques
  • qPCR quantitative polymerase chain reaction
  • ELISA enzyme-linked immunosorbent assays
  • a cell is considered to “express” a gene or protein of interest if one or more, or all, of the above events can be detected in the cell or in a medium in which the cell resides.
  • a gene or protein of interest is considered to be “expressed” by a cell or population of cells if one can detect (i) production of a corresponding RNA transcript, such as an mRNA template, by the cell or population of cells (e.g., using RNA detection procedures described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5’ cap formation, and/or 3’ end processing, such as using RNA detection procedures described herein); (iii) translation of the RNA template into a protein product (e.g., using protein detection procedures described herein); and/or (iv) post-translational modification of the protein product (e.g., using protein detection procedures described herein).
  • target refers to generating an antisense strand so as to anneal the antisense strand to a region within the mRNA transcript of interest in a manner that results in a reduction in translation of the mRNA into the protein product.
  • nucleotide analog As used herein, the terms “chemically modified nucleotide,” “nucleotide analog,” “altered nucleotide,” and “modified nucleotide” refer to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function.
  • RNA molecules that contain ribonucleotides that have been chemically modified in order to decrease the rate of metabolism of an RNA molecule that is administered to a subject.
  • exemplary modifications include 2’-hydroxy to 2’-O- methoxy or 2’-fluoro, and phosphodiester to phosphorothioate.
  • phosphorothioate refers to a phosphate group of a nucleotide that is modified by substituting one or more of the oxygens of the phosphate group with sulfur.
  • nucleoside and “internucleotide” refer to the bonds between nucleosides and nucleotides, respectively.
  • antiagomirs refers to nucleic acids that can function as inhibitors of miRNA activity.
  • the term “gapmers” refers to chimeric antisense nucleic acids that contain a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage. The deoxynucleotide block is flanked by ribonucleotide monomers or ribonucleotide monomers containing modifications.
  • the term “mixmers” refers to nucleic acids that contain a mix of locked nucleic acids (LNAs) and DNA.
  • guide RNAs refers to nucleic acids that have sequence complementarity to a specific sequence in the genome immediately or 1 base pair upstream of the protospacer adjacent motif (PAM) sequence as used in CRISPR/Cas9 gene editing systems.
  • guide RNAs may refer to nucleic acids that have sequence complementarity (e.g., are antisense) to a specific messenger RNA (mRNA) sequence.
  • mRNA messenger RNA
  • a guide RNA may also have sequence complementarity to a “passenger RNA” sequence of equal or shorter length, which is identical or substantially identical to the sequence of mRNA to which the guide RNA hybridizes.
  • branched siRNA refers to a compound containing two or more double-stranded siRNA molecules covalently bound to one another.
  • Branched siRNA molecules may be “di-branched,” also referred to herein as “di-siRNA,” wherein the siRNA molecule includes 2 siRNA molecules covalently bound to one another, e.g., by way of a linker.
  • Branched siRNA molecules may be “tri-branched,” also referred to herein as “tri-siRNA,” wherein the siRNA molecule includes 3 siRNA molecules covalently bound to one another, e.g., by way of a linker.
  • Branched siRNA molecules may be “tetra-branched,” also referred to herein as “tetra-siRNA,” wherein the siRNA molecule includes 4 siRNA molecules covalently bound to one another, e.g., by way of a linker.
  • branch point moiety refers to a chemical moiety of a branched siRNA structure of the disclosure that may be covalently linked to a 5’ end or a 3’ end of an antisense strand or a sense strand of an siRNA molecule and which may support the attachment of additional single- or double-stranded siRNA molecules.
  • branch point moieties suitable for use in conjunction with the disclosed methods and compositions include, e.g., phosphoroamidite, tosylated solketal, 1 ,3-diaminopropanol, pentaerythritol, and any one of the branch point moieties described in US 10,478,503.
  • phosphate moiety refers to a terminal phosphate group that includes phosphates as well as modified phosphates.
  • the phosphate moiety may be located at either terminus but is preferred at the 5'-terminal nucleoside.
  • the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R’) or alkyl where R’ is H, an amino protecting group or unsubstituted or substituted alkyl.
  • the 5' and or 3' terminal group may include from 1 to 3 phosphate moieties that are each, independently, unmodified (di- or tri-phosphates) or modified.
  • the term “5' phosphorus stabilizing moiety” refers to a terminal phosphate group that includes phosphates as well as modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates).
  • the phosphate moiety may be located at either terminus but is preferred at the 5'-terminal nucleoside.
  • the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R’), or alkyl where R’ is H, an amino protecting group, or unsubstituted or substituted alkyl.
  • the 5' and or 3' terminal group may include from 1 to 3 phosphate moieties that are each, independently, unmodified (di- or tri-phosphates) or modified.
  • the phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 10:117-21 , 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 10:333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev. 11 :317-25, 2001 ; Vorobjev et al., Antisense Nucleic Acid Drug Dev.
  • Certain of the above- referenced modifications preferably decrease the rate of hydrolysis of, for example, polynucleotides including said analogs in vivo or in vitro.
  • Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs.
  • a proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.”
  • Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • Percent (%) sequence complementarity with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity.
  • a given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs.
  • Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine- guanine base pairs.
  • a proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.”
  • Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared.
  • the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, is calculated as follows:
  • a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • nucleic acid sequence or a portion thereof that need not be fully complementary (e.g., 100% complementary) to a target region or a nucleic acid sequence or a portion thereof that has one or more nucleotide mismatches relative to the target region but that is still capable of hybridizing to the target region under specified conditions.
  • the nucleic acid may be, e.g., 95% complementary, 90%, complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, but still form sufficient base pairs with the target so as to hybridize across its length.
  • Hybridization or “annealing” of nucleic acids is achieved when one or more nucleoside residues within a polynucleotide base pairs with one or more complementary nucleosides to form a stable duplex.
  • the base pairing is typically driven by hydrogen bonding events.
  • Hybridization includes Watson-Crick base pairs formed from natural and/or modified nucleobases.
  • the hybridization can also include non-Watson-Crick base pairs, such as wobble base pairs (guanosineuracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs. Nucleic acids need not be 100% complementary to undergo hybridization.
  • one nucleic acid may be, e.g., 95% complementary, 90%, complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, relative to another nucleic acid, but the two nucleic acids may still form sufficient base pairs with one another so as to hybridize.
  • the "stable duplex" formed upon the annealing/hybridization of one nucleic acid to another is a duplex structure that is not denatured by a stringent wash.
  • exemplary stringent wash conditions include temperatures of about 5° C less than the melting temperature of an individual strand of the duplex and low concentrations of monovalent salts, such as monovalent salt concentrations (e.g., NaCI concentrations) of less than 0.2 M (e.g., 0.2 M, 0.19 M, 0.18 M, 0.17 M, 0.16 M, 0.15 M, 0.14 M, 0.13 M, 0.12 M, 0.11 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M, or less).
  • monovalent salt concentrations e.g., NaCI concentrations
  • gene silencing refers to the suppression of gene expression, e.g., endogenous gene expression of TARDBP, which may be mediated through processes that affect transcription and/or through processes that affect post-transcriptional mechanisms.
  • gene silencing occurs when an RNAi molecule initiates the inhibition or degradation of the mRNA transcribed from a gene of interest in a sequence-specific manner by way of RNA interference, thereby preventing translation of the gene's product.
  • overactive disease driver gene refers to a gene having increased activity and/or expression that contributes to or causes a disease state in a subject (e.g., a human).
  • the disease state may be caused or exacerbated by the overactive disease driver gene directly or by way of an intermediate gene(s).
  • ethylene glycol chain refers to a carbon chain with the formula ((CH 2 OH) 2 ).
  • alkyl refers to a saturated hydrocarbon group. Alkyl groups may be acyclic or cyclic and contain only C and H when unsubstituted. When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, and isobutyl.
  • alkyl examples include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • alkyl may be substituted.
  • Suitable substituents that may be introduced into an alkyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.
  • alkenyl may be substituted.
  • Suitable substituents that may be introduced into an alkenyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.
  • alkynyl refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula CEC). Alkynyl groups contain only C and H when unsubstituted. When an alkynyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “pentynyl” is meant to include n-pentynyl, secpentynyl, /so-pentynyl, and te/Y-pentynyl.
  • alkynyl examples include -CECH and -CEC-CH3. In some embodiments, alkynyl may be substituted. Suitable substituents that may be introduced into an alkynyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.
  • phenyl denotes a monocyclic arene in which one hydrogen atom from a carbon atom of the ring has been removed.
  • a phenyl group may be unsubstituted or substituted with one or more suitable substituents, wherein the substituent replaces an H of the phenyl group.
  • benzyl refers to monovalent radical obtained when a hydrogen atom attached to the methyl group of toluene is removed.
  • a benzyl group generally has the formula of phenyl-CH2-.
  • a benzyl group may be unsubstituted or substituted with one or more suitable substituents.
  • the substituent may replace an H of the phenyl component and/or an H of the methylene (-CH2-) component.
  • amide refers to an alkyl, alkenyl, alkynyl, or aromatic group that is attached to an amino-carbonyl functional group.
  • triazole refers to heterocyclic compounds with the formula (C2H3N3), having a five-membered ring of two carbons and three nitrogens, the positions of which can change resulting in multiple isomers.
  • terminal group refers to the group at which a carbon chain or nucleic acid ends.
  • amino acid refers to a molecule containing amine and carboxyl functional groups and a side chain specific to the amino acid.
  • the amino acid is chosen from the group of proteinogenic amino acids.
  • the amino acid is an L-amino acid or a D-amino acid.
  • the amino acid is a synthetic amino acid (e.g., a beta-amino acid).
  • lipophilic amino acid refers to an amino acid including a hydrophobic moiety (e.g., an alkyl chain or an aromatic ring).
  • target of delivery refers to the organ or part of the body to which it is desired to deliver the branched oligonucleotide compositions.
  • the term “between X and Y” is inclusive of the values of X and Y.
  • “between X and Y” refers to the range of values between the value of X and the value of Y, as well as the value of X and the value of Y.
  • the terms “subject’ and “patient” are used interchangeably and refer to an organism, such as a mammal (e.g., a human), that experiences a neurodegenerative disease or disorder (e.g., amyotrophic lateral sclerosis of frontotemporal dementia) and/or contains a gain-of- function TARDBP variant allele.
  • a mammal e.g., a human
  • a neurodegenerative disease or disorder e.g., amyotrophic lateral sclerosis of frontotemporal dementia
  • a gain-of- function TARDBP variant allele e.g., amyotrophic lateral sclerosis of frontotemporal dementia
  • TARDBP refers to the gene encoding transactive response DNA binding protein 43 kDa (TDP-43), including any native TARDBP gene from any source.
  • TDP-43 transactive response DNA binding protein 43 kDa
  • the term encompasses “full-length,” unprocessed TARDBP as well as any form of TARDBP that results from processing in the cell.
  • the term also encompasses naturally occurring variants of TARDBP, e.g., splice variants or allelic variants.
  • the nucleic acid sequence of an exemplary TARDBP gene is shown in European Nucleotide Archive (ENA) Accession No. U23731.1.
  • ENA European Nucleotide Archive
  • U23731.1 The amino acid sequence of an exemplary protein encoded by an TARDBP gene is shown in UNIPROTTM Accession No. Q13148.
  • Neuroinflammation refers to a range of immune responses in the central nervous system (e.g., in microglia). Neuroinflammation may be brain-derived or result from a systemic inflammatory response.
  • neurodegenerative disease and “neurodegenerative disorder” are used interchangeably to refer to any condition that is in some way caused by a loss of function or death of cells of the central nervous system or peripheral nervous system.
  • exemplary neurodegenerative diseases are Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, and spinocerebellar ataxias.
  • the terms “treat,” “treated,” and “treating” mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent, ameliorate, or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results.
  • Beneficial or desired clinical results include, but are not limited to, a reduction in a patient’s reliance on pharmacological treatments; alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical, cognitive, or behavioral (e.g., depressive behavior or apathy) parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • clinical benefits in the context of a subject having ALS administered an siRNA molecule or siRNA composition of the disclosure include, without limitation, a reduction in involuntary movements, muscle cramps, weakness, loss of motor control.
  • clinical benefits in the context of a subject having FTD administered an siRNA molecule or siRNA composition of the disclosure include, without limitation, a reduction in memory problems, behavioral problems, and language problems.
  • “Benefit” and “response” are also used interchangeably to refer to, for example, a reduction in wild type TARDBP transcripts, mutant TARDBP transcripts, variant TARDBP transcripts, splice isoforms of TARDBP transcripts, and/or overexpressed TARDBP transcripts.
  • siRNA molecules with sequence homology to a TAR DNA-binding protein-43 (TARDBP) gene and methods for administering said siRNA molecules to a subject.
  • the siRNA molecules described herein may be composed as branched siRNA structures, such as di-branched, tri-branched, and tetrabranched siRNA structures and may further include specific patterns of chemical modifications (e.g., 2’ ribose modifications or internucleoside linkage modifications) to improve resistance against nuclease enzymes, toxicity profile, and physicochemical properties (e.g., thermostability).
  • Small interfering RNA molecules are short, double-stranded RNA molecules. They are capable of mediating RNA interference (RNAi) by degrading mRNA with a complementary nucleotide sequence, thus preventing the translation of the target gene.
  • RNAi RNA interference
  • the siRNA molecules of the disclosure may exhibit, for example, robust gene-specific suppression of TARDBP, relative to other human genes.
  • the siRNA molecules of the disclosure may feature an antisense strand having a nucleic acid sequence that is complementary to a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.
  • the degree of complementarity of the antisense strand to the region of the TARDBP mRNA transcript may be sufficient for the antisense strand to anneal over the full length of the region of the TARDBP mRNA transcript.
  • the antisense strand may have a nucleic acid sequence that is at least 60% complementary (e.g., 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the region of the TARDBP mRNA transcript.
  • 60% complementary e.g., 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%
  • the siRNA molecules of the disclosure feature an antisense strand having the nucleic acid sequence of any one of SEQ ID NOs: 385-576, or a nucleic acid sequence that is at least 60% identical thereto.
  • the siRNA molecules of the disclosure may feature an antisense strand having a nucleic acid sequence that is at least 60% identical (e.g., 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-576.
  • the siRNA molecules of the disclosure feature a sense strand having the nucleic acid sequence of any one of SEQ ID NOs: 193-384, or a nucleic acid sequence that is at least 60% identical thereto.
  • the siRNA molecules of the disclosure may feature a sense strand having a nucleic acid sequence that is at least 60% identical (e.g., 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
  • siRNA molecules of the disclosure are those shown in Table 1 , below.
  • Table 1 summarizes the antisense strands, sense strands, and corresponding regions of a TARDBP mRNA transcript that are targeted by each antisense strand.
  • the siRNA molecules of the disclosure may be in the form of a single-stranded (ss) or double- stranded (ds) oligonucleotide structure.
  • the siRNA molecules may be dibranched, tri-branched, ortetra-branched molecules.
  • the siRNA molecules of the disclosure may contain one or more phosphodiester internucleoside linkages and/or an analog thereof, such as a phosphorothioate internucleoside linkage.
  • the siRNA molecules of the disclosure may further contain chemically modified nucleosides having 2’ sugar modifications.
  • siRNAs consist of a ribonucleic acid, including a ss- or ds- structure, formed by a first strand (i.e., antisense strand), and in the case of a ds-siRNA, a second strand (i.e., sense strand).
  • the first strand includes a stretch of contiguous nucleotides that is at least partially complementary to a target nucleic acid.
  • the second strand also includes a stretch of contiguous nucleotides where the second stretch is at least partially identical to a target nucleic acid.
  • the first strand and said second strand may be hybridized to each other to form a double-stranded structure. The hybridization typically occurs by Watson Crick base pairing.
  • the hybridization or base pairing is not necessarily complete or perfect, which means that the first and second strand are not 100% basepaired due to mismatches.
  • One or more mismatches may also be present within the duplex without necessarily impacting the siRNA RNAi activity.
  • the first strand contains a stretch of contiguous nucleotides which is essentially complementary to a target nucleic acid.
  • the target nucleic acid sequence is, in accordance with the mode of action of interfering ribonucleic acids, a ss-RNA, preferably an mRNA.
  • a ss-RNA preferably an mRNA.
  • Such hybridization occurs most likely through Watson Crick base pairing but is not necessarily limited thereto.
  • the extent to which the first strand has a complementary stretch of contiguous nucleotides to a target nucleic acid sequence may be between 80% and 100%, e.g., 80%, 85%, 90%, 95%, or 100% complementary.
  • siRNA molecules described herein may employ modifications to the nucleobase, phosphate backbone, ribose core, 5'- and 3'-ends, and branching, wherein multiple strands of siRNA may be covalently linked.
  • any length, known and previously unknown in the art, may be employed for the current invention.
  • potential lengths for an antisense strand of the siRNA molecules of the present disclosure is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleo
  • the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.
  • the sense strand of the siRNA molecules of the present disclosure is between 12 and 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides).
  • the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides.
  • the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.
  • the present disclosure may include ss- and ds- siRNA molecule compositions including at least one (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or more) nucleosides having 2’ sugar modifications.
  • Possible 2'-modifications include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • the modification includes a 2’-O-methyl (2’-O-Me) modification.
  • Other potential sugar substituent groups include: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • the modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE).
  • the modification includes 2'- dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylamino-ethoxy-ethyl or 2'- DMAEOE), i.e., 2'-O-CH 2 OCH 2 N(CH 3 ) 2 .
  • 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • the 2'-arabino modification is 2'-F.
  • Similar modifications may also be made at other positions on the siRNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • the siRNA molecules of the disclosure may also include nucleosides or other surrogate or mimetic monomeric subunits that include a nucleobase (often referred to in the art simply as “base” or “heterocyclic base moiety”).
  • the nucleobase is another moiety that has been extensively modified or substituted and such modified and or substituted nucleobases are amenable to the present disclosure.
  • "unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in US 3,687,808, those disclosed in Kroschwitz, J. I., ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition 30:613, 1991 ; and those disclosed by Sanghvi, Y.S., Chapter 16, Antisense Research and Applications, CRC Press, Gait, M.J.
  • siRNA molecules of the present disclosure may also include polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties.
  • polycyclic heterocyclic compounds A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand.
  • Representative cytosine analogs that make three hydrogen bonds with a guanosine in a second strand include 1 ,3-diazaphenoxazine-2-one (Kurchavov ef al., Nucleosides and Nucleotides, 16:1837-46, 1997), 1 ,3-diazaphenothiazine-2-one (Lin et al. Am. Chem. Soc., 117:3873-4, 1995), and 6,7,8,9-tetrafluoro-l,3-diazaphenoxazine-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998).
  • RNA phosphate backbone Another variable in the design of the present disclosure is the internucleoside linkage making up the phosphate backbone of the siRNA molecule.
  • the natural RNA phosphate backbone may be employed here, derivatives thereof may be used which enhance desirable characteristics of the siRNA molecule.
  • protecting parts, or the whole, of the siRNA molecule from hydrolysis is phosphorothioates. Any portion or the whole of the backbone may contain phosphate substitutions (e.g., phosphorothioates).
  • the internucleoside linkages may be between 0 and 100% phosphorothioate, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40 and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphorothioate linkages.
  • 0 and 100% phosphorothioate e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and
  • the internucleoside linkages may be between 0 and 100% phosphodiester linkages, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100% 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40 and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphodiester linkages.
  • 0 and 100% phosphodiester linkages e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100% 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%,
  • oligonucleotides containing modified e.g., non-naturally occurring internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • a preferred phosphorus containing modified internucleoside linkage is the phosphorothioate internucleoside linkage.
  • the modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3
  • Exemplary U.S. patents describing the preparation of phosphorus-containing linkages include but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321 ,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541 ,316; 5,550,111 ; 5,563,253; 5,571 ,799; 5,587,361 ; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,5
  • the modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • patents that teach the preparation of non-phosphorus backbones include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141 ; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541 ,307; 5,561 ,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.
  • the following section provides a set of exemplary scaffolds into which the siRNA molecules of the disclosure may be incorporated.
  • the siRNA may contain an antisense strand including a region represented by Formula I, wherein Formula I is, in the 5’-to-3’ direction
  • Formula I wherein A is represented by the formula C-P 1 -D-P 1 ; each A’ is represented by the formula C-P 2 -D-P 2 ; B is represented by the formula C-P 2 -D-P 2 -D-P 2 ; each C is a 2’-O-methyl (2’-O-Me) ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-fluoro (2’-F) ribonucleoside; each D is a 2’-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7).
  • j is an integer
  • the antisense strand includes a structure represented by Formula A1 , wherein Formula A1 is, in the 5’-to-3’ direction: A-S-B-S-A-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-S-A-S-B-S-A Formula A1 ; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • A represents a 2’-O-Me ribonucleoside
  • B represents a 2’-F ribonucleoside
  • O represents a phosphodiester internucleoside linkage
  • S represents a phosphorothioate internu
  • the siRNA may contain an antisense strand including a region represented by Formula II, wherein Formula II is, in the 5’-to-3’ direction:
  • A-B-(A’)j-C-P 2 -D-P 1 -(C-P 1 )k-C’ Formula II; wherein A is represented by the formula C-P 1 -D-P 1 ; each A’ is represented by the formula C-P 2 -D-P 2 ; B is represented by the formula C-P 2 -D-P 2 -D-P 2 ; each C is a 2’-O-methyl (2’-O-Me) ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-fluoro (2’-F) ribonucleoside; each D is a 2’-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1 , 2, 3, 4,
  • the antisense strand includes a structure represented by Formula A2, wherein Formula A2 is, in the 5’-to-3’ direction:
  • the sense strand includes a structure represented by Formula III, wherein Formula III is, in the 5’-to-3’ direction:
  • E-(A’)m-F Formula III wherein E is represented by the formula (C-P 1 )2; F is represented by the formula (C-P 2 )3-D-P 1 -C-P 1 -C, (C-P 2 ) 3 -D-P 2 -C-P 2 -C, (C-P 2 ) 3 -D-P 1 -C-P 1 -D, or (C-P 2 ) 3 -D-P 2 -C-P 2 -D; A’, C, D, P 1 , and P 2 are as defined in Formula I; and m is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4.
  • the sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.
  • the sense strand includes a structure represented by Formula S1 , wherein Formula S1 is, in the 5’-to-3’ direction:
  • Formula S1 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand includes a structure represented by Formula S2, wherein Formula S2 is, in the 5’-to-3’ direction:
  • Formula S2 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand includes a structure represented by Formula S3, wherein Formula S3 is, in the 5’-to-3’ direction:
  • Formula S3 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand includes a structure represented by Formula S4, wherein Formula S4 is, in the 5’-to-3’ direction:
  • Formula S4 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the siRNA may contain an antisense strand including a region represented by Formula IV, wherein Formula IV is, in the 5’-to-3’ direction:
  • the antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.
  • the antisense strand includes a structure represented by Formula A3, wherein Formula A3 is, in the 5’-to-3’ direction:
  • A represents a 2’-O-Me ribonucleoside
  • B represents a 2’-F ribonucleoside
  • O represents a phosphodiester internucleoside linkage
  • S represents a phosphorothioate internucleoside linkage.
  • the siRNA of the disclosure may have a sense strand represented by Formula V, wherein Formula V is, in the 5’-to-3’ direction:
  • E-(A’) m -C-P 2 -F Formula V wherein E is represented by the formula (C-P 1 )2; F is represented by the formula D-P 1 -C-P 1 -C, D-P 2 - C-P 2 -C, D-P 1 -C-P 1 -D, or D-P 2 -C-P 2 -D; A’, C, D, P 1 , and P 2 are as defined in Formula IV; and m is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5.
  • the sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.
  • the sense strand includes a structure represented by Formula S5, wherein Formula S5 is, in the 5’-to-3’ direction:
  • Formula S5 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand includes a structure represented by Formula S6, wherein Formula S6 is, in the 5’-to-3’ direction:
  • the sense strand includes a structure represented by Formula S7, wherein Formula S7 is, in the 5’-to-3’ direction:
  • Formula S7 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the sense strand includes a structure represented by Formula S8, wherein Formula S8 is, in the 5’-to-3’ direction:
  • Formula S8 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the siRNA may contain an antisense strand including a region represented by Formula VI, wherein Formula VI is, in the 5’-to-3’ direction:
  • Formula VI wherein A is represented by the formula C-P 1 -D-P 1 ; each B is represented by the formula C-P 2 ; each C is a 2’-O-Me ribonucleoside; each O’, independently, is a 2’-O-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’-F ribonucleoside; each E is represented by the formula D-P 2 -C-P 2 ; F is represented by the formula D-P 1 -C-P 1 ; each G is represented by the formula C-P 1 ; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); and I is
  • the antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.
  • the antisense strand includes a structure represented by Formula A4, wherein Formula A4 is, in the 5’-to-3’ direction:
  • the siRNA may contain a sense strand including a region represented by Formula VII, wherein Formula VII is, in the 5’-to-3’ direction: H-Bm-ln-A’-Bo-H-C
  • Formula VII wherein A’ is represented by the formula C-P 2 -D-P 2 ; each H is represented by the formula (C-P 1 )2; each I is represented by the formula (D-P 2 ); B, C, D, P 1 , and P 2 are as defined in Formula VI; m is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3.
  • the sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.
  • the sense strand includes a structure represented by Formula S9, wherein Formula S9 is, in the 5’-to-3’ direction:
  • Formula S9 wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
  • the siRNA may contain an antisense strand including a region that is represented by Formula VIII:
  • siRNA molecules of the disclosure can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
  • the siRNA agent can be prepared using solution-phase or solid-phase organic synthesis or both.
  • Organic synthesis offers the advantage that the oligonucleotide including unnatural or modified nucleotides can be easily prepared.
  • siRNA molecules of the disclosure can be prepared using solution-phase or solid-phase organic synthesis or both. Further, it is contemplated that for any siRNA agent disclosed herein, further optimization could be achieved by systematically either adding or removing linked nucleosides to generate longer or shorter sequences.
  • such optimized sequences can be adjusted by, e.g., the introduction of modified nucleosides, and/or modified internucleoside linkages as described herein or as known in the art, including alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and/or targeting to a particular location or cell type).
  • modified nucleosides, and/or modified internucleoside linkages as described herein or as known in the art, including alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and/or targeting to a particular location or cell type).
  • a 5'-phosphorus stabilizing moiety may be employed.
  • a 5'-phosphorus stabilizing moiety replaces the 5'-phosphate to prevent hydrolysis of the phosphate. Hydrolysis of the 5'-phosphate prevents binding to RISC, a necessary step in gene silencing. Any replacement for phosphate that does not impede binding to RISC is contemplated in this disclosure. In some embodiments, the replacement for the 5'-phosphate is also stable to in vivo hydrolysis.
  • Each strand of a siRNA molecule may independently and optionally employ any suitable 5'-phosphorus stabilizing moiety.
  • Formulas IX-XVI Some exemplary endcaps are demonstrated in Formulas IX-XVI.
  • Nuc in Formulas IX-XVI represents a nucleobase or nucleobase derivative or replacement as described herein.
  • X in formula IX-XVI represents a 2’-modification as described herein.
  • Some embodiments employ hydroxy as in Formula IX, phosphate as in Formula X, vinylphosphonates as in Formula XI and XIV, 5’-methyl- substitued phosphates as in Formula XII, XIII, and XVI, methylenephosphonates as in Formula XV, or vinyl 5'-vinylphsophonate as a 5'-phosphorus stabilizing moiety as demonstrated in Formula XI. Hydrophobic Moieties
  • the present disclosure further provides siRNA molecules having one or more hydrophobic moieties attached thereto.
  • the hydrophobic moiety may be covalently attached to the 5’ end or the 3’ end of the siRNA molecules of the disclosure.
  • Non-limiting examples of hydrophobic moieties suitable for use with the siRNA molecules of the disclosure may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid or any combination of the aforementioned hydrophobic moieties with PC.
  • siRNA molecules of the disclosure may be branched.
  • the siRNA molecules of the disclosure may have one of several branching patterns, as described herein.
  • the siRNA molecules disclosed herein may be branched siRNA molecules.
  • the siRNA molecule may not be branched, or may be di-branched, tri-branched, or tetra-branched, connected through a linker.
  • Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 separate RNA single- or double-strands.
  • the branch points on the linker may stem from the same atom, or separate atoms along the linker.
  • the siRNA molecule is a branched siRNA molecule.
  • the branched siRNA molecule is di-branched, tri-branched, ortetra-branched.
  • the di-branched siRNA molecule is represented by any one of Formulas XVII-XIX, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety (e.g., phosphoroamidite, tosylated solketal, 1 ,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US 10,478,503).
  • a branch point moiety e.g., phosphoroamidite, tosylated solketal, 1 ,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US 10,478,503.
  • the tri-branched siRNA molecule represented by any one of Formulas XX-XXIII, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.
  • the tetra-branched siRNA molecule represented by any one of Formulas XXIV-XXVIII, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.
  • Linkers include ethylene glycol chains of 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits), alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, and others.
  • any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent.
  • the linker is a poly-ethylene glycol (PEG) linker.
  • PEG linkers suitable for use with the disclosed compositions and methods include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEGs, linear forked PEGs, or branched forked PEGs.
  • the PEG linker may have a weight that is between 5 and 500 Daltons. In some embodiments, a PEG linker having a weight that is between 500 and 1 ,000 Dalton may be used. In some embodiments, a PEG linker having a weight that is between 1 ,000 and 10,000 Dalton may be used. In some embodiments, a PEG linker having a weight that is between 200 and 20,000 Dalton may be used. In some embodiments, the linker is covalently attached to a sense strand of the siRNA. In some embodiments, the linker is covalently attached to an antisense strand of the siRNA. In some embodiments, the PEG linker is a triethylene glycol (TrEG) linker. In some embodiments, the PEG linker is a tetraethylene glycol (TEG) linker.
  • TrEG triethylene glycol
  • TEG linker tetraethylene glycol
  • the linker is an alkyl chain linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker is a DNA linker.
  • Linkers may covalently link 2, 3, 4, or 5 unique siRNA strands.
  • the linker may covalently bind to any part of the siRNA oligomer.
  • the linker attaches to the 3' end of nucleosides of each siRNA strand.
  • the linker attaches to the 5' end of nucleosides of each siRNA strand.
  • the linker attaches to a nucleoside of an siRNA strand (e.g., sense or antisense strand) by way of a covalent bond-forming moiety.
  • the covalent-bond-forming moiety is selected from the group consisting of an alkyl, ester, amide, carbonate, carbamate, triazole, urea, formacetal, phosphonate, phosphate, and phosphate derivative (e.g., phosphorothioate, phosphoramidate, etc.).
  • the linker has a structure of Formula L1 :
  • the linker has a structure of Formula L2:
  • the linker has a structure of Formula L3:
  • the linker has a structure of Formula L4:
  • the linker has a structure of Formula L5:
  • the linker has a structure of Formula L6:
  • the linker has a structure of Formula L7, as is shown below:
  • the linker has a structure of Formula L8:
  • the linker has a structure of Formula L9:
  • the selection of a linker for use with one or more of the branched siRNA molecules disclosed herein may be based on the hydrophobicity of the linker, such that, e.g., desirable hydrophobicity is achieved for the one or more branched siRNA molecules of the disclosure.
  • a linker containing an alkyl chain may be used to increase the hydrophobicity of the branched siRNA molecule as compared to a branched siRNA molecule having a less hydrophobic linker or a hydrophilic linker.
  • siRNA agents disclosed herein may be synthesized and/or modified by methods well established in the art, such as those described in Beaucage, S. L. et al. (edrs.), Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, N.Y., 2000, which is hereby incorporated herein by reference.
  • the TARDBP-targetmg siRNA molecules of the disclosure may be delivered to a subject, thereby treating a neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), and/or mitigating phenotypes associated with the disorders.
  • a neurodegenerative disease such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)
  • the siRNA molecules may be delivered to a subject, thereby treating ALS and/or mitigating ALS disease associated phenotypes (e.g., involuntary movements, muscle cramps, weakness, loss of motor control).
  • the TARDBP -targeting siRNA molecules of the disclosure may be delivered to a subject, thereby treating FTD and/or mitigating FTD-associated phenotypes (e.g., memory problems, behavioral problems, and language problems).
  • siRNA molecules of the disclosure may also be delivered to a subject having a variant of the TARDBP gene for which siRNA-mediated gene silencing of the TARDBP variant gene reduces the expression level of TARDBP transcript, thereby treating a neurodegenerative disease (e.g., ALS or FTD) or other TARDBP-re ⁇ ated diseases or disorders.
  • a neurodegenerative disease e.g., ALS or FTD
  • other TARDBP-re ⁇ ated diseases or disorders e.g., ALS or FTD
  • the disclosure provides methods of treating a subject by way of TARDBP gene silencing with one or more of the siRNA molecules described herein.
  • the gene silencing may be performed in a subject to silence wild type TARDBP transcripts, mutant TARDBP transcripts, splice isoforms of TARDBP transcripts, and/or overexpressed TARDBP transcripts thereof, relative to a healthy subject.
  • the method may include delivering to the CNS or affected tissues of the subject (e.g., a human) the siRNA molecules of the disclosure or a pharmaceutical composition containing the same by any appropriate route of administration (e.g., intracerebroventricular, intrathecal injection, intrastriatal injection, intra-cisterna magna injection by catheterization, intraparenchymal injection, intravenous injection, subcutaneous injection, or intramuscular injection).
  • the active compound can be administered in any suitable dose.
  • the actual dosage amount of a composition of the present disclosure administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredients) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, and for as long as necessary.
  • Subjects may be adult or pediatric humans, with or without comorbid diseases.
  • Subjects that may be treated with the siRNA molecules disclosed herein are subjects in need of treatment of, for example, a neurodegenerative disease (e.g., ALS or FTD) and/or any other medical risk(s) associated with gain of function mutations in the TARDBP gene.
  • a neurodegenerative disease e.g., ALS or FTD
  • Subjects that may be treated with the siRNA molecules disclosed herein may include, for example, humans, monkeys, rats, mice, pigs, and other mammals containing at least one orthologous copy of the TARDBP gene.
  • Subjects may be adult or pediatric humans, with or without comorbid diseases.
  • the siRNA molecules in the present disclosure may be formulated into a pharmaceutical composition for administration to a subject in a biologically compatible form suitable for administration in vivo. Accordingly, the present disclosure provides a pharmaceutical composition containing a siRNA molecule of the disclosure in admixture with a suitable diluent, carrier, or excipient.
  • the siRNA molecules may be administered, for example, directly into the CNS or affected tissues of the subject (e.g., by way of intracerebroventricular, intrastriatally, intrathecal injection, intra-cisterna magna injection by catheterization, intraparenchymal injection, intravenous injection, subcutaneous injection, or intramuscular injection).
  • a pharmaceutical composition may contain a preservative, e.g., to prevent the growth of microorganisms.
  • Pharmaceutical compositions may include sterile aqueous solutions, dispersions, or powders, e.g., for the extemporaneous preparation of sterile solutions or dispersions. In all cases the form may be sterilized using techniques known in the art and may be fluidized to the extent that may be easily administered to a subject in need of treatment.
  • a pharmaceutical composition may be administered to a subject, e.g., a human subject, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which may be determined by the solubility and/or chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.
  • a physician having ordinary skill in the art can readily determine an effective amount of the siRNA molecule for administration to a mammalian subject (e.g., a human) in need thereof.
  • a physician could start prescribing doses of one the siRNA molecules of the disclosure at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a physician may begin a treatment regimen by administering one of the siRNA molecules of the disclosure at a high dose and subsequently administer progressively lower doses until reaching a minimal dosage at which a therapeutic effect is achieved (e.g., a reduction in expression of a target gene sequence).
  • a suitable daily dose of one of the siRNA molecules of the disclosure will be an amount of the siRNA molecule which is the lowest dose effective to produce a therapeutic effect.
  • the ss- or ds-siRNA molecules of the disclosure may be administered by injection, e.g., intrathecally, intracerebroventricularly, by intra- cisterna magna injection by catheterization, intraparenchymally, intravenously, subcutaneously, or intramuscularly.
  • a daily dose of a therapeutic composition of the siRNA molecules of the disclosure may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for the siRNA molecules of the disclosure to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents.
  • the method of the disclosure contemplates any route of administration tolerated by the therapeutic composition.
  • Some embodiments of the method include injection intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, or by intra-cisterna magna injection by catheterization.
  • Intrathecal injection is the direct injection into the spinal column or subarachnoid space.
  • the siRNA molecules of the disclosure have direct access to cells (e.g., neurons and glial cells) in the spinal column and a route to access the cells in the brain by bypassing the blood brain barrier.
  • Intracerebroventricular (ICV) injection is a method to directly inject into the CSF of the cerebral ventricles. Similar to intrathecal injection, ICV is a method of injection which bypasses the blood brain barrier. Using ICV allows the advantage of access to the cells of the brain and spinal column without the danger of the therapeutic being degraded in the blood.
  • Intrastriatal injection is the direct injection into the striatum, or corpus striatum.
  • the striatum is an area in the subcortical basal ganglia in the brain. Injecting into the striatum bypasses the blood brain barrier and the pharmacokinetic challenges of injection into the blood stream and allows for direct access to the cells of the brain.
  • Intraparenchymal administration is the direct injection into the parenchyma (e.g., the brain parenchyma). Injection into the brain parenchyma allows for injection directly into brain regions affected by a disease or disorder while bypassing the blood brain barrier.
  • parenchyma e.g., the brain parenchyma
  • Intra-cisterna magna injection by catheterization is the direct injection into the cisterna magna.
  • the cisterna magna is the area of the brain located between the cerebellum and the dorsal surface of the medulla oblongata. Injecting into the cisterna magna results in more direct delivery to the cells of the cerebellum, brainstem, and spinal cord.
  • the therapeutic composition may be delivered to the subject by way of systemic administration, e.g., intravenously, intramuscularly, or subcutaneously.
  • IV injection is a method to directly inject into the bloodstream of a subject.
  • the IV administration may be in the form of a bolus dose or by way of continuous infusion, or any other method tolerated by the therapeutic composition.
  • Intramuscular (IM) injection is injection into a muscle of a subject, such as the deltoid muscle or gluteal muscle. IM may allow for rapid absorption of the therapeutic composition. Subcutaneous injection is injection into subcutaneous tissue. Absorption of compositions delivered subcutaneously may be slower than IV or IM injection, which may be beneficial for compositions requiring continuous absorption.
  • This Example describes the results of a series of experiments undertaken to investigate the ability of siRNA molecules complementary to specific regions within a human TARDBP mRNA transcript to effectuate reduced expression of the TARDBP gene.
  • HeLa cells were actively transfected with TARDBP siRNA at either 20 nM or 0.5 nM concentration. After 24 hours, cells were lysed, and mRNA levels of TARDBP and a housekeeping gene (ATP5B) were assessed via reverse transcription-quantitative polymerase chain reaction (RT- qPCR), using standard reagents and Applied Biosystems TaqMan Assays. Results are presented as the percent residual TARDBP mRNA relative to untreated control cells in the same assay.
  • RT- qPCR reverse transcription-quantitative polymerase chain reaction
  • siRNA molecule of the disclosure having an antisense strand and a sense strand as shown in Tables 3 and 4, below.
  • the knockdown efficiency of the siRNA molecule was measured as the residual mRNA expression as a percent of untreated at 20 nM and 0.5 nM.
  • the knockdown results are reported in Table 3 (20 nM) and Table 4 (0.5 nM).
  • siRNA molecules of the disclosure can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
  • the siRNA agent can be prepared using solution-phase or solid-phase organic synthesis or both.
  • Organic synthesis offers the advantage that the oligonucleotide including unnatural or modified nucleotides can be easily prepared.
  • Specific examples of siRNA molecules, with the nucleotide sequence of the sense and antisense strand, as well as the TARDBP mRNA target sequence, are shown in Table 1 , above. It is appreciated that one of skill in the art could anneal the antisense (AS) strand to the corresponding sense (S) strand to yield a ds-siRNA molecule. Alternatively, one of skill in the art could derive a ss-siRNA molecule using antisense strand only.
  • siRNA small interfering RNA
  • modifications to the siRNA may further optimize the molecule’s efficacy or biophysical properties (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and/or targeting to a particular location or cell type).
  • Such optimization could be achieved by systematically either adding or removing linked nucleosides to generate longer or shorter sequences.
  • Further siRNA optimization could include the incorporation of, for example, one or more alternative nucleosides, alternative 2’ sugar moieties, and/or alternative internucleoside linkages.
  • such optimized siRNA molecules may include the introduction of hydrophobic and/or stabilizing moieties at the 5’ and/or 3’ ends.
  • the siRNA molecules may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further optimization of the siRNA molecules of the disclosure may include nucleobases disclosed in US 3,687,808; Kroschwitz, J. I., ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.
  • optimization of the siRNA molecules of the disclosure may include one or more of the following 2’ sugar modifications: 2’-O-methyl (2’-O-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and/or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'- O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2.
  • 2’-O-methyl (2’-O-Me 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2
  • RNA molecules of the disclosure include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • the 2'-arabino modification is 2'-F.
  • Similar modifications may also be made at other positions on the siRNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • optimization of the siRNA molecules of the disclosure may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
  • siRNA Optimization with Hydrophobic Moieties siRNA Optimization with Hydrophobic Moieties
  • optimization of the siRNA molecules of the disclosure may include hydrophobic moieties covalently attached to the 5’ end or the 3’ end.
  • hydrophobic moieties suitable for use with the siRNA molecules of the disclosure may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid or any combination of the aforementioned hydrophobic moieties with PC.
  • optimization of the siRNA molecules of the disclosure may include a 5’-phosphorous stabilizing moiety that protects the siRNA molecules from degradation.
  • a 5'-phosphorus stabilizing moiety replaces the 5'-phosphate to prevent hydrolysis of the phosphate. Hydrolysis of the 5'- phosphate prevents binding to RISC, a necessary step in gene silencing. Any replacement for phosphate that does not impede binding to RISC is contemplated in this disclosure. In some embodiments, the replacement for the 5'-phosphate is also stable to in vivo hydrolysis.
  • Each siRNA strand may independently and optionally employ any suitable 5'-phosphorus stabilizing moiety.
  • Nonlimiting examples of 5’ stabilizing moieties suitable for use with the siRNA molecules of the disclosure may include those demonstrated by Formulas IX-XVI above.
  • optimization of the siRNA molecules of the disclosure may include the incorporation of branching patterns, such as, for example, di-branched, tri-branched, ortetra-branched siRNAs connected by way of a linker.
  • branching patterns such as, for example, di-branched, tri-branched, ortetra-branched siRNAs connected by way of a linker.
  • Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 separate RNA single- or double-strands.
  • the branch points on the linker may stem from the same atom, or separate atoms along the linker.
  • the siRNA composition of the disclosure may be optimized to be in the form of: di-branched siRNA molecules, as represented by any one of Formulas XVII-XIX; tri-branched siRNA molecules, as represented by any one of Formulas XX-XXIII; and/or tetra-branched siRNA molecules, as represented by any one of Formulas XXIV-XXVIII, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety (e.g., phosphoroamidite, tosylated solketal, 1 ,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US 10,478,503).
  • a branch point moiety e.g., phosphoroamidite, tosylated solketal, 1 ,3-diaminopropanol, pentaerythritol
  • the siRNA molecules in the present disclosure may be formulated into a pharmaceutical composition for administration to a subject in a biologically compatible form suitable for administration in vivo.
  • the siRNA molecules of the disclosure may be administered in a suitable diluent, carrier, or excipient, and may further contain a preservative, e.g., to prevent the growth of microorganisms.
  • a suitable diluent, carrier, or excipient may further contain a preservative, e.g., to prevent the growth of microorganisms.
  • Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 nd ed. and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).
  • the method of the disclosure contemplates any route of administration to the subject that is tolerated by the siRNA compositions of the disclosure.
  • siRNA injections into the CNS include intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, or intracisterna magna injection by catheterization.
  • systemic administration include intravenous, intramuscular, and subcutaneous injection. A physician having ordinary skill in the art can readily determine an effective route of administration.
  • a subject in need of treatment of a neurodegenerative disease such as amyotrophic lateral sclerosis type 3 (ALS) or frontotemporal dementia (FTD) is treated with a dosage of the siRNA molecule or siRNA composition of the disclosure, formulated as a salt, at frequency determined by a practitioner.
  • a dosage of the siRNA molecule or siRNA composition of the disclosure formulated as a salt, at frequency determined by a practitioner.
  • a physician having ordinary skill in the art can readily determine an effective amount of the siRNA molecule for administration to a mammalian subject (e.g., a human) in need thereof.
  • a physician could start prescribing doses of one of the siRNA molecules of the disclosure at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a physician may begin a treatment regimen by administering one of the siRNA molecules of the disclosure at a high dose and subsequently administer progressively lower doses until a minimum dose that produces a therapeutic effect (e.g., a reduction in expression of TARDBP mRNA or suitable biomarker or a reduction of ALS- or FTD- associated phenotypes) is achieved.
  • a suitable daily dose of one of one of the siRNA molecules of the disclosure will be an amount which is the lowest dose effective to produce a therapeutic effect.
  • the ss- or ds-siRNA molecules of the disclosure may be administered by injection, e.g., intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, intravenously, intramuscularly, subcutaneously, or by intra-cisterna magna injection via catheterization.
  • a daily dose of a therapeutic composition of one of the siRNA molecules of the disclosure may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms.
  • siRNA molecules of the disclosure While it is possible for any of the siRNA molecules of the disclosure to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents. Dosage and frequency are determined based on the subject’s height, weight, age, sex, and other disorders.
  • the siRNA molecule(s) of the disclosure is selected by the practitioner for compatibility with the subject.
  • Single- or double-stranded siRNA molecules e.g., non-branched siRNA, di-branched siRNA, tri-branched siRNA, tetra-branched siRNA
  • the siRNA molecule chosen has an antisense strand and may have a sense strand with a sequence and RNA modifications (e.g., natural and non-natural internucleoside linkages, modified sugars, 5'-phosphorus stabilizing moieties, hydrophobic moieties, and/or branching structures) best suited to the patient.
  • the siRNA molecule is delivered by the route best suited the patient (e.g., intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, intravenously, intramuscularly, subcutaneously, or by intra-cisterna magna injection via catheterization) and condition at a rate tolerable to the patient until the subject has reached a maximum tolerated dose, or until symptoms are ameliorated satisfactorily.
  • the route best suited the patient e.g., intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, intravenously, intramuscularly, subcutaneously, or by intra-cisterna magna injection via catheterization

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Abstract

La présente divulgation concerne des molécules d'ARN interférent simple ou double brin (par exemple, ARNsi) qui ciblent un gène de protéine de liaison à l'ADN TAR (TARDBP). Les molécules d'ARN interférent peuvent présenter des profils spécifiques de modifications de nucléosides et de modifications de liaisons internucléosides sous forme de compositions pharmaceutiques les contenant. Les molécules d'ARNsi peuvent être des molécules d'ARNsi ramifiées, telles que des molécules d'ARNsi di-ramifiées, tri-ramifiées ou tétra-ramifiées. Les molécules d'ARNsi divulguées peuvent en outre présenter une fraction de stabilisation phosphore 5' et/ou une fraction hydrophobe. De plus, la divulgation concerne des procédés d'administration de la molécule d'ARNsi de la divulgation au système nerveux central d'un sujet, tel qu'un sujet identifié comme ayant une maladie neurodégénérative (par exemple, la sclérose latérale amyotrophique ou la démence frontotemporale).
PCT/US2023/075425 2022-09-28 2023-09-28 Compositions et procédés pour le traitement de maladies neurodégénératives WO2024073604A2 (fr)

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