[go: up one dir, main page]

WO2024091286A1 - Compositions et procédés pour le traitement d'oligonucléotides antisens (aso) de la maladie de huntington - Google Patents

Compositions et procédés pour le traitement d'oligonucléotides antisens (aso) de la maladie de huntington Download PDF

Info

Publication number
WO2024091286A1
WO2024091286A1 PCT/US2023/017275 US2023017275W WO2024091286A1 WO 2024091286 A1 WO2024091286 A1 WO 2024091286A1 US 2023017275 W US2023017275 W US 2023017275W WO 2024091286 A1 WO2024091286 A1 WO 2024091286A1
Authority
WO
WIPO (PCT)
Prior art keywords
per
aso
composition
sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/017275
Other languages
English (en)
Inventor
Douglas J. Macneil
Brigitte ANGENIEUX
Yacoub HABIB
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ophidion Inc
Original Assignee
Ophidion Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ophidion Inc filed Critical Ophidion Inc
Priority to EP23883269.5A priority Critical patent/EP4608980A1/fr
Publication of WO2024091286A1 publication Critical patent/WO2024091286A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • ASO Anti-sense oligonucleotides
  • knockdown e.g., reduction
  • knockout e.g., substantially complete reduction
  • therapeutic use of ASOs has been limited by poor bioavailability, non-specific targeting, and immunogenic effects.
  • ASO anti-sense oligonucleotide
  • HTT Huntingtin
  • HD Huntington’s disease
  • the ASO compositions are complexed with a carrier to facilitate intravascular administration and delivery to targets across the blood brain barrier (BBB).
  • BBB blood brain barrier
  • One or more embodiments of the present disclosure provide a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and/or mRNA levels in a target, the composition including: an anti-sense oligonucleotide -1- 119239542.1 (ASO) including an ASO sequence selected from SEQ ID NOs:18 to 86 or a variant thereof; and a carrier peptide including a base peptide sequence of SEQ ID NO:1 or a variant thereof.
  • the ASO sequence includes a common sub-sequence selected from SEQ ID NOs:18 to 28.
  • the ASO sequence is a 20-mer selected from SEQ ID NOs:29 to 125, for example SEQ ID NOs:29 to 86. In some embodiments, the ASO sequence is selected from SEQ ID NOs: 87, 97, 99, 100, 125 and 41. [0009] In some embodiments, the ASO sequence includes one or more chemical modifications selected from 2'-O-methylation of A, G, C, and/or U, and substitution of phosphorothioate in a phosphodiester backbone unit. In some embodiments, the ASO sequence is 2'-O-methylated at positions 1-5 and 16-20, and phosphorothioated between positions 1-3, 6-16, and 18-20.
  • the carrier peptide further includes a linker sequence of SEQ ID NO:14 prepended to the base sequence.
  • the carrier peptide is selected from SEQ ID NOs:16 and 17.
  • the ASO is conjugated with the carrier peptide.
  • a ratio of the ASO and the carrier peptide is 1:5 to 1:25.
  • One or more embodiments of the present disclosure provide a method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and/or mRNA levels in a target, the method including: administering a composition including an ASO sequence selected from SEQ ID NOs:18 to 86.
  • the ASO sequence includes a common sub-sequence selected from SEQ ID NOs:18 to 28. [0013] In some embodiments, the ASO sequence includes a 20-mer selected from SEQ ID NOs:29 to 125, for example SEQ ID NOs:29 to 86. In some embodiments, the ASO sequence is selected from SEQ ID NOs: 87, 97, 99, 100, 125 and 41. [0014] In some embodiments, the ASO sequence includes one or more chemical modifications selected from 2'-O-methylation of A, G, and/or U; and substitution of phosphorothioate in a phosphodiester backbone unit.
  • the ASO sequence is 2'-O-methylated at positions 1-5 and 16-20, and phosphorothioated between positions 1-3, 6-16, and 18-20.
  • One or more embodiments of the present disclosure provide a method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and/or HTT mRNA levels in a target, the method including: administering the composition of any one of claims 1-6 to the target. -2- 119239542.1
  • the composition is prepared by mixing the ASO or the variant thereof and the carrier peptide or the variant thereof in a ratio of 1:5 to 1:25 in a solution that is acceptable for parenteral administration.
  • the solution is a saline solution including 0.1 wt% to 1 wt% PEG 8000. In some embodiments, the solution is a dextrose solution including 0.01 wt% to 1.5 wt% Polysorbate 80.
  • the administering of the composition occurs in a human subject.
  • the target is a cell, and the cell is selected from the group consisting of neurons, neuronal cells, brain cells, glial cells, astrocytes, neuronal supporting cells, and cells of the central nervous system (CNS).
  • the method further includes conjugating the ASO to the carrier peptide, prior to administering the composition.
  • the carrier peptide further includes a linker sequence of SEQ ID NO:14 prepended to the base sequence, and the ASO is conjugated with the carrier peptide via the linker sequence.
  • the HTT mRNA levels are decreased by greater than 20%, for example, greater than 25%, or 20% to 90%.
  • One or more embodiments of the present disclosure provide a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and/or mRNA levels in a target, the composition including: a 16 to 27-mer anti-sense oligonucleotide (ASO) including one of SEQ ID NOs:18 to 28; and a carrier peptide including a base peptide sequence of SEQ ID NO:1 or a variant thereof.
  • the 16 to 27-mer ASO is complementary to a HTT mRNA sequence.
  • the ASO is an 18 to 24-mer.
  • the ASO is a 20-mer.
  • the ASO sequence includes one or more chemical modifications selected from 2'-O-methylation of A, G, C, and/or U, and substitution of phosphorothioate in a phosphodiester backbone unit.
  • the carrier peptide further includes a linker sequence of SEQ ID NO:14, prepended to the base sequence.
  • the carrier peptide is selected from SEQ ID NOs:16 and 17.
  • the ASO is conjugated with the carrier peptide via the linker sequence.
  • Figure 1 is a flowchart diagram summarizing the in silico procedure used to design and select ASO sequences for Huntingtin (HTT) knockdown.
  • Figure 2 is a bar chart summarizing the change in measured mRNA levels in SHSY5Y cells treated with Peptide 1 (SEQ ID NO:16):ASO or Peptide 4 (SEQ ID -3- 119239542.1 NO:17):ASO conjugates (ASOs 1 to 5, SEQ ID NOS: 87, 97, 99, 100, and 125), as normalized against the change in HTT1 mRNA in a DMEMF12 growth media-only control.
  • Figure 3 is a bar chart summarizing the fold-change in measured mRNA levels in SHSY5Y cells treated with Peptide 1 (SEQ ID NO:16):ASO conjugates (ASOs 6, 3 and 2, SEQ ID NOs: 41, 99 and 97), as normalized against synuclein alpha (SNCA) mRNA levels and relative to a dextrose-0.6% only control.
  • Peptide 1 SEQ ID NO:16
  • ASO conjugates ASOs 6, 3 and 2, SEQ ID NOs: 41, 99 and 97
  • Figure 4 is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the hippocampus (HP) and striatum (ST) of wild-type C57bl6 mice treated with Peptide 4 (SEQ ID NO:17):ASO3 (SEQ ID NO:99) complexes, as normalized against synuclein alpha (SNCA) mRNA levels and relative to a saline control.
  • Figure 5A is a graph of Open Field Total Rearing showing total rearing bouts in the three groups of mice studied in Example 4. N values represent the number of animals at the time of open field assessment. Statistics showing the total rearing bouts for the three groups of mice studied in Example 4.
  • Figure 5B is a graph of Open Field Total Distance Traveled, showing the total distance for the three groups of mice studied in Example 4. N values represent the number of animals at the time of open field assessment. Statistics showing the total distance traveled for the three groups of mice studied in Example 4.
  • Figure 6A is a graph comparing the soluble expanded mHTT levels in the cortex (CTX) according to Assay 6 (2B7/MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.
  • Figure 6B is a graph comparing the aggregated mHTT levels in the cortex (CTX) according to Assay 45 (MW8/4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.
  • Figure 6C is a graph comparing the expanded mHTT levels in the striatum (ST) according to Assay 6 (2B7/MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.
  • Figure 6D is a graph comparing the aggregated mHTT levels in the striatum (ST) according to Assay 45 (MW8/4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.
  • Figure 7A is a graph of Open Field Total Rearing showing total rearing bouts in the three groups of mice studied in Example 5. N values represent the number of animals at the time of open field assessment. Statistics showing the total rearing bouts for the three groups of mice studied in Example 5.
  • Figure 7B is a graph of Open Field Statistics Total Distance Traveled showing total distance traveled in the three groups of mice studied in Example 5. N values represent the number of animals at the time of open field assessment. Statistics showing the total distance traveled for the three groups of mice studied in Example 5.
  • Figure 8A is a graph comparing the soluble expanded mHTT levels in the cortex (CTX) according to Assay 6 (2B7/MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 5.
  • Figure 8B is a graph comparing the aggregated mHTT levels in the cortex (CTX) according to Assay 45 (MW8/4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 5.
  • Figure 8C is a graph comparing the soluble expanded mHTT levels in the striatum (ST) according to Assay 6 (2B7/MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 5.
  • Figure 8D is a graph comparing the aggregated mHTT levels in the striatum (ST) according to Assay 45 (MW8/4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 5.
  • Figure 9 is a side-by-side comparison of the Total Rearing Frequency graph of Figure 5A and Total Distance Traveled graph of Figure 5B with the Total Rearing Frequency graph of Figure 7A and the Total Distance Traveled graph of Figure 7A, providing a comparison of these functional measures between the 25 ⁇ g dose of Example 4 and the 2.5 ⁇ g dose of Example 5.
  • DETAILED DESCRIPTION Selected definitions [0041] Abbreviations for nucleic acids and associated structures are used throughout this disclosure and follow the standard IUPAC nomenclature known in the art.
  • Adenine is Ade or A
  • Cytosine is Cyt or C
  • Guanine is Gua or G
  • Thymine is Thy or T
  • Uracil is Ura or U.
  • Alanine is Ala or A; Arginine is Arg or R; Asparagine is Asn or N; Aspartic Acid is Asp or D; Cysteine is Cys or C; Glutamic acid is Glu or E; Glutamine is Gln or Q; Glycine is Gly or G; Histidine is His or H; Isoleucine is Ile or I; Leucine is Leu or L; Lysine is Lys or K; Methionine is Met or M; Phenylalanine is Phe or F; Proline is Pro or P; Serine is Ser or S; Threonine is Thr or T; Tryptophan is Trp or W; Tyrosine is Tyr or Y; and Valine is Val or V.
  • carrier peptides may refer to various peptides, including those described by SEQ ID NOS:16 and 17, combinations of one of SEQ ID NOS:1 to 7 and 9 to 13 with one of SEQ ID NOS:14 and 15, and variants thereof, that can be used as targeting or “carrier” molecules to facilitate delivery of an active molecule or effector agent to a target in the brain.
  • delivery of an effector agent to a target may be limited when the effector agent is administered alone, but may be increased in the presence of the carrier peptide, for example when the effector agent is conjugated or complexed with the carrier peptide.
  • effector agent refers to any molecule that imparts an effect on a target inside the blood brain barrier (BBB), for example, within tissues of the central nervous system (CNS) or brain or spinal column.
  • BBB blood brain barrier
  • the effector agent may include an ASO sequence, including those ASO sequences described herein and variants thereof.
  • the effector agent may further include, for example: short hairpin or stem loop RNA (shRNA); microRNA, double stranded RNA (dsRNA); strand template RNA (stRNA); oligonucleotides (DNA or RNA); modified oligonucleotides (DNA or RNA); aptamers; analogs and combinations of DNA and RNA; antisense oligomers (ASO); triplex-forming oligonucleotides (TFO); genes; peptides including antibody and antigen fragments; proteins, including antibodies and antigens; small chemical molecules; large chemical molecules; viral particles; liposomes; endosomes; exosomes; nanoparticles; dendrimers such as poly(amidoamine) (PAMAM); positron emission tomography (PET) ligands; eukaryotic cells; prokaryotic cells; microspheres; nanogels; and/or bionanocapsules.
  • shRNA short hairpin or stem loop RNA
  • the effector agent can be conjugated or linked to a carrier, such as a carrier peptide or carrier peptide variant.
  • a carrier such as a carrier peptide or carrier peptide variant.
  • ASO antisense oligonucleotide
  • antisense therapeutic are used interchangeably to refer to a non-coding, single stranded oligonucleotide sequence, typically 16-27 (or e.g., 18-22) nucleotides in length.
  • the ASO sequence is selected to be complementary to and capable of binding or hybridizing with a target mRNA sequence.
  • the hybridization of the ASO with the mRNA may trigger cleavage of the mRNA via one or more cellular mechanisms.
  • an ASO based on single stranded DNA ssDNA
  • ssRNA single stranded RNA
  • RISC RNA-induced silencing complex
  • references to ASOs and/or ASO variants will be understood to encompass DNA-based as well as RNA-based counterparts or equivalents, unless otherwise clear from the context. Further, the terms explicitly include ASO molecules that have been chemically modified as described below, for example, to increase their stability and/or bioavailability, and/or to decrease their immunostimulatory effects.
  • triplex-forming oligonucleotides and “triplex-forming oligonucleotide therapeutics” refer to a non-coding, single stranded oligonucleotide sequence, typically 10-30 nucleotides in length, or a mimic thereof, such as peptide nucleic acids (PNAs) and/or polyamides.
  • PNAs peptide nucleic acids
  • the oligonucleotide sequence or TFO is selected to be capable of binding within the major groove of a target genomic DNA sequence to thereby form a triple helix (triplex) structure.
  • TFOs When binding of the TFO outcompetes binding of transcription factors or polymerases to the DNA (e.g., within regulatory sequences or coding sequences, respectively), gene transcription can be controlled or blocked. Additional information about TFOs is disclosed in e.g., Ohkubo et al., “Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs,” Nucl. Acids Res., 2015, 43(12),5675-5686, and Jain et al, “DNA Triple Helices: biological consequences and therapeutic potential,” Biochimie.2008, 90(8), 1117-1130, the entire content of each of which is incorporated herein by reference.
  • a "fragment" of a polynucleotide or polypeptide refers to a smaller set (e.g., a contiguous sub-set) of nucleotides or peptides with respect to the referenced polynucleotide or polypeptide.
  • the length of the fragment may range from two to N-1 nucleotides or peptides (where N is the number of residues in the referenced parent), unless further specified.
  • a fragment of a carrier peptide (described below) may refer to a peptide including any sub-sequence or truncation of the reference carrier peptide.
  • the fragment may retain at least about 80%, 85%, 90%, 95%, or 99% of the same biochemical functions as the reference.
  • the peptide fragment may be identical to the corresponding sub-sequence in the reference carrier peptide, or may further include additional amino acid mutations or chemical derivatizations (e.g., may also be a mutant or a chemical derivative), as described herein.
  • a “mutant” of a peptide refers to a polypeptide that includes one or more point mutations (amino acid mutations, e.g., residue additions, substitutions, and/or deletions) compared to a reference polypeptide, but still achieves similar biological functions.
  • a "mutant " of a polypeptide or carrier peptide may have a sequence and/or structural homology of at least about 80%, 85%, 90%, 95%, or 99% to the reference polypeptide; and may retain at least about 80%, 85%, 90%, 95%, or 99% of the same biochemical functions as the reference.
  • amino acid mutations refers to one or more residue additions, substitutions, and/or deletions with respect to a reference polypeptide.
  • a "chemical derivative" of a peptide is a modification of a referenced peptide to include a chemical modification at one or more positions, for example, to improve stability, mediate intermolecular interactions, facilitate visual localization, etc.
  • the modification may include replacement of standard amino acids with non-standard residues and/or addition of new functional groups (e.g., methylation at a hydroxyl group).
  • Non-limiting examples of chemical modifications of peptides include the products of techniques such as ubiquitination, methylation, fluorination, fluorescent -7- 119239542.1 labeling, phosphorescent labeling, and PEGylation (e.g., derivatization with ubiquitin, methyl, fluorine, a fluorescent dye, a phosphorescent dye, and polyethylene glycol, respectively).
  • the point(s) of modification e.g., position or starting functional group
  • chemical derivatizations refers to one or more of the above-described chemical modifications.
  • a "chemical derivative" of a polynucleotide is a modification of a referenced polynucleotide to include a chemical modification at one or more positions, for example, to improve stability, mediate intermolecular interactions, facilitate visual localization, etc.
  • the modification may include replacement of reference functional groups (e.g., replacement of a phosphodiester with a phosphorothioate) and/or addition of new functional groups (e.g., methylation at a hydroxyl group).
  • the point(s) of modification is (are) not particularly limited, and for example, may include the phosphates and ribose hydroxides of the backbone, and the aromatic rings of the nucleobases.
  • Examples of such chemical modifications include moieties added to the 2’ position of one or more ribose (sugar) units; substitution or modification of internucleotide phosphodiesters in the backbone with phosphorothioates, phosphorodithioates, triazoles, amides, boranophosphates, etc.; nucleobase modifications to incorporate fluorescent groups or replace standard bases with difluorotoluene, dichlorobenzene, halogenated variants, etc.; and modification of terminus or conjugate groups with aromatic compounds, PEG, triphosphate groups, etc. Selected example modifications are disclosed in Selvam et al., “Therapeutic potential of chemically modified siRNA: Recent trends,” Chem. Biol. Drug.
  • a molecule may be described as being a “functional equivalent” of another molecule if both molecules possess a similar biological activity (e.g., fulfill at least about 80%, 85%, 90%, 95%, or 99% of the same biological or biochemical role(s)), and do so at similar levels (e.g., the functional equivalent may possess at least about 80%, 85%, 90%, 95%, or 99% of the biochemical activity of the reference, and in some embodiments greater than 100% of the biochemical activity of the reference).
  • a similar biological activity e.g., fulfill at least about 80%, 85%, 90%, 95%, or 99% of the same biological or biochemical role(s)
  • the functional equivalent may possess at least about 80%, 85%, 90%, 95%, or 99% of the biochemical activity of the reference, and in some embodiments greater than 100% of the biochemical activity of the reference.
  • the molecule may possess a similar activity as quantified above and be considered a functional equivalent as that term is used herein, even if, for example, the sequence of residues (e.g., primary structure) is not identical, or different chemical modifications are made to a shared basic sequence.
  • a functional equivalent may be a fragment, mutant, or chemical derivative (as those terms are defined herein) of a reference, and may therefore possess certain structural similarities with respect to the reference.
  • fragments, mutants, chemical derivatives, and functional equivalents (e.g., as defined above) of a carrier peptide may be collectively referred to as -8- 119239542.1 “carrier peptide variants,” “carrier-derived peptides,” or “Peptide n-derived peptides,” where “n” is any integer and is used to refer to a disclosed peptide having a specified sequence.
  • ASO variants or “ASO-derived sequences.”
  • References to a sequence identification number (SEQ ID NO) will be understood as disclosing and encompassing variants thereof, for example, variants having 80% to 99% sequence identity (e.g., homology or similarity) when compared to the named sequence, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity.
  • conjugate refers to the attachment of two or more entities to form one entity (molecule).
  • embodiments of the present disclosure provide for a composition including an ASO effector agent conjugated with a carrier peptide.
  • the attachment can be by means of chemical modification, protein fusion, covalent bonds, or non-covalent bonds (e.g., ionic or intramolecular attractions), and in some embodiments may include the use of intermediary linkers, including peptide linkers, chemical (e.g., functional group or small molecule) linkers, or any means known to one skilled in the art.
  • the joining can be permanent or reversible.
  • linkers can be enzymatically cleavable, acid cleavable, photocleavable, and/or heat sensitive (e.g., thermally cleavable). In some embodiments, several linkers can be included according to the desired properties of each linker and each component in the conjugate. Flexible linkers and linkers that increase the solubility of the conjugates are contemplated for use alone, or in combination with other linkers disclosed herein. Methods for conjugation are well known by persons skilled in the art. For example, peptide linkers can be attached and deployed by prepending and/or appending a DNA sequence encoding the linker to one or more proteins in the conjugate (e.g., as a recombinant protein).
  • target refers to any cell that is intended to receive a carrier peptide and/or an effector agent.
  • the target may be entirely within the central nervous system (CNS) tissue, which is isolated from the intervascular system by the BBB. In other embodiments, the target may be or include cells located in tissues outside the BBB.
  • CNS central nervous system
  • target cells as used herein may also refer to cells expressing the alpha ( ⁇ ) subunit and/or the beta ( ⁇ ) subunit of the nicotinic acetylcholine receptor (nAChR).
  • Carrier peptides that bind to the ⁇ -subunit of the nicotinic acetylcholine receptor can thus be used to selectively bind target cells expressing the ⁇ subunit of the nicotinic acetylcholine receptor.
  • Examples of cells that express the ⁇ subunit of the nicotinic acetylcholine receptor include, for example, the neurons, glial cells, and endothelial cells comprising the BBB.
  • Target cells of the present disclosure also include cells whose endogenous milieu is separated by the BBB, for example, cells in the CNS, including brain cells, spinal cord cells, glial cells, and other cells supporting neurons, e.g., astrocytes or "nursing cells".
  • the target cells may include any cell expressing the alpha subunit of the nicotinic acetylcholine receptor or a homologue -9- 119239542.1 thereof, such as but not limited to neuronal cells in a subject (i.e. in vivo), neuronal cells ex vivo, or cultured neuronal cells (i.e.
  • the target cells are neuronal precursor or neuronal progenitor cells, such as neuronal progenitor stem cells that express an alpha subunit of the nicotinic acetylcholine receptor or a homologue thereof.
  • the target is present within a subject, for example a mammalian subject, such as a human subject or patient.
  • the target is ex vivo, and in further embodiments, the target is in a biological sample, for example in vitro.
  • the effector agent can be transported to various target cells or tissues.
  • the effector agent can be transported to any nerve cell, e.g., a nerve cell in the central nervous system, olfactory, or visual system.
  • the effector agent can also be transported to a neurologically related target cell or tissue, e.g., cells or tissues that interact with or are targets of the nervous system.
  • All nucleotides and peptides disclosed herein, including ASO sequences and carrier peptides may be constructed by using any suitable method available in the art.
  • peptides may be synthesized using a peptide synthesizer (Applied Biosystems Model 433) or can be synthesized recombinantly by methods well known in the art, such as those described in Merrifield, "Solid Phase Synthesis," J. Am. Chem. Soc., 1963, 83:2149-2154.
  • Nucleotides may be synthesized using an oligonucleotide sequencer or by using in vitro transcription, PCR cassette, or expression vector methods well known in the art. The method by which a peptide or nucleotide of the present disclosure is synthesized does not limit the present disclosure.
  • an effector agent or therapeutic agent for example an ASO as disclosed herein, can be prepared to be delivered in a "prodrug” form.
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide, and may be inclusive of all coding segment sequences (exons) as well as (optionally) non-coding intervening sequences (introns).
  • gene may refer to the coding sequence of a gene product, or the coding sequence together with the non-coding regions of the gene product, including the 5'UTR and 3'UTR regions, introns, and promoters or enhancers of the gene product.
  • gene product(s) may encompass the RNA (e.g., mRNA) transcribed from a gene, and/or the polypeptide (e.g., protein) produced by translation of the RNA.
  • promoter is a region of a genomic nucleic acid sequence at which initiation and rate of transcription are controlled.
  • RNA polymerase and other transcription factors can contain elements at which regulatory proteins and -10- 119239542.1 molecules can bind (such as RNA polymerase and other transcription factors) to initiate the transcription of a specific nucleic acid sequence.
  • the term "enhancer” refers to a cis- acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence, e.g., to increase transcription of a particular gene product.
  • An enhancer can function in either orientation and can be included upstream or downstream of the promoter.
  • some of the above definitions generally refer to a single-stranded molecule (oligonucleotides), in some embodiments and contexts the terms and usage may also encompass one or more additional strands that are partially, substantially, or fully complementary to the single-stranded molecule.
  • a nucleic acid can encompass a double-stranded molecule or a double- or multi-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence.
  • a single stranded nucleic acid can be denoted by the prefix "ss”, a double stranded nucleic acid by the prefix "ds”, and a triple stranded nucleic acid by the prefix "ts.”
  • compositionss compositions administered for therapeutic, diagnostic, or prophylactic purposes, and which usually comprise one or more excipients, such as a pharmaceutically acceptable carrier (e.g., solvent or diluent) in the art that is suitable for administration to mammals, and for example, humans or human cells.
  • a pharmaceutically acceptable carrier e.g., solvent or diluent
  • Cells that are administered a composition as disclosed herein can be part of a living subject, animal, or human, including a transgenic animal for research purposes.
  • the cells can also be cultured, for example as cells as part of an assay for screening potential pharmaceutical compositions.
  • the compositions also can include stabilizers, preservatives, lubricants, and other adjuvants and excipients.
  • stabilizers and adjuvants see Genarro, AR., Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed., the entire content of which is incorporated herein by reference. Those having ordinary skill in the art are capable of choosing suitable formulations depending on the administration route, as described herein.
  • pharmaceutically acceptable carrier refers to any pharmaceutically acceptable means to mix and/or deliver an effector agent or pharmaceutical composition to a subject.
  • the term may refer to any pharmaceutically acceptable material, formulation, or vehicle (such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material) involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • the carrier is "acceptable” in the sense of being compatible with the other ingredients of the formulation as well as being compatible with administration to a subject, for example a human.
  • the carrier can be in the form of a solid, semi-solid, or liquid diluent, cream, or capsule.
  • compositions disclosed herein can be administered by any convenient route, including sublingual, parenteral, enteral, mucosal, topical, subcutaneous, intravascular, intravenous, intraarterial, intramuscular, intraperitoneal, transdermal, rectal, vaginal, intranasal, intraocular, intraspinal, or intracerebroventricular. In some embodiments, the compositions as disclosed herein are not topically administered.
  • the delivery is by intranasal administration of the composition, especially for use in therapy of the brain and related organs (e.g., meninges and spinal cord). Along these lines, intraocular administration is also possible.
  • the delivery means is by intravenous (IV) administration of the composition, which is especially advantageous when a longer-lasting IV formulation is desired. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), the entire content of each of which is incorporated herein by reference.
  • parenteral administration and “administered parenterally” as used herein refer to modes of administration other than enteral and topical administration.
  • the term refers to injection, but may include, without limitation, intravenous, intraarterial, intramuscular, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.
  • compositions as disclosed herein can be administered in prophylactically or therapeutically effective amounts.
  • a “prophylactically or therapeutically effective amount” refers to an amount necessary to at least partially attain a desired effect, e.g., to delay the onset of, inhibit the progression of, or halt altogether the onset or progression of the particular disease or disorder being treated (e.g., Huntington’s disease).
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, and individual patient parameters including age, physical condition, size, weight, co-morbidities, and concurrent medications or treatment protocols. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose can be administered for medical reasons, psychological reasons, or for virtually any other reason.
  • the compositions disclosed herein may be administered to a patient or human subject in an amount of 0.1 mg per kg to 100 mg per kg (mpk) of subject body weight, for example, 0.1 mpk to 50 mpk, 0.2 mph to 20 mpk, 0.3 mpk to 10 mpk, or 0.5 mpk to 5 mpk.
  • the compositions may be administered using any suitable dose volume, for example, a dose volume of 100 ⁇ L to 1000 mL, 200 ⁇ L to 750 mL, 500 ⁇ L to 500 mL, 1 mL to 200 mL, or 5 mL to 100 mL.
  • the compositions may be administered in an amount of 0.1 to 10 mpk and in a dose volume of 100 ⁇ L to 5 mL.
  • the compositions disclosed herein may be particularly effective in lower doses. Without being bound by any particular theory, and without limiting the scope of the present disclosure, it is believed that since the compositions and methods disclosed herein rely on receptors, high doses of the effector agents (e.g., the anti-sense oligonucleotides) in the compositions according to embodiments of the present disclosure may – in some instances – lead to receptor down regulation and limited efficacy.
  • the effector agents e.g., the anti-sense oligonucleotides
  • the conjugates, complexes and compositions according to the present disclosure can enable more effective treatment in lower doses of the effector agent (e.g., the anti-sense oligonucleotides), i.e., doses of the effector agent lower than 1,000 ⁇ g per kg (body weight), 600 ⁇ g per kg (body weight) or lower, lower than 4,000 ⁇ g per kg (body weight), or lower than 100 ⁇ g per kg (body weight).
  • the effector agent e.g., the anti-sense oligonucleotides
  • the compositions disclosed herein may be administered to an animal subject (e.g., a mouse) in an amount sufficient to deliver an amount of the effector agent (e.g., anti-sense oligonucleotide) of 1 ⁇ g per kg to 4,000 ⁇ g per kg (or 0.001 mg per kg to 4 mg per kg) of subject body weight, for example, 1 ⁇ g per kg to 3,000 ⁇ g per kg, 1 ⁇ g per kg to 2,000 ⁇ g per kg, 1 ⁇ g per kg to 1,000 ⁇ g per kg, 1 ⁇ g per kg to 600 ⁇ g per kg, 2 ⁇ g per kg to 4,000 ⁇ g per kg, 2 ⁇ g per kg to 3,000 ⁇ g per kg, 2 ⁇ g per kg to 2,000 ⁇ g per kg, 2 ⁇ g per kg to 1,000 ⁇ g per kg, 2 ⁇ g per kg to 600 ⁇ g per kg, 10 ⁇ g per kg to 4,000 ⁇ g per kg, 10 ⁇ g per kg to 4,000 ⁇ g per kg, 10
  • compositions disclosed herein may be administered to an animal subject (e.g., a mouse) in an amount sufficient to deliver an amount of the effector agent (e.g., anti-sense oligonucleotide) of 1 ⁇ g per kg, 2 ⁇ g per kg, 10 ⁇ g per kg, 20 ⁇ g per kg, 40 ⁇ g per kg, 100 ⁇ g per kg, 200 ⁇ g per kg, 350 ⁇ g per kg, 600 ⁇ g per kg, 1,000 ⁇ g per kg, or 2000 ⁇ g per kg.
  • the effector agent e.g., anti-sense oligonucleotide
  • any and all ranges between the end points and specific amounts described above are also within this disclosure, including any and all sub-ranges subsumed in the ranges above, and other ranges that may span between or across the disclosed ranges, sub-ranges or specific amounts.
  • Those of ordinary skill in the art would be capable of discerning the human equivalent dose from the animal (e.g., mouse) doses described above.
  • the same doses (per kg of body weight) described above may be used in human patients when scaling by body weight, or doses 1.7-fold higher may be used in human patients when scaling by brain weight.
  • the compositions disclosed herein may be administered to a human subject or patient in an amount sufficient to deliver an amount of effector agent (e.g., anti-sense oligonucleotide) of 1.7 ⁇ g per kg to 6,800 ⁇ g per kg of subject body weight, for example, 1.7 ⁇ g per kg to 5,100 ⁇ g per kg, 1.7 ⁇ g per kg to 3,400 ⁇ g per kg, 1.7 ⁇ g per kg to 1,700 ⁇ g per kg, 1.7 ⁇ g per kg to 1,000 ⁇ g per kg, 3.4 ⁇ g per kg to 6,800 ⁇ g per kg, 3.4 ⁇ g per kg to 5,100 ⁇ g per kg, 3.4 ⁇ g per kg to 3,400 ⁇ g per kg, 3.4 ⁇ g per kg to 1,700 ⁇ g per kg, 3.4 ⁇ g per kg to 1,000 ⁇ g per kg, 17 ⁇ g per kg to 6,800 ⁇ g per kg, 17 ⁇ g per kg to 5,100 ⁇ g per kg per
  • the compositions disclosed herein when scaling by brain weight, may be administered to a human subject or patient in an amount sufficient to deliver an amount of the effector agent (e.g., anti-sense oligonucleotide) of 1.7 ⁇ g per kg, 3.4 ⁇ g per kg, 17 ⁇ g per kg, 34 ⁇ g per kg, 68 ⁇ g per kg, 85 ⁇ g per kg, 170 ⁇ g per kg, 340 ⁇ g per kg, 595 ⁇ g per kg, 1,700 ⁇ g per kg, 1,000 ⁇ g per kg, or 3,400 ⁇ g per kg.
  • the effector agent e.g., anti-sense oligonucleotide
  • the amount of the conjugate or complex (or composition) including the carrier peptide and the effector agent (e.g., anti-sense oligonucleotide) needed to yield these ranges of the dose of the effector agent (e.g., anti-sense oligonucleotide) will vary depending on the ratio (or relative amounts) of the carrier peptide to the effector agent (e.g., anti-sense oligonucleotide).
  • conjugates or complexes having ratios of effector agent (e.g., anti-sense oligonucleotide) to carrier peptide of 1:4 and 1:12, respectively, would require different total amounts of the composition as a whole to achieve the same effector agent dose.
  • effector agent e.g., anti-sense oligonucleotide
  • carrier peptide 1:4 and 1:12
  • administering may refer to the placement of the pharmaceutical composition including a described effector agent or ASO composition into an in vivo subject or in vitro culture by a method or route that results in at least partial localization of the agents at a desired site (e.g., target cells and/or molecules).
  • the agents of the present disclosure can be administered by any appropriate route that results in an effective treatment in the subject.
  • ASO compositions [0072] Huntingtin (HTT) is a protein encoded by the HTT gene in humans (NCBI Ref. NP_002102) and mice (NP_034544).
  • HTT is known to be highly expressed in neuronal cells and involved in axonal transport. Further, HTT is essential for normal, successful embryonic development.
  • the 5’ end of a normal (wild-type) HTT gene typically includes 5 to 35 CAG repeats (SEQ ID NO: 148).
  • mutations in the HTT gene that result in 36 or more CAG repeats e.g., resulting in transcription of 36 or more N-terminus Glu residues
  • a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) levels includes an ASO as an effector agent, and a carrier peptide for delivery to a target. Each component is described in more detail below.
  • the ASO may be a ssDNA sequence. Hybridization of the ssDNA to the HTT mRNA forms a DNA-RNA heteroduplex, which may then trigger cleavage of the mRNA by RNA H enzymes (including RNA H1 and RNA H2).
  • the ASO may be a ssRNA sequence. Hybridization of the ssRNA to the HTT mRNA may then trigger cleavage of the mRNA by the RNA-Induced Silencing Complex (RISC).
  • RISC RNA-Induced Silencing Complex
  • embodiments of the present disclosure are not limited thereto, and may include other mechanisms of transcriptional control. It will be understood that when an embodiment is described as including an ASO with a ssDNA sequence (for example, as described in a sequence listing), an alternative embodiment including an ASO with the corresponding ssRNA sequence (i.e., with uracil residues in place of the thymine residues) is also disclosed and within the scope of the present disclosure.
  • the ASO may include a combination or mixture of DNA and RNA and modified bases, e.g., as a result of being chemically modified according to the modifications described above.
  • the ASO may be a single-stranded polynucleotide including a mixture of modified RNA and DNA bases, which may facilitate improved stability and in vivo half-life.
  • the ASO may include a sequence in which a sequence of DNA bases is sandwiched between two shorter sequences of modified RNA bases, e.g., as in a “gapmer” configuration.
  • the ASO may be a “gapmer” ASO including a central DNA oligomer block (“gap”), flanked by two blocks of modified RNA bases (“wings”) at the 5’- and 3’-terminal ends.
  • the modified RNA bases in the wings may be 2’-O modified RNA bases, which is described in more detail below.
  • each wing may include 2 to 7 bases or 3 to 5 bases, and the gap may include 8 to 16 bases or 10 to 12 bases.
  • the ASO may be a 20-mer gapmer having a sequence including 5 modified RNA bases, 10 DNA bases, and 5 modified RNA bases, in that order.
  • embodiments of the present disclosure are not limited thereto.
  • RNA, DNA and modified bases may be used, and that when an embodiment is described as including an ASO with a given polynucleotide sequence, alternative embodiments including ASOs with any analogous sequence of mixed modified RNA and DNA bases are also disclosed and within the scope of the present disclosure.
  • an apparent disclosure of an ssDNA sequence (for example, as described in a sequence listing) will be understood to encompass disclosure of additional embodiments including chemical modifications of that ssDNA sequence in which one or more deoxyribonucleotide(s) is (are) replaced with the corresponding ribonucleotide(s), etc., as in the gapmer configurations.
  • a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) levels in a target includes an ASO and a carrier peptide, each described in more detail below.
  • the ASO included in the ASO compositions may be 16 to 27 nucleobases in length (e.g., may be a 16-mer to 27-mer).
  • the ASO may be an 18-mer to 22-mer, or a 19-mer, 20-mer, or 21-mer.
  • the ASO may be a 20-mer.
  • Certain HTT mRNA sub-sequences that are as long as or longer than the ASO according to embodiments of the present disclosure may serve as suitable mRNA target regions for ASO-induced transcriptional arrest.
  • target regions may be located within various exons of the HTT mRNA.
  • the target region within the HTT mRNA may be located within exon 2, 8-9, 12-13, 17, 27, 30, 36, 36-37, 42, 42-43, 54, 63, or 67, where the notation “i-j” indicates that the target region spans adjacent portions of the two exons, numbered i and j.
  • the ASO embodiments may be capable of binding to one of the above-listed target regions.
  • multiple ASOs according to embodiments of the present disclosure may be capable of binding within the same target region at different starting base positions.
  • Such ASOs may thus be related to each other by a series of frame shifts along a DNA (or RNA) sequence that is complementary to the HTT mRNA target region.
  • the related ASOs may share a common sub-sequence of between 1 to 18 nucleobases in length, for example, 6 to 16 or 8 to 14 nucleobases in length, where the common sub- sequence is included at a different starting base position (position number) in each member of the group of related ASOs.
  • ASOs may thus include a common sub-sequence represented by one of SEQ ID NOS: 18-28, as listed in Table 1 below, which also lists the starting position and exon of the corresponding sequence in the HTT mRNA. -17- 119239542.1 Table 1 SEQ ID Common sub-sequence HTT mRNA HTT mRNA [0080]
  • the common sub-sequence of the ASOs may be, for example, 11 to 16 nucleobases in length, and may be selected from SEQ ID NOs:18, 21, and 25-28.
  • the common sub-sequence of the ASOs may be, for example, 11 to 13 nucleobases in length, and may be selected from SEQ ID NOs:18, 25, 27, and 28.
  • the ASO may be a 16 to 27-mer, for example, an 18 to 24-mer or a 20-mer including a common sub-sequence selected from SEQ ID NOS: 18- 28.
  • the ASO may be complementary to one HTT mRNA target region regardless of its specific length.
  • the ASO included in the ASO compositions may be designed or selected to have a reduced risk of cross-reactivity (e.g., with non-HTT mRNA) and immunotoxicity.
  • the ASO may meet the following sequence-related criteria: [0083] 1) The ASO is complementary to an HTT mRNA exon sequence; [0084] 2) The ASO is not complementary to any mRNA sequence having one or more nucleotide polymorphisms with an occurrence frequency of >1%; [0085] 3) The ASO is not complementary to the mRNA of any human genes (e.g., any sequences in the target transcriptome) other than HTT.
  • the ASO does not appreciably bind to other mRNA sequences due to the presence of at least 3 mismatches; -18- 119239542.1 [0086] 4)
  • the ASO does not have a sequence that lends itself to formation of a strong secondary structure (such as a loop or pseudoknot); and [0087] 5)
  • the ASO does not include any known toxic motifs (e.g., motifs that trigger a hepatotoxic physiological response), such as tgc, tcc, and gc.
  • the ASO compositions according to embodiments of the present disclosure may exclude ASO sequences that: 1) are not complementary to an HTT mRNA exon sequence; 2) are complementary to any HTT mRNA sequence having one or more nucleotide polymorphisms with an occurrence frequency of greater than 1%; 3) are complementary to the mRNA of any transcriptome sequence other than HTT; 4) has a strong secondary structure; and/or 5) include any of the above-described toxic motifs.
  • the ASO may have a total guanine and cytosine content of 20% to 60% with respect to the total number of bases, for example, 25% to 55%, 30% to 50%, or 35% to 45%.
  • the ASO may be selected from particular 20-mer ASO sequences and variants thereof that meet the preceding combined criteria.
  • the ASO may be selected from the ASO sequences listed in Table 2 below (SEQ ID NOS:29 to 86), which also lists the starting position and exon of the corresponding sequence in the HTT mRNA.
  • the ASO sequences may be selected from the Table 2 sequences and variants thereof, including sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.
  • the ASO sequences may be selected from “gapmer” modified variants of SEQ ID NOS:29 to 86, respectively referred to as SEQ ID NOS:87 to 144, also shown in Table 2.
  • Table 2 SEQ ID ASO Sequence HTT mRNA HTT SEQ Gapmer variant u u u u 119239542.1 37 gacaatgattcacacggtct 436 2 95 gacaatgattcacacggucu 38 agacaatgattcacacggtc 437 2 96 agacaatgattcacacgguc g a c g a uu gu ug uuuu c u g a g ag u g -20- 119239542.1 77 ggtaaatgatggagggggtg 8752 63 135 gguaaatgatggaggggggtg 8752 63 135 gguaaatgatggaggggggtg 87
  • sequences may be alternatively referred to herein by the names in Table 3: Table 3 SEQ SEQ ID NO Gapmer variant u ag c c c O6 (SEQ O: ). SO6 aso as d erent wng mod catons t an t ose s own beow with respect to wing modified SEQ ID NO.99. Specifically, ASO6 has 2-methoxyethoxy modified RNA on the wing RNA instead of the 2-methoxy modified RNA shown below in SEQ ID NO:145.
  • ASO1, ASO2, ASO4 and ASO5 refer to the gapmer variant sequences SEQ ID NOs:87, 97, 100 and 125, while the SEQ ID NOs:29, 39, 42 and 67 are provided to show the variants. -21- 119239542.1 [0092]
  • the modified RNA bases at positions 1-5 and 16-20 in the gapmer variant sequences may be 2'-O-methylated, and the bases at positions 1-3, 6-15, and 18-20 and may be connected via phosphorothioate linkages, as described in more detail in the following section.
  • ASO sequences [0093] It is well known that unmodified ASOs (e.g., ASO sequences consisting only of standard nucleobases) have short half-lives in vivo due to rapid degradation, which limits their utility as therapeutics. Accordingly, in some embodiments, the ASO may be chemically modified at one or more bases to provide improved in vivo stability and therapeutic efficacy. Examples of such chemical modifications include those described above in connection with the term “chemical derivatives,” but are not limited thereto.
  • the ASO or ASO variant may be a 20-mer having a “gapmer” configuration as described above, e.g., so that the first five bases and last five bases of the sequence are RNA or modified RNA bases, and the middle (ten) bases are DNA bases.
  • the ASO or ASO variant includes one or more chemical modifications selected from 2'-O-methylation of A, G, C, T, and/or U; and substitution of a phosphodiester backbone unit with a phosphorothioate unit.
  • the ASO includes one or more instance of both types of chemical modifications.
  • the 20-mer ASO may have a sequence is 2'-O-methylated at positions 1-5 and 16-20, and phosphorothioated between positions 1-3, 6-16, and 18-20.
  • the position number n refers to the nth base within the sequence starting from the 5’-end.
  • the ASO variant may be represented by the following generic sequence showing the positions and types of chemical modifications to each strand: [0097] mN*mN*mN mNmN N*N*N*N*N*N*N*N*N*N*mNmN mNmN*mN* [0098]
  • mN indicates a 2'-O-methyl modified base
  • * indicates a phosphorothioate linkage (e.g., in place of a standard phosphodiester linkage).
  • the underlying or original base sequence may be the same as any ASO sequence according to embodiments of the present disclosure, for example, as represented by any of SEQ ID NOS:29 to 86.
  • a 20-mer ASO may be an ASO variant having a gapmer sequence of 5 RNA bases, 10 DNA bases, and 5 RNA bases, where the sequence is 2'- O-methylated at each of the RNA bases, and phosphorothioated at each of the middle DNA bases and within the 2 inter-base linkages at each end of the sequence.
  • the ASO may be a variant of ASO3 (SEQ ID NO:99) that includes the following set of modifications: [00101] mG*mU*mC mAmG T*T*C*A*T*A*A*C*C*mUmG mGmA*mC* (wing modified SEQ ID NO: 145) DNA and * indicates a phosphorothioate linkage (e.g., in place of a standard phosphodiester linkage).
  • the first two bases are phosphorothiate 2'-O-methyl RNA bases
  • the next 3 bases are phosphorodiester 2'-O-methyl RNA bases
  • the next 10 bases are phosphorothioate DNA bases
  • the next 3 bases are phosphorodiester 2'-O-methyl RNA bases
  • the last two bases are phosphorothiate 2'-O-methyl RNA bases.
  • the ASO may be a variant of any of the ASO sequences of SEQ ID NO:29 to 86, including substantially the same modifications (e.g., with respect to position within the 20-mer sequence) as detailed for SEQ ID NO:67, as represented by corresponding SEQ ID NOS:87 to 144 (e.g., including the described 2'-O-methylation and phosphorothioate modifications at the indicated positions).
  • the ASO may be an ASO variant with fewer phosphodiester linkages.
  • the 4x phosphodiester linkages may, in some instances, reduce the potential for toxicity, but such 4x phosphodiester linkages may, in some instances, result in a metabolically unstable ASO.
  • more stable variants may incorporate 1 to 3 phosphodiester linkages instead of 4 phosphodiester linkages.
  • the ASO may be a variant of any of SEQ ID NOs: 29, 39, 41, and 42 including substantially the same modifications (e.g., with respect to position within the 20-mer sequence) as detailed for SEQ ID NO: 67 and its gapmer variant sequence SEQ ID NO:125 (ASO5) (for example, represented by SEQ ID NOs: 87 (ASO1), 97 (ASO2), 99 (ASO 3), or 100 (ASO4).
  • ASO5 for example, represented by SEQ ID NOs: 87 (ASO1), 97 (ASO2), 99 (ASO 3), or 100 (ASO4).
  • ASO5 for example, represented by SEQ ID NOs: 87 (ASO1), 97 (ASO2), 99 (ASO 3), or 100 (ASO4
  • ASO4 the Example data presented herein were collected using ASO compositions that were chemically modified in this manner.
  • additional embodiments may include ASO fragments, extensions (e.g., longer ASO sequences complementary to the HTT mRNA target regions), and/or functional equivalents having at least about 80%, 85%, 90%, 95%, or 99% sequence and/or structural homology to ASO sequences described herein, as well as chemical derivatives of all of the above.
  • Carrier Peptide -23- 119239542.1 [00107]
  • the composition further includes a carrier peptide (e.g., carrier-derived peptide) to facilitate delivery of the ASO or another effector agent to a suitable target (e.g., a cell or tissue in which HTT is expressed).
  • the carrier peptide may target (e.g., bind to) a receptor included on a particular cell or tissue of interest, and may subsequently facilitate passage of the ASO payload into that cell or tissue.
  • the carrier peptide may include or be formed of two oligomers: a base sequence; and a linker sequence.
  • the linker sequence may facilitate conjugation of the carrier peptide to the ASO or effector agent (e.g., may be the point of contact between the effector agent and the carrier peptide), and the base sequence may facilitate delivery of the conjugate to the specific target.
  • Lynx1 is a protein that binds to neuronal nicotinic receptors (NNRs), also referred to as nicotinic acetylcholine receptors (nAChRs). Detailed information about lynx1 is disclosed in Ibanez-Tallon et al., 2002, Neuron, 33:893-903, the entire content of which is incorporated herein by reference.
  • NNRs neuronal nicotinic receptors
  • nAChRs nicotinic acetylcholine receptors
  • nAChRs are frequently expressed in target cells of the blood brain barrier (BBB), and the loop2 region of lynx1 is the putative binding domain for nAChRs.
  • BBB blood brain barrier
  • lynx1-loop2-derived base sequences e.g., base sequences derived from the loop2 domain of human lynx1, in lieu of the full lynx1 protein
  • Cellular uptake and transport of the effector agent across the BBB via the interaction of lynx1-loop2-derived base sequences with nAChRs can be determined by, for example, cellular imaging or confocal microscopy of relevant tissue sections.
  • Lynx1- loop2-derived peptides are described in more detail in e.g., U.S.8,629,114, issued on January 14, 2014; U.S.9,522,193, issued on December 20, 2016; U.S.9,913,915, issued on March 13, 2018; U.S.10,328,156, issued on June 25, 2019; U.S.10,772,966, issued on September 15, 2020, and U.S.17/020,613, filed on September 14, 2020, the entire content of each of which is incorporated herein by reference.
  • the terms “lynx1-loop2- derived peptide” and “base sequence” may be interchangeably used herein.
  • the composition may include a carrier peptide including a base sequence having the general sequence of SEQ ID NO:1, X1-X2- X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -R-X 12 -K-X 14 -X 15 -X 16 , in which for SEQ ID NO:1: [00112] X 1 is M or T; [00113] X 2 is T or I; -24- 119239542.1 [00114] X3 is T or W; [00115] X4 is R or C; [00116] X5 is T or D; [00117] X 6 is Y, I, or G; [00118] X 7 is F or Y; [00119] X8 is T or C; [00120] X9 is P, N, or S; [00121] X10 is Y, T, or S; [00122] X 12 is
  • the carrier peptide may include a base sequence that is similar to or conserved with a sequence in an analogue lynx1 protein of another (e.g., non-human) species.
  • the base sequence may be represented by any of MTTRTYFTPYRMKVRK (SEQ ID NO:2), MTTRTYYTPTRMKVSK (SEQ ID NO:3), MTWCDYFTPSRGKVRKS (SEQ ID NO:4), or MTTRTYFTPYRGKVRK (SEQ ID NO:5).
  • the base sequence may be selected from SEQ ID NOS:2 to 5 and sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.
  • the carrier peptide may include a lynx1-loop2-derived base sequence with the sequence MTTRTYATPYRMKVRKS (SEQ ID NO:6), which is similar to SEQ ID NO:2 except that the phenylalanine (F) at position 7 is substituted with alanine (A).
  • the carrier peptide may include a lynx1-loop2-derived base sequence with the sequence MTTRTYFTPYAMADRKS (SEQ ID NO:7), which is similar to SEQ ID NO:2 except that the RMKV residues at positions 11-14 are respectively substituted with AMAD.
  • the base sequence may be selected from SEQ ID NOS:6 and 7 and sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc. [00128]
  • additional amino acids may be added (appended, prepended, or inserted) to the lynx1-loop2-derived base sequence.
  • MPENPRPGTP may be added to a suitable position within the 16-mer peptide of SEQ ID NO:1.
  • MPENPRPGTP (SEQ ID NO: 8) is added between residues X3 and X4 of SEQ ID NO:1 as defined above, resulting in X 1 X 2 X 3 MPENPRPGTPX 4 X 5 X 6 X 7 X 8 X 9 X 10 RX 12 KX 14 X 15 X 16 (SEQ ID NO:9).
  • the carrier peptide may include a lynx1-loop2-derived base sequence with the sequence MTTMPENPRPGTPRTYFTPYRMKVRKS (SEQ ID NO:10), or a sequence having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.
  • the carrier peptide may include the lynx1-loop2-derived base sequence of SEQ ID NO:4.
  • the lynx1-loop2-derived base sequence may be a 12- mer peptide (e.g.
  • peptide fragment having a sequence derived from SEQ ID NO:1, and for example may be SEQ ID NO:11, X3-X4-X5-X6-X7-X8-X9-X10-R11-X12-K13-X14, in which for SEQ ID NO:11: [00131] X 3 is T or W; [00132] X4 is R or C; [00133] X5 is T or D; [00134] X6 is any amino acid; [00135] X 7 is F or Y; [00136] X 8 is any amino acid; [00137] X9 is any amino acid; [00138] X10 is any amino acid; [00139] X12 is M or G; and [00140] X 14 is V or R.
  • a SEQ ID NO:1-derived 12-mer base sequence has the sequence of SEQ ID NO:12, X3-X4-X5-X6-X7-X8-X9-X10-R-X12-K-X14, in which for SEQ ID NO:12: [00142] X 3 is T or W; [00143] X 4 is R or C; [00144] X 5 is T or D; -26- 119239542.1 [00145] X6 is Y, G or I; [00146] X7 is F or Y; [00147] X8 is T or C; [00148] X 9 is P, N, or S; [00149] X 10 is Y, T, or S; [00150] X12 is M or G; and [00151] X14 is V.
  • a SEQ ID NO:1-derived 12-mer base sequence has the sequence of SEQ ID NO:13, X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -R 11 -X 12 -K 13 -X 14 , in which for SEQ ID NO:13: [00153] X3 is T or W; [00154] X4 is R or C; [00155] X5 is T or D; [00156] X 6 is Y, G or I; [00157] X 7 is F or Y; [00158] X8 is T; [00159] X9 is P; [00160] X10 is Y or T; [00161] X 12 is M or G; and [00162] X 14 is V.
  • individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids may be made to a lynx1-loop2-derived base sequence of the present disclosure (e.g., any of SEQ ID NOs:1 to 7 or 9 to 13).
  • Such substitutions, insertions, and/or deletions may typically be in the range of about 1 to 5 amino acids.
  • the choice of amino acids for conservative substitutions may be selected according to the location of the amino acid to be substituted in the peptide.
  • a conservative substitution for an amino acid on the exterior of the peptide, which is exposed to solvents and/or potential intermolecular interactions may differ from a conservative substitution for an amino acid in the interior of the peptide, which is not exposed to solvents and intermolecular interactions.
  • Such context-sensitive selection of conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol. Biol., 1999, 217, 721-739; Taylor et al, J. Theor. Biol., 119 (1986);205-218; and S. French and B. Robson, J. Mol. Evol., 19 (1983)171, the entire content of each of which is incorporated herein by reference.
  • conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide may include the following substitutions: substitution of Y with F; T with S, K, or A; P with A; E with D or Q; N with D or G; R with K; G with N or A; T with S, K, or A; D with N or E, I with L or V, F with Y or L; S with T or A, R with K, G with N or A, K with R; A with S, K, P, G, T, or V; W with Y; and M with L.
  • the carrier peptide may include a lynx1-loop2-derived base sequence variant with a sequence selected from SEQ ID NOs: 1 to 7 or 9 to 13 that is modified to include one or more conservative substitutions.
  • embodiments of the present disclosure are not limited thereto, and for example, compositions according to embodiments of the present disclosure may incorporate lynx1-loop2 base sequence variants, other base sequences having no sequence relationship to the lynx1-loop2 base sequence, or other components and methods of transporting the ASO across the BBB.
  • the linker sequence may include any suitable peptide sequence that is capable of mediating or facilitating an inter- or intramolecular association between the carrier peptide and the effector agent. Stated another way, the linker sequence of the carrier peptide may participate in one or more conjugate-forming interactions with the effector agent. Non-limiting examples of such interactions include electrostatic attraction, hydrogen bonding, Van der Waals interactions, covalent or ionic bond formation, and combinations thereof.
  • the carrier peptide may be suitably conjugated with other effector agents, such as drugs, antibodies, and oligonucleotides.
  • the carrier peptide may include a linker sequence including 1 to 4 C-terminal glycine residues (SEQ ID NO: 147). [00167] In some embodiments, the carrier peptide may include a linker sequence including 1 to 4 C-terminal glycine residues (SEQ ID NO: 147) (for example, 1, 2, 3, or 4 Gly residues), followed by a 3 to 12 residue sub-sequence capable of facilitating an interaction with an effector agent.
  • SEQ ID NO: 147 for example, 1, 2, 3, or 4 Gly residues
  • the carrier peptide residue sub-sequence may include or consist of -28- 119239542.1 positively charged residues so that an electrostatic attraction can be formed with the negatively charged effector agent (e.g., siRNA, ASO, or TFO).
  • the effector agent e.g., siRNA, ASO, or TFO
  • the carrier peptide residue sub-sequence may include or consist of -28- 119239542.1 positively charged residues so that an electrostatic attraction can be formed with the negatively charged effector agent (e.g., siRNA, ASO, or TFO).
  • the effector agent e.g., siRNA, ASO, or TFO
  • the effector agent may be conjugated with the carrier peptide via the linker sequence due an electrostatic attraction between the positively charged linker sequence and the negatively charged effector agent.
  • the sub-sequence that is capable of facilitating an electrostatic interaction between the linker sequence and a negatively charged effector agent may include 3 to 11 amino acids, for example, 5 to 10 amino acids, or 6 to 9 amino acids as described above (e.g., such that at least 3 are selected from Arg and Lys).
  • the linker sequence may be represented by GGGG-X 5 -X 6 -X 7 -X 8 - X9-X10-X11-X12-X13-X14-X15-X16 (SEQ ID NO: 14), where the glycine residues at positions 2 to 4 are optional (e.g., may be present or absent), and the amino acids of X5 to X16 are selected from the standard amino acids or are absent, provided that at least three of these residues are present and are selected from Arg and Lys.
  • the linker sequence may be represented by (G) n -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 (SEQ ID NO: 149), where n is an integer of 1 to 4 and the amino acids of X5 to X16 are the same as above.
  • the 3 to 12 residue sub-sequence may only include positively charged amino acids, and for example, all of the 3 to 12 amino acids may be independently selected from arginine and lysine.
  • the amino acids of X5 to X16 are selected from Arg and Lys or are absent, provided that at least of these three residues are present.
  • the linker sequence may be represented by (G) n -(X 5 ) m (SEQ ID NO: 146), where n is an integer of 1 to 4, each instance of X 5 is independently selected from arginine and lysine, and m is an integer of 3 to 12.
  • the carrier peptide may include a linker sequence including 4 C-terminal glycine residues followed by a 7 to 9 residue sub-sequence capable of facilitating an electrostatic interaction between the linker sequence and the ASO (SEQ ID NO: 15).
  • the 7 to 9 amino acids may include or consist of arginine or lysine residues.
  • the 7 to 9 amino acids may include or consist of all arginine residues, or all lysine residues.
  • the linker sequence may be represented by GGGG-X5-X6-X7-X8- X 9 -X 10 -X 11 -X 12 -X 13 (SEQ ID NO: 15), where the amino acids of X 5 to X 16 are selected from -29- 119239542.1 Arg and Lys or are absent, provided that at least seven of these residues are present.
  • the linker sequence may be represented by (G)n-(X5)m (SEQ ID NO: 150), where n is an integer of 1 to 4, each instance of X5 is independently selected from arginine and lysine, and m is an integer of 7 to 9.
  • the carrier peptide may include a linker sequence including 4 C-terminal glycine residues followed by 7 or 9 arginine residues (SEQ ID NO: 151).
  • the carrier peptide (which includes the base sequence and the linker sequence) may have a sequence of MTWCDYFTPSRGKVRKSGGGGRRRRRRRRR (SEQ ID NO:16).
  • the carrier peptide may have a sequence of MTWCDYFTPSRGKVRKSGGGGRRRRRRR (SEQ ID NO: 17).
  • the carrier peptide may be selected from SEQ ID NOS:16 and 17 and variant sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.
  • Each of the amino acids in the linker sequence may have any suitable stereochemistry.
  • the linker sequences according to embodiments of the present disclosure includes one or more arginine and/or lysine residues, the arginine and/or lysine residues may each independently be L- or D-residues.
  • the linker sequence may include, consist essentially of, or consist of a poly-L-arginine peptide having 7, 8, or 9 arginine residues (SEQ ID NO: 152). In some embodiments, the linker sequence may include, consist essentially of, or consist of a poly-D-arginine peptide having 7, 8, or 9 arginine residues (SEQ ID NO: 153). It will be understood that the descriptions herein of carrier peptides encompass embodiments including carrier peptide variants as defined above.
  • additional embodiments may include carrier peptide fragments, mutants, and/or functional equivalents having at least about 80%, 85%, 90%, 95%, or 99% sequence and/or structural homology to peptide sequences described herein, as well as chemical derivatives of all of the above.
  • dose refers to a specified or measured amount of a therapeutic agent to be taken or given at any one given time
  • dosage refers to both the size of the dose to be taken as well as the timing regimen for taking multiple doses.
  • a dosage of the conjugate, complex and/or composition may include any number of doses administered according to a dosage plan prescribed by a physician, without limitation.
  • the dosage plan may include a single dose of the conjugate and/or -30- 119239542.1 composition administered only once. In some embodiments, however, the dosage plan may include multiple doses of the conjugate, complex and/or composition administered according to a time interval prescribed by a physician.
  • the dosage plan may include periodic doses, with subsequent doses administered a prescribed interval from the first dose.
  • the prescribed interval is not particularly limited, and may be any interval prescribed by a physician. In some embodiments, however, the prescribed interval may be 1 hour to 6 months, for example, 1 hour to 3 months, 1 hour to 1 month, 1 hour to 1 week, or 1 hour to 1 day.
  • Each dose of the conjugate, complex and/or composition may comprise a pharmaceutical composition including the conjugate, complex and/or composition and one or more pharmaceutically acceptable excipients, such as (but not limited to) pharmaceutically acceptable carriers.
  • the amount of the conjugate, complex and/or composition in each dose may vary depending on the prescription of the physician.
  • each dose includes a low dose or reduced dose of the effector agent in the conjugate, complex and/or composition compared to a conventional dose unit.
  • each dose may include a pharmaceutical composition including the conjugate in an amount sufficient to deliver an amount of the effector agent of lower than 0.1 mg per kg of body weight of the intended subject (or recipient), for example, lower than 4,000 ⁇ g per kg body weight of the intended subject, or lower than 1,000 ⁇ g per kg body weight of the intended subject.
  • a method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and/or mRNA levels in a target includes: administering a composition including any of the above-described ASO sequences, ASO variants, or chemical derivatives thereof to the target.
  • the ASO may be a sequence of SEQ ID NOs: 29 to 86, as described above.
  • the target may be in vivo or in vitro.
  • the administering the ASO composition may include administering a composition comprising the ASO conjugated to a carrier peptide.
  • the administering of the ASO composition may occur in a mouse or human subject (e.g., a patient with Huntington’s disease).
  • the target may be a cell selected from the group consisting of neurons, neuronal cells, brain cells, glial cells, astrocytes, neuronal supporting cells, and cells of the central nervous system (CNS).
  • the target e.g., cell
  • the target includes or expresses a receptor that is targeted by the carrier peptide, for example, a nicotinic acetylcholine receptor.
  • the target may be within or across the BBB.
  • the administering the ASO composition to the target includes delivery of the ASO across the BBB.
  • Any suitable method or manner of delivering the ASO across the BBB may be used without limitation.
  • delivery across the BBB may be accomplished by conjugating the ASO to one of the carrier peptides disclosed herein.
  • the relative amounts of effector agent (e.g., ASO) and carrier peptide in the ASO composition may be selected to maximize or increase the amount of ASO that is conjugated to carrier peptide and therefore available for delivery to the target.
  • the concentration of the conjugate is related to the equilibrium constant for association (Ka) between the two molecules.
  • the relative concentrations of the ASO and the carrier peptide may be modified or selected to account for various intermolecular forces (including electrostatic attraction) affecting the Ka.
  • a molar ratio of the effector agent to the carrier peptide may be 1:30 to 1:1, 1:25 to 1:1, 1:22 to 1:1, 1:20 to 1:1, 1:18 to 1:1, 1:15 to 1:1, 1:12 to 1:1, 1:10 to 1:1, 1:8 to 1:1, 1:6 to 1:1, 1:4 to 1:1, or 1:2 to 1:1.
  • the molar ratio of the ASO and the carrier peptide may be 1:5 to 1:30, for example, 1:7 to 1:25; 1:7 to 1:22, 1:10 to 1:20, or 1:14 to 1:18. In some embodiments, the ratio of the ASO and the carrier peptide may be 1:15 or 1:19. And in some example embodiments, the ratio of the effector agent (e.g., anti-sense oligonucleotide) and the carrier peptide may be 1: greater than 10, for example, 1: greater than 10 to 1:30, 1: greater than 10 to 1:25, 1: greater than 10 to 1:22, 1: greater than 10 to 1:20, 1: greater than 10 to 1:18, 1: greater than 10 to 1:15.
  • the ratio of the effector agent e.g., anti-sense oligonucleotide
  • the carrier peptide may be 1: greater than 10, for example, 1: greater than 10 to 1:30, 1: greater than 10 to 1:25, 1: greater than 10 to 1:22, 1: greater than 10 to
  • the ratio of the effector agent and the carrier peptide may be 1: lower than 10, for example, 1:4 to 1: lower than 10, 1:2 to 1: lower than 10, or 1:7 to 1: lower than 10. [00186] Additionally, in some embodiments, the ratio of the effector agent and the carrier peptide may be greater than 1:1.
  • the ratio of the effector agent and the carrier peptide may be about 1:30 to 5:1, 1:25 to 5:1, 1:22 to 5:1, 1:20 to 5:1, 1:18 to 5:1, 1:15 to 5:1, 1:12 to 5:1, 1:10 to 5:1, 1:8 to 5:1, 1:6 to 5:1, 1:4 to 5:1, 1:2 to 5:1, 1:30 to 3:1, 1:25 to 3:1, 1:22 to 3:1, 1:20 to 3:1, 1:18 to 3:1, 1:15 to 3:1, 1:12 to 3:1, 1:10 to 3:1, 1:8 to 3:1, 1:6 to 3:1, 1:4 to 3:1, 1:2 to 3:1, 1:30 to 2:1, 1:25 to 2:1, 1:22 to 2:1, 1:20 to 2:1, 1:18 to 2:1, 1:15 to 2:1, 1:12 to 2:1, 1:10 to 2:1, 1:8 to 2:1, 1:6 to 2:1, 1:4 to 2:1, 1:2 to 2:1, 1:30 to 1.5:1, 1:25 to 1.5:1, 1:22 to 1.5:1, 1:20 to 1.5:1, 1:1
  • the composition is a pharmaceutical composition.
  • the ASO and the carrier peptide may be dissolved or suspended in any suitable aqueous carrier or solution that is acceptable for parenteral administration (such as a saline and/or dextrose solution).
  • the solution may be a 0.5 wt% to 5 wt% saline solution (e.g., a 0.5 wt% to 3 wt%, or 0.9 wt% to 2 wt% saline solution) or a 1 wt% to 10 wt% dextrose solution (e.g., a 2 wt% to 8 wt%, or 3 wt% to 5 wt% dextrose solution).
  • a 0.5 wt% to 5 wt% saline solution e.g., a 0.5 wt% to 3 wt%, or 0.9 wt% to 2 wt% saline solution
  • a 1 wt% to 10 wt% dextrose solution e.g., a 2 wt% to 8 wt%, or 3 wt% to 5 wt% dextrose solution.
  • the solution may include additional stabilizing agents in the art, such as PEG8000 (e.g., at a concentration of 0.01 wt% to 1 wt%, for example, 0.05 wt% to 0.5 wt%, 0.1 wt% to 0.4 wt%, or 0.2 wt% to 0.3 wt%) and/or Polysorbate 80 (e.g., at a concentration of 0.01 wt% to 1.5 wt%, for example, 0.05 wt% to 1.2 wt%, 0.1 wt% to 1 wt, 0.15 wt% to 0.8 wt%, 0.2 wt% to 0.7 wt%, or 0.3 wt% to 0.6 wt%).
  • PEG8000 e.g., at a concentration of 0.01 wt% to 1 wt%, for example, 0.05 wt% to 0.5 wt%, 0.1 wt% to 0.4 wt%, or 0.2 w
  • the ASO may be included in the pharmaceutical solution in an amount of 0.005% to 1% by weight, for example, 0.015% to 0.5%, 0.03% to 0.25%, 0.04% to 0.1%, or 0.05% to 0.08% by weight.
  • a pharmaceutical solution may include about 2.5 to 5.0 ⁇ g of effector agent (about 15 to 30 ⁇ g of conjugate) in 100 ⁇ L of solution.
  • the pharmaceutical solution may include about 5.0 ⁇ g of the effector agent (about 30 ⁇ g of conjugate) in 100 ⁇ L of solution.
  • the pharmaceutical solution may include about 2.5 ⁇ g of the effector agent (about 15 ⁇ g of conjugate) in 100 ⁇ L of solution.
  • the method may further include conjugating the ASO to the carrier peptide, prior to administering the composition.
  • the chemical or biochemical steps used to achieve such a conjugation depend on the particular linker or interaction between the two molecules, and those having ordinary skill in the art are capable of selecting suitable methods, parameters, etc., to achieve the conjugation.
  • Administration of the composition to the target may result in an observable decrease in HTT mRNA levels and/or HTT protein expression.
  • the decrease in HTT mRNA levels in targeted cells and tissues can be measured by extracting RNA from a suitable sample of the target (e.g., from cells including a receptor that can be targeted by the carrier peptide, such as a nACHR receptor), generating cDNA from a set or predetermined volume of the RNA extract, and subjecting the cDNA to PCR.
  • a suitable sample of the target e.g., from cells including a receptor that can be targeted by the carrier peptide, such as a nACHR receptor
  • generating cDNA from a set or predetermined volume of the RNA extract
  • the relative decrease (e.g., knock-down) of HTT mRNA with respect to an untreated control can be quantified by comparing the change in HTT mRNA levels in the presence and absence of the ASO:carrier peptide composition with an unrelated mRNA that is expected to remain at a constant level even in the presence of the ASO:carrier peptide composition.
  • the unrelated mRNA standard may be the synuclein alpha (SNCA) protein, which is also expressed in neuronal cells.
  • the method of treating HD according to embodiments of the present disclosure may result in a statistically significant decrease in (e.g., knock- down of) HTT mRNA levels (e.g., with a P-value of less than 0.05, and in some embodiments less than 0.01).
  • the methods according to embodiments of the present disclosure may result in a decrease in HTT mRNA levels of greater than 20%, for example, a decrease of 20% to 90%, 30% to 90%, 40% to 85%, 50% to 80%, or 60% to 75%.
  • HTT mRNA levels may be decreased by 25% to 90%, for example, 35% to 85%, 45% to 75%, or 55% to 70% relative to untreated (control) levels.
  • HTT mRNA levels may be decreased by 40% to 65% in the hippocampus, for example, 45% to 60%, or 50% to 55%, and by 15% to 40% in the striatum, for example, 20% to 35% or 25% to 30% relative to untreated (control) levels.
  • Example 1 Selection of lead ASOs
  • Figure 1 is a flowchart diagram summarizing the selection procedure. As shown in box 10 of the flowchart, 13,456 complementary 20-mer oligonucleotides to human HTT mRNA were identified, and then filtered to include only the 711 sequences that are conserved between humans and mice.
  • Oligomers predicted to form secondary structures with a MFE less than -4 kcal/mol were eliminated, resulting in 380 remaining candidates.
  • candidates with known toxic motifs such as tgc, tcc, and gc
  • toxic motifs are further described in e.g., Burdick et al., “Sequence motifs associated with hepatotoxicity of locked nucleic acid – modified antisense oligonucleotides,” Nucleic Acids Res., 2014, 42(8), pg.4882-4891, the entire content of which is incorporated herein by reference.
  • the ASO candidates of (SEQ ID NOS:29 to 86) had a total guanine and cytosine content of between 20% to 60%.
  • Five ASO candidates (ASO1 to ASO5 having SEQ ID NOs: 87, 97, 99, 100 and 125), each having a total guanine and cytosine content of 35% to 45%, were selected for further study.
  • -34- 119239542.1 Example 2.
  • the selected HTT ASO candidates were initially evaluated for HTT knockdown efficacy and specificity in vitro, using lynx1-loop2-derived carrier peptides to mediate delivery into human neuronal cells.
  • the key genes involved in lynx1-loop2-mediated delivery via the nACHRs include the nicotinic receptor alpha7 subunit (CHRNA7), nicotinic receptor alpha4 subunit (CHRNA4), nicotinic receptor beta2 subunit (CHRNB2), and NACHO. Because differentiated SHSY5Y human cells express all such genes of interest, they were selected to test for knockdown of HTT mRNA. The HTT mRNA knockdown efficiency was assessed by comparison to synuclein alpha (SNCA), which is a neuronally expressed gene that is regulated independently of HTT.
  • SNCA synuclein alpha
  • ASO1 to ASO5 were tested with Peptide 1 (SEQ ID NO:16) or Peptide 4 (SEQ ID NO:17) as follows.
  • Peptide 1 SEQ ID NO:16
  • Peptide 4 SEQ ID NO:17
  • ASO complexes were mixed in DMEMF12 media and incubated for 15 min at room temperature (e.g., 25 oC).
  • room temperature e.g. 25 oC
  • 0.1 mL of the differentiation media was replaced with 0.1 mL of the peptide:ASO complexes.
  • the final concentrations in the cell growth media were 15 ⁇ M peptide:1 ⁇ M ASO (15:1 ratio).
  • the KIT QPCR FASTMIX II ROX was used for the qPCR reaction, and triplicates were run on the Applied BiosystemsTM 7500 Real-Time PCR System.
  • HTT1 Taqman assay Hs00918174_m1
  • Hs00240907_m1 alpha-synuclein
  • the qPCR values of the HTT and SNCA gene were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Taqman assay Hs02786624_g1) and peptidylprolyl isomerase (PPIA, Taqman assay Hs04194521_s1).
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • PPIA peptidylprolyl isomerase
  • the cells showed a decrease in HTT mRNA expression upon exposure to ASO1, ASO2, ASO3, ASO4, and ASO5 (SEQ ID NOS: 87, 97, 99, 100, and 125) when complexed with either of Peptide 1 (SEQ ID NO:16) or Peptide 4 (SEQ ID NO:17), but also showed a simultaneous decrease in SNCA mRNA expression, indicating that these ASOs had comparatively lower specificity for HTT.
  • Table 4 summarizes the change in measured mRNA levels as normalized against the change in HTT1 mRNA in the DMEMF 12-only control; values in bold highlight statistically significant knockdowns.
  • Figure 2 presents the same data in bar chart form; the error bars represent the standard deviation.
  • ASO3 was further tested in vivo for their efficacy in specific brain regions, discussed below. -37- 119239542.1 [00209] To assess differences in knockdown according to differences in concentration of the ASO, selected ASOs were evaluated for HTT knockdown efficacy and specificity in vitro, using different ratios of the lynx1-loop2-derived carrier peptides to the ASOs.
  • the protocol was generally as described above, except that the final concentrations of the peptide:ASO mixtures in the cell growth media were varied between a 15:1 ratio (15 ⁇ M peptide:1 ⁇ M ASO), a 10:1 ratio (10 ⁇ M peptide:1 ⁇ M ASO), and a 4:1 ratio (4 ⁇ M peptide:1 ⁇ M ASO). Also, the final peptide:ASO mixtures (having different peptide:ASO ratios) were prepared in a solution of dextrose with 0.6% polysorbate 80.
  • HTT3 Taqman assay Hs00918178_m1 (HTT3) for HTT.
  • Result Differentiated SHSY5Y cells showed a statistically significant decrease of HTT mRNA expression upon exposure to all tested ASOs (including those with ASO3 (SEQ ID NO:99), ASO6 (SEQ ID NO:41) and ASO2 (SEQ ID NO:97)) when conjugated to Peptide 1 (SEQ ID NO:16).
  • ASO6 (SEQ ID NO:41) conjugates exhibited similar knockdown at all tested ratios (i.e., 15:1, 10:1 and 4:1). But while all ASO3 (SEQ ID NO:99) conjugates exhibited significant knockdown, the 15:1 and 10:1 ratios exhibited better knockdown than the 4:1 ratio. Similarly, while all ASO2 (SEQ ID NO:97) conjugates exhibited significant knockdown, the 15:1 ratio exhibited better knockdown than the 4:1 ratio. Exposure to Dextrose-0.6% alone (e.g., in the absence of any ASO or peptide) did not show any statistically significant effect on HTT mRNA levels.
  • ASOs were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). Carrier peptides were synthesized by Lifetein (Hillsborough, NJ). [00214] ASO3 (SEQ ID NO: 99) was administrated to each animal using the following procedure.
  • the KIT QPCR FASTMIX II ROX (Quanta, VWR, # 97065-998) was used for the qPCR reaction; triplicates were run on the Applied BiosystemsTM 7500 Real-Time PCR System. Knockdown of HTT target mRNA was assessed by comparison to SNCA using suitable primers for RT-qPCR: Taqman assay Mm01213763 for HTT, and Taqman assay Mm01188700_m1 for SNCA.
  • Figure 4 presents the same data in bar chart form; the error bars represent the standard deviation.
  • a 48% (P ⁇ .01) reduction of HTT mRNA was observed in the HP of mice injected with Peptide 4 (SEQ ID NO:17):ASO3 (SEQ ID NO:99), and a 26% (P ⁇ .01) reduction was observed in the ST.
  • Table 6 Fold Change Standard HTT mRNA knockdown in wild-type Deviation p value Avera e CA 00 00 00 00 EQ ID NO: 99.
  • mice Three groups of 7-8 mice each were tested in this study, including: 1) ASO6 conjugate treated mice; 2) vehicle control mice; and 3) Wild-Type (WT) mice. Each mouse was 12 weeks old at the initiation of the therapy, and was administered one dose every two weeks for a total of 6 months.
  • Each dose included an ASO6 (SEQ ID NO:41) conjugate administrated to each animal using the following procedure. Briefly, 25 ⁇ g of ASO6 (SEQ ID NO:41) and Peptide 1 (SEQ ID NO:16) were mixed in a dextrose-0.6% polysorbate 80 solution to produce a 10:1 molar ratio of peptide to ASO, and incubated for 15 minutes at room temperature.
  • the peptide:ASO conjugates were then delivered with a dose of 25 ⁇ g of ASO6 (SEQ ID NO:41) as a 100 ⁇ L injection per mouse via the lateral tail -41- 119239542.1 vein.
  • An identical volume of a dextrose-0.6% polysorbate 80 solution was injected in vehicle control animals.
  • CTX cortex
  • ST striatum
  • Table 7 Distance Traveled (25 ⁇ g study) Group Total Distance in cm (average ⁇ SD)
  • Table 8 Rearing Frequency (25 ⁇ g study)
  • Table 9 Expanded mHTT in CTX in 25 ⁇ g study (Assay 6: 2B7/MW1-ST)
  • Group Expanded mHTT % Expanded mHTT % mHTT KD % ol 119239542.1 protein; average ⁇ SD
  • Table 10 Aggregated mHTT in CTX in 25 ⁇ g study (Assay 45: MW8/4C9-ST)
  • Example 5 Chronic Dosage of 2.5 ⁇ g in Humanized zQ175 Huntington’s mice
  • Methods Male and female zQ175 mice at PsychoGenics (Paramus, New Jersey) were acclimated for several days before the start of the experiment. Fasting was not required for this study.
  • the ASOs and carrier peptides were obtained as in Example 3.
  • Each dose included an ASO6 (SEQ ID NO:41) or ASO4 (SEQ ID NO:100) conjugate administrated to each animal using the following procedure. Briefly, 2.5 ⁇ g of ASO6 (SEQ ID NO:41) or ASO4 (SEQ ID NO:100) and Peptide 1 (SEQ ID NO:16) were mixed in a dextrose-0.6% polysorbate 80 solution to produce a 10:1 molar ratio of peptide to ASO, and incubated for 15 minutes at room temperature.
  • the peptide:ASO conjugates were then delivered with a dose of 2.5 ⁇ g of ASO6 (SEQ ID NO:41) or ASO4 (SEQ ID NO:100) as a 100 ⁇ L injection per mouse via the lateral tail vein.
  • An identical volume of a dextrose-0.6% polysorbate 80 solution was injected in vehicle control animals.
  • Total rearing frequency, total distance traveled, and post-treatment expanded and aggregated mutant HTT levels in the cortex (CTX) and striatum (ST) were assessed. Expanded mutant HTT levels in both the CTX and the ST were assessed via Assay 6 (2B7/MW1-ST), and aggregated mutant HTT levels in both the CTX and ST were assessed via Assay 45 (MW8/4C9-ST).
  • Table 13 Distance Traveled (2.5 ⁇ g study) Group Total Distance in cm (average ⁇ SD)
  • Table 14 Rearing Frequency (2.5 ⁇ g study) Group Total Rearing Bouts (average ⁇ SD)
  • Table 15 Expanded mHTT in Cortex in 2.5 ⁇ g study (Assay 6: 2B7/MW1-ST) Grou Ex anded mHTT % Ex anded mHTT % mHTT KD % ol -45- 119239542.1
  • Table 16 Aggregated mHTT in Cortex in 2.5 ⁇ g study (Assay 45: MW8/4C9-ST) Group Aggregated mHTT % Aggregated % mHTT KD (fmol/mg total mHTT based on based on 100% ol
  • Table 17 Soluble Expanded mHTT in Striatum 2.5 ⁇ g study (Assay 6: 2B7/MW1-ST) Group Expanded m
  • compositions according to embodiments of the present disclosure exhibit significant improvements in functional measures, demonstrating a reversal of phenotype.
  • Figure 9 which compares results of both studies
  • the ASO6 treated mice showed a Total Rearing Frequency after treatment of 91% of WT and no improvement in Total Distance Traveled.
  • the ASO6 treated mice showed a Total Rearing Frequency of 97% of WT in addition to a Total Distance Traveled of 81% of WT.
  • the lower dose (2.5 ⁇ g) surprisingly and unexpectedly improved the function with respect to both Total Distance Travelled and Total Rearing Frequency in the amount of 25% and 50% relative to the vehicle control group
  • the high dose (25 ⁇ g) only improved the Total Rearing Frequency relative to the vehicle control group in the amount of 39%.
  • the Total Rearing Frequency was improved by 97% for the lower dose (2.5 ⁇ g) and 91% for the higher dose (25 ⁇ g) – both demonstrating a reversal of the phenotype of the zQ175 mice back to WT as noted above.
  • the lower dose (2.5 ⁇ g) exhibited greater reduction of the aggregated protein in the cortex in the ASO6 treated group (statistically significant KD of 16%) compared to no statistically significant KD in the cortex in the high dose (25 ⁇ g) ASO6 treated group.
  • the lower dose (2.5 ⁇ g) exhibited greater reduction of the soluble protein in the striatum in the ASO6 treated group (statistically significant KD of 14%) compared to no statistically significant KD in the striatum in the high dose (25 ⁇ g) ASO6 treated group.
  • any numerical range recited herein is intended to include all sub-ranges of the same numerical precision subsumed within the recited range.
  • a range of "1.0 to 10.0" is intended to include all subranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 10.0, that is, having a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 10.0, such as, for example, 2.4 to 7.6.
  • Any maximum numerical limitation recited herein is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Composition pour le traitement de la maladie de Huntington (MH) par réduction des niveaux endogènes de Huntingtine (HTT) dans une cible comprenant un ASO comprenant une séquence d'ASO choisie parmi les SEQ ID NO : 18 à 86, et un peptide porteur comprenant une séquence peptidique de SEQ ID NO : 1. Le peptide porteur peut cibler le récepteur nicotinique de l'acétylcholine (nAChR) des cellules neuronales afin d'administrer l'ASO à travers la barrière hémato-encéphalique (BHE). Dans certains modes de réalisation, l'ASO peut être conjugué au peptide porteur. Un procédé de réduction des niveaux de HTT endogènes pour traiter la HD dans la cible peut comprendre l'administration d'une composition comprenant une séquence ASO choisie parmi SEQ ID NO : 18 à 86 de la cible.
PCT/US2023/017275 2022-10-24 2023-04-03 Compositions et procédés pour le traitement d'oligonucléotides antisens (aso) de la maladie de huntington Ceased WO2024091286A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP23883269.5A EP4608980A1 (fr) 2022-10-24 2023-04-03 Compositions et procédés pour le traitement d'oligonucléotides antisens (aso) de la maladie de huntington

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263418945P 2022-10-24 2022-10-24
US63/418,945 2022-10-24

Publications (1)

Publication Number Publication Date
WO2024091286A1 true WO2024091286A1 (fr) 2024-05-02

Family

ID=90831513

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/017275 Ceased WO2024091286A1 (fr) 2022-10-24 2023-04-03 Compositions et procédés pour le traitement d'oligonucléotides antisens (aso) de la maladie de huntington

Country Status (2)

Country Link
EP (1) EP4608980A1 (fr)
WO (1) WO2024091286A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110135607A1 (en) * 2008-05-23 2011-06-09 John Sinclair Viral nucleic acid for the treatment of neurodegenerative disorders
US20130005794A1 (en) * 2005-05-06 2013-01-03 Medtronic, Inc. Methods and sequences to suppress primate huntington gene expression
US20200277598A1 (en) * 2011-05-05 2020-09-03 Sarepta Therapeutics, Inc. Peptide Oligonucleotide Conjugates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130005794A1 (en) * 2005-05-06 2013-01-03 Medtronic, Inc. Methods and sequences to suppress primate huntington gene expression
US20110135607A1 (en) * 2008-05-23 2011-06-09 John Sinclair Viral nucleic acid for the treatment of neurodegenerative disorders
US20200277598A1 (en) * 2011-05-05 2020-09-03 Sarepta Therapeutics, Inc. Peptide Oligonucleotide Conjugates

Also Published As

Publication number Publication date
EP4608980A1 (fr) 2025-09-03

Similar Documents

Publication Publication Date Title
JP7438103B2 (ja) アポリポタンパク質Ｃ－ＩＩＩ（ＡＰＯＣ３）の発現を阻害するためのＲＮＡｉ剤および組成物
EP2751128B1 (fr) Peptides pénétrants de cellules avec un domaine hydrophobe central
US20250270555A1 (en) Organic compositions to treat beta-catenin-related diseases
CN104126010A (zh) 嵌合的双链核酸
TW201018471A (en) Multiple exon skipping compositions for DMD
JP2011510678A (ja) Ｄｎａ反復不安定性関連遺伝性障害を治療するための方法及び手段
JP7441455B2 (ja) 筋ジストロフィーの処置のためのチオモルホリノオリゴヌクレオチド
KR20180104075A (ko) IL4Rα, TRPA1, 또는 F2RL1을 표적화하는 RNA 복합체를 사용한 아토피 피부염 및 천식의 치료
TW202400190A (zh) 活化補體因子h表現的寡核苷酸調節劑
CN120752341A (zh) 靶向sod1的寡核苷酸
JP2011504375A (ja) 細胞の中に核酸を導入する医薬組成物及びその方法
US9217149B2 (en) Pharmaceutical composition for treating cancer
CN118043461A (zh) 方法
WO2024091286A1 (fr) Compositions et procédés pour le traitement d'oligonucléotides antisens (aso) de la maladie de huntington
WO2025090567A1 (fr) Traitements de la maladie de huntington par arnsi à faible dose
WO2024186688A2 (fr) Compositions et procédés de distribution d'agents effecteurs à faible dose
CN119365598A (zh) 用于治疗单基因神经发育障碍的组合物和方法
EP4366785A1 (fr) Traitement de troubles associés à la dégradation de la tyrosine
EP2776450B1 (fr) Modulateurs oligonucléotidiques antisens du récepteur 2c de la sérotonine et leurs utilisations
WO2024169770A1 (fr) Arnsi inhibant l'expression du gène scn9a, composition pharmaceutique et son utilisation
WO2025242147A1 (fr) Oligonucléotide ciblant le gène de la protéine-22 de la myéline périphérique et utilisation associée
TW202532088A (zh) 抑制亨廷頓蛋白（htt）表達的組合物和方法
WO2024169907A1 (fr) Arnsi pour la régulation de l'expression du complément c3, son conjugué, sa composition pharmaceutique et son utilisation
WO2025190276A1 (fr) Molécule polynucléotidique pour la suppression de l'expression de dmpk et son utilisation
TW202345873A (zh) 調節ｓｃａｐ活性之組合物及方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23883269

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023883269

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023883269

Country of ref document: EP

Effective date: 20250526

WWP Wipo information: published in national office

Ref document number: 2023883269

Country of ref document: EP