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WO2024092256A2 - Oligonucléotides antisens-peptides et leur utilisation pour le traitement de troubles neurodégénératifs - Google Patents

Oligonucléotides antisens-peptides et leur utilisation pour le traitement de troubles neurodégénératifs Download PDF

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WO2024092256A2
WO2024092256A2 PCT/US2023/078121 US2023078121W WO2024092256A2 WO 2024092256 A2 WO2024092256 A2 WO 2024092256A2 US 2023078121 W US2023078121 W US 2023078121W WO 2024092256 A2 WO2024092256 A2 WO 2024092256A2
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Prior art keywords
peptide
antisense oligonucleotide
oligonucleotide conjugate
conjugate
nucleotides
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PCT/US2023/078121
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WO2024092256A3 (fr
Inventor
Vinod VATHIPADIEKAL
Branko MITASEV
John Wang
Courtney EASLEY-NEAL
Hyeong Wook Choi
Francis G. Fang
Praveen Vemula
Jung Hwa Lee
Jeffrey Henry
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Eisai R&D Management Co., Ltd.
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Publication of WO2024092256A2 publication Critical patent/WO2024092256A2/fr
Publication of WO2024092256A3 publication Critical patent/WO2024092256A3/fr

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    • 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
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
    • A61K47/54—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 an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • 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
    • C12N2310/35—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
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • CPP-ASOs novel peptide-antisense oligonucleotides comprising cell penetrating peptides conjugated to antisense oligonucleotides
  • Neurodegenerative disorders are a group of disorders characterized by the decline of central nervous system and peripheral nervous system structure and function. While neurodegenerative disorders exhibit heterogeneous symptoms, they can share similar features.
  • One neurodegenerative disease, Alzheimer’s Disease is a neurodegenerative disorder characterized by buildup of amyloid beta plaques and neurofibrillary tangles. It is also the leading cause of dementia.
  • LOAD late-onset Alzheimer’s Disease
  • CD33 also known as Siglec-3.
  • Griciuc et al., Alzheimer’s Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta, 78 NEURON 631 (2013).
  • CD33 is expressed in myeloid-derived cells, including macrophages such as microglia, and encodes the CD33 protein.
  • Microglia account for approximately 10% of the cells in the brain and represent the first line of immunological defense. Microglia modulate several important activities in the brain, such as homeostasis, cognition, and neurogenesis. Augusto-Oliveira et al., What Do Microglia Really Do in Healthy Adult Brain?, 8 CELLS 1293 (2019).
  • Microglia cells are known to contribute to neurodegeneration by releasing proinflammatory substances in the central nervous system. Wojtera et al., Microglial cells in neurodegenerative disorders, 43 FOLIA NEUROPATHOLOGY 311 (2005).
  • CD33 is a transmembrane receptor protein that has an extracellular receptor that binds the ligand sialic acid.
  • the intracellular immunoreceptor tyrosine-based inhibition motif recruits phosphatases upon phosphorylation of its tyrosine residues, leading to suppression of immune cell activity such as phagocytosis.
  • CD33 has been found to inhibit microglial uptake of amyloid beta protein, which suggests that therapies targeting CD33 could be potential LOAD treatment options.
  • Griciuc et al., Alzheimer’s Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta, 78 NEURON 631 (2013).
  • rs3865444 SNP comes in two forms, rs3865444-C and rs3865444-A.
  • the first form results in normal length CD33 protein.
  • the second form, rs3865444-A modulates splicing of CD33 pre-m RNA, resulting in skipping of Exon-2 and a CD33 protein lacking the sialic acid binding domain.
  • the noncoding introns are excised from the pre-m RNA transcript and the coding exons are spliced together to form mRNA. If an intron is left in the final mRNA transcript or an exon is left out, the mRNA reading frame may be disrupted during translation of the mRNA. This may result in a non-functional polypeptide sequence or a premature stop codon.
  • the splicing process is further complicated by alternative splicing, where the same pre-mRNA sequence can be spliced into different exon combinations to form multiple mRNA sequences.
  • RNA sequences are the 5’ splice site, 3’ splice site, and the branch site. Will & Luhrmann, Spliceosome Structure and Function, 3 COLD SPRING HARB. PERSPECT. BIOL. 1 (2011 ).
  • Splicing begins with the 2’ OH group of the branch site binding to the 5’ splice site via a nucleophilic attack, causing cleavage of the 5’ exon at the 5’ splice site and forming a lariat. Then the 3’ OH group of the 5’ exon attacks the 3’ exon at the 3’ splice site, ligating the 5’ and 3’ exons and cleaving the intron lariat. Will & Luhrmann, Spliceosome Structure and Function, 3 COLD SPRING HARB. PERSPECT. BIOL. 1 (2011). Because the splicing process involves spliceosome recognition sites, 5’ and 3’ splice sites, and the branch site, a mutation in any one of these sites can disrupt the splicing process.
  • ASOs are polynucleotides designed to bind with specificity to a target nucleotide sequence, thereby affecting one or more aspects of gene expression, such as transcription, splicing, stability, and/or translation.
  • ASOs may be directed to either RNA or DNA.
  • ASOs directed to RNA can bind to target mRNA sequences, affecting mRNA stability or translation at the ribosome.
  • ASOs that bind to target sequences in pre-m RNA transcripts can affect the splicing process.
  • ASOs may be used to induce exon skipping during pre-mRNA splicing.
  • DMD Duchenne Muscular Dystrophy
  • ASOs may be utilized to correct the reading frame by inducing skipping of an exon during splicing. Removing an exon of the correct number of base pairs results in a shorter mRNA transcript, but the reading frame may be corrected.
  • dystrophin RNA consists of 79 exons, skipping one or several exons during splicing still results in a partly functional protein.
  • Echigoya et al. Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges, 8 J. PERS. MED. 41 (2016).
  • the FDA approved an exon-skipping drug called Exondys 51 (eteplirsen) for treatment of DMD in 2016. Dowling, Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line, 12 NATURE RE . NEUROLOGY 675 (2016).
  • ASOs may be used to prevent or reduce exon skipping during pre-mRNA splicing.
  • the ASO drug nusinersen (Spinraza®) reduces Exon-7 skipping during splicing of the SMN2 gene to treat spinal muscular atrophy.
  • Son & Yokota Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy, in EXON SKIPPING & INCLUSION THERAPIES, 57-68 (2018).
  • the rs3865444-A variant that induces Exon-2 skipping of CD33 conveys protection against LOAD.
  • CPP-ASOs Disclosed herein are CPP-ASOs, methods of using such CPP-ASOs to induce exon skipping during pre-mRNA splicing, pharmaceutical compositions that comprise such CPP-ASOs, and methods of using such compositions to treat neurodegenerative disease.
  • a peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO:1 , and wherein the peptide-antisense oligonucleotide conjugate has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for the antisense oligonucleotide.
  • the peptide-antisense oligonucleotide conjugate comprises a cellpenetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide comprises all or a portion of SEQ ID NO:2, SEQ ID NO: 12, or SEQ ID NO:224.
  • the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25- 30 nucleotides in length.
  • the antisense oligonucleotide is 21-
  • the antisense oligonucleotide is 21-
  • the antisense oligonucleotide is 18-
  • the antisense oligonucleotide is 18-
  • the antisense oligonucleotide is 25-
  • the antisense oligonucleotide is 21 or 25 nucleotides in length. In some embodiments, the antisense oligonucleotide is 25 nucleotides in length. [15] in some embodiments, disclosed herein is a peptide-antisense oligonucleotide conjugate, wherein the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.
  • the antisense oligonucleotide comprises one or more non- natural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for PMO ASOs. In some embodiments, the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).
  • MOE methoxyethyl ribose oligomer
  • the antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for MOE ASOs.
  • the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.
  • the one or more non-natural internucleotide linkages comprise one or more phosphorodiamidate linkages and/or one or more phosphorothioate linkages.
  • all of the one or more non-natural internucleotide linkages have an Sp configuration.
  • all of the one or more non-natural internucleotide linkages have an Rp configuration.
  • the antisense oligonucleotide comprises one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases.
  • composition comprising a peptide-antisense oligonucleotide conjugate and optionally a pharmaceutically acceptable carrier or excipient.
  • a peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to all or a portion of PMO-002 (SEQ ID NO:2), MOE-012 (SEQ ID NO:12), or PMO-424 (SEQ ID NO:224).
  • the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide selected from the group consisting of PMO-002 (SEQ ID NO:2), MOE-012 (SEQ ID NO: 12), and PMO-424 (SEQ ID NO:224).
  • the peptide comprises at least one proteogenic amino acid, at least one non-proteogenic amino acid, or at least one proteogenic amino acid and at least one non-proteogenic amino acid. In some embodiments, the peptide comprises 5-25 amino acids. In some embodiments, the non-proteogenic amino acid comprises a modified proline residue, a lipophilic group, and/or a lactam group. In some embodiments, the peptide is a linear peptide. In some embodiments, the linear peptide comprises at least a portion of Pip6a, ApoE, and/or a neurotensin-based peptide. In some embodiments, the peptide is a cyclic peptide. In some embodiments, the cyclic peptide is CPP9
  • the peptide comprises a lipoic acid group.
  • the lipoic acid group is an (R)-lipoic acid group.
  • the lipoic acid group is an (S)-lipoic acid group.
  • the peptide comprises an oxadiazole linkage.
  • the peptide is conjugated directly to the antisense oligonucleotide.
  • the peptide is conjugated to the antisense oligonucleotide using a chemical reaction.
  • the chemical reaction is a strain-promoted azide-alkyne cycloaddition reaction, a strained alkene-tetrazine cycloaddition reaction, or an amide bond reaction.
  • the peptide is indirectly conjugated to the antisense oligonucleotide, wherein a linker is conjugated between the peptide and antisense oligonucleotide.
  • the peptide-antisense oligonucleotide conjugate further comprises one or more nuclear localization sequences, wherein the one or more nuclear localization sequences are independently conjugated to the peptide and/or the antisense oligonucleotide.
  • a method of inducing Exon-2 skipping in the CD33 gene during pre-mRNA splicing comprising introducing a peptide-antisense oligonucleotide conjugate into a cell, wherein the peptide- antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO:1 , and wherein the peptide-antisense oligonucleotide conjugate has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for the antisense oligonucleotide.
  • the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide, and wherein the antisense oligonucleotide comprises all or a portion of SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:224.
  • the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length.
  • the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more non-natural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).
  • PMO phosphorodiamidate morpholino oligomer
  • the peptide-antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for PMO ASOs.
  • the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).
  • the antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for MOE ASOs.
  • the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.
  • all of the one or more non-natural internucleotide linkages have an Sp configuration In some embodiments, all of the one or more non-natural internucleotide linkages have an Rp configuration. In some embodiments, the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the peptide-antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient.
  • a method of inducing Exon-2 skipping in the CD33 gene during pre-mRNA splicing comprising introducing a peptide-antisense oligonucleotide conjugate into a cell, wherein the peptide- antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and wherein the antisense oligonucleotide comprises all or a portion of PMO-002 (SEQ ID NO:2), MOE-012 (SEQ ID NO: 12) or PMO-424 (SEQ ID NO:224).
  • the cell is an animal cell. In some embodiments, the cell is a human cell.
  • a method of treating a subject having a neurodegenerative disease comprising administering to said subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense nucleotide conjugated to a cell-penetrating peptide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO:1 , and wherein the peptide-antisense oligonucleotide conjugate has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for the antisense oligonucleotide.
  • the antisense oligonucleotide is complementary to all or a portion of SEQ ID NO:2, SEQ ID NO:12 or SEQ ID NO:224. In some embodiments, the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length.
  • the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more non-natural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).
  • PMO phosphorodiamidate morpholino oligomer
  • the peptide-antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for PMO ASOs.
  • the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).
  • the antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for MOE ASOs.
  • the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.
  • all of the one or more non-natural internucleotide linkages have an Sp configuration. In some embodiments, all of the one or more non-natural internucleotide linkages have an Rp configuration. In some embodiments, the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient.
  • a method of treating a subject having a neurodegenerative disease comprising administering to said subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and wherein the antisense oligonucleotide comprises all or a portion of PMO-002 (SEQ ID NO:2), MOE-012 (SEQ ID NO:12), or PMO-424 (SEQ ID NO:224).
  • a method of treating a subject having a neurodegenerative disease comprising administering to said subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and wherein the antisense oligonucleotide is selected from the group consisting of PMO-002 (SEQ ID NO:2), MQE-012 (SEQ ID NO:12), or PMO-424 (SEQ ID NO:224)
  • the subject is a human subject.
  • the neurodegenerative disease is Alzheimer’s Disease.
  • a peptide-antisense oligonucleotide conjugate according to claim 1 for use in a method of inducing Exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing into a cell the peptide- antisense oligonucleotide conjugate of claim 1 , wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces Exon-2 skipping during pre-mRNA splicing of the CD33 gene.
  • a peptide-antisense oligonucleotide conjugate for use in a method of inducing Exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces Exon-2 skipping during pre-mRNA splicing of the CD33 gene.
  • the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length.
  • the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.
  • the antisense oligonucleotide comprises one or more modified sugar moieties.
  • the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).
  • PMO phosphorodiamidate morpholino oligomer
  • the peptide-antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for PMO ASOs.
  • the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).
  • MOE methoxyethyl ribose oligomer
  • the antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for MOE ASOs.
  • the antisense oligonucleotide comprises one or more non-natural internucleotide linkages. In some embodiments, all of the one or more non-natural internucleotide linkages have an Sp configuration. In some embodiments, all of the one or more non-natural internucleotide linkages have an Rp configuration In some embodiments, the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient.
  • a peptide-antisense oligonucleotide conjugate for use in a method of inducing Exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces Exon-2 skipping during pre-mRNA splicing of the CD33 gene.
  • a peptide-antisense oligonucleotide conjugate for use in a method of inducing Exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing into a cell the peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces Exon-2 skipping during pre-mRNA splicing of the CD33 gene.
  • the cell is an animal cell. In some embodiments, the cell is a human cell.
  • a peptide-antisense oligonucleotide conjugate for use in a method of treating a subject having a neurodegenerative disease, comprising administering to said subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate.
  • the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length.
  • the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more non-natural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).
  • PMO phosphorodiamidate morpholino oligomer
  • the peptide-antisense oligonucleotide conjugate has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for PMO ASOs.
  • the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).
  • the antisense oligonucleotide has a CD33 Exon-2 skipping efficiency of 30% or greater according to a Standard Exon-Skipping Efficiency Assay for MOE ASOs.
  • the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.
  • all of the one or more non-natural internucleotide linkages have an Sp configuration. In some embodiments, all of the one or more non-natural internucleotide linkages have an Rp configuration. In some embodiments, the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration.
  • the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the peptide-antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the neurodegenerative disease is Alzheimer’s Disease.
  • a peptide comprising a cyclic peptide comprising a lipoic acid group is an (R)-lipoic acid group. In some embodiments, the lipoic acid group is an (S)- lipoic acid group. In some embodiments, the cyclic peptide comprises 4 to 40 amino acids, optionally wherein the cyclic peptide comprises 6 to 10 amino acids. In some embodiments, the cyclic peptide comprises 1 to 5 arginine residues, optionally wherein the cyclic peptide comprises 2 to 4 arginine residues. In some embodiments, the cyclic peptide comprises 1-5 aromatic hydrophobic amino acids, optionally wherein the cyclic peptide comprises 2-4 aromatic hydrophobic amino acids. In some embodiments, the cyclic peptide is chosen from:
  • the cyclic peptide comprises two or more lipoic acid groups.
  • the two or more lipoic acid groups comprise two or more (R)-lipoic acid groups, two or more (S)-lipoic acid groups, and/or one or more (R)-lipoic acid groups and one or more (S)-lipoic acid groups.
  • a peptide comprising a cyclic lactam group.
  • the peptide is a cell-penetrating peptide.
  • the peptide is a cyclic peptide.
  • the peptide comprises 4 to 40 amino acids, optionally wherein the peptide comprises 6 to 10 amino acids.
  • at least one amino acid of the peptide comprises the cyclic lactam group.
  • the cyclic lactam group is an eight, nine, or ten-membered ring.
  • the cyclic lactam group has a structure according to Formula III: wherein: R 1 and R 2 are each independently selected from the group consisting of H, an aryl group, a heteroaryl group, an alkylaryl group, an arylalkyl group, a linear alkyl group, a branched alkyl group, and a guanidine-comprising group, wherein each of R 1 and R 2 is optionally substituted with one or more substituents; and n is an integer from 1 to 3.
  • the cyclic lactam comprises at least one side chain group.
  • the side chain group is R 1 or R 2 .
  • R 1 and/or R 2 independently comprise a substituted or unsubstituted aryl group
  • the side chain group comprises a natural or non-natural group comprising aromatic group, a linear alkyl group, a branched alkyl group, a functionalized alkyl group, a guanidine group, a proline group, a lipophilic group, or an arginine group.
  • the aryl group is selected from the group consisting of a phenyl group, a benzyl group, and a naphthyl group.
  • R 1 and/or R 2 independently comprise a substituted or unsubstituted guanidine-comprising group.
  • the guanidine-comprising group is -(CFb ⁇ CNsFL
  • the peptide comprises 1-5 arginine residues, optionally wherein the peptide comprises 2-4 arginine residues.
  • cyclic peptide comprising at least one amino acid having a structure according to Formula IV:
  • R 1 comprises an aryl group or a guanidine-comprising group, wherein R 1 is optionally substituted with one or more substituents.
  • the aryl group is selected from the group consisting of a benzyl group, a phenyl group, and a naphthyl group.
  • the guanidine-comprising group is -(CH 2 ) 2 - CN 3 H 4 .
  • a cyclic peptide comprising at least one oxadiazole linkage having a structure according to Formula V: wherein R comprises a substituted or unsubstituted aryl group.
  • the aryl group is selected from the group consisting of a phenyl group, a benzyl group, a naphthyl group, and a methyl naphthyl group.
  • Fig. 1 shows the levels of CD33 mRNA in plasma and cerebrospinal fluid in patients relative to the rs3865444 SNP.
  • C rs3865444-C
  • A rs3865444-A.
  • FIG. 2 shows various cognitive results in patients with the rs3865444-A allele vs. patients with the rs201074739 indel frameshift allele.
  • FIG. 3 shows various physiological results in patients with the rs3865444-A allele vs. patients with the rs201074739 indel allele.
  • Fig. 4 shows the levels of CD33 mRNA in plasma and cerebrospinal fluid in patients relative to the rs201074739 indel.
  • Fig. 5 shows HPLC chromatogram and HRMS trace of PMO-424.
  • Fig. 6 shows HPLC chromatogram and HRMS trace of PMO-324.
  • Fig. 7 shows Tm of PMO-324, PMO-424, and PMO-224.
  • Fig. 8 shows HPLC chromatogram and HRMS trace of PMO-502.
  • Fig. 9 shows HPLC chromatogram and HRMS trace of PMO-402.
  • Fig. 10 shows Tm of PMO-402, PMO-502, and PMO-002.
  • FIG. 11 shows chromatogram of PMO-424 with N3’ -trityl group (resin cleaved).
  • Fig. 12 shows the melting temperature of MOE-012, MOE-277, and MOE-278.
  • FIG. 13 shows the HPLC elution profile of stereopure ASOs MOE-288 to
  • Fig. 14 shows in vitro Exon-2 skipping efficiencies for peptide-ASO conjugates at several doses using mouse bone-marrow derived macrophages.
  • Fig. 15 shows the in vivo activity of Compound 30 at a 30 pg dose and PMO- 002 at 30 pg, 100 pg and 300 pg doses.
  • Fig. 16 shows the duration of in vivo skipping activity of Compound 30 with a single 30 pg ICV dose.
  • Fig. 17 shows the brain concentration of Compound 30 and naked PMO-002 after a single 30 pg ICV dose.
  • Fig. 18 shows the in vivo skipping activity in the cortex of Compound 31 at 3 pg, 10 pg, 30 pg and 60 pg and Sp-PMO-424 at 30 pg and 100 pg ICV dose.
  • FIG. 19 shows the in vivo skipping activity in the cortex and hippocampus of lipoic acid-containing peptides Compound 32, Compound 33, and Compound 34 at a 10 pg ICV dose, with 10 pg Compound 30 for comparison.
  • Fig. 20 shows examples of cyclic lactam amino acids.
  • Fig. 21 shows examples of cell penetrating peptides with lactam building blocks.
  • Fig. 22 shows an exemplary synthesis of unsaturated amino acids.
  • Fig. 23 shows an exemplary synthesis of 8-membered lactams with phenyl and 2-naphthalene side chains.
  • Fig. 24 shows an exemplary synthesis of lactams with guanidine side chains.
  • Fig. 25 shows an exemplary synthesis of lactams via consecutive Claisen rearrangements.
  • Fig. 26 shows an exemplary synthesis of 9- and 10-membered lactam amino acid rings.
  • Fig. 27 shows an exemplary synthesis of 9- and 10-membered lactam amino acid rings with guanidine side chains.
  • Fig. 28 shows examples of cell penetrating peptides with modified proline residues.
  • Fig. 29 shows examples of cell penetrating peptides containing 1 ,3,4- oxadiazole linkages.
  • Fig. 30 shows in vitro cellular uptake data for Compound 160, Compound 161, and Compound 162.
  • Fig. 31 shows the in vivo activity of Compound 166/167 and Compound 168/169 at a 30 pg dose.
  • Fig. 32 shows pharmacodynamic data for Compound 31 and Compound 33 in a 90-day duration study with transgenic hCD33 mice.
  • Fig. 33 shows pharmacokinetic data for Compound 31 and Compound 33 in a 90-day duration study with transgenic hCD33 mice.
  • Fig. 34 shows efficacy data for Compound 31 in 5XFAD hCD33 mice.
  • oligonucleotide is used herein to refer to a nucleotide sequence comprising at least ten DNA or RNA nucleotides.
  • antisense oligonucleotide is used herein to refer to a nucleotide sequence comprising an antisense sequence that is sufficiently complementary to a target nucleotide sequence in order to form a stable double stranded hybrid with the target nucleotide sequence.
  • the target nucleotide sequence is an RNA nucleotide sequence.
  • ASOs represented herein are displayed in the 5' to 3' orientation.
  • peptide is used herein to refer to a compound comprising two or more proteogenic or non-proteogenic amino acids.
  • amino acid is used herein to refer to a chemical compound containing an amine group and a carboxylic acid group in the same compound.
  • proteogenic amino acid is used herein to refer to an amino acid that is naturally included in a peptide.
  • non-proteogenic amino acid is used herein to refer to an amino acid that is not naturally included in a peptide.
  • Non-proteogenic amino acids include naturally occurring amino acids or synthesized amino acids.
  • cell penetrating peptide or “CPP” is used herein to refer to a peptide that can penetrate a cell membrane, and can penetrate a cell membrane when conjugated to an antisense oligonucleotide.
  • nucleobase is used herein to refer to a base that is a component of a nucleoside.
  • Example nucleobases include adenine, guanine, thymine, cytosine, and uracil.
  • nucleoside is used herein to refer to a nucleobase covalently linked to a sugar. Examples of naturally occurring and non-natural nucleosides are described below.
  • nucleotide is used herein to refer to a nucleoside covalently linked to a phosphate group. Examples of naturally occurring nucleotides include adenosine, thymidine, uridine, cytidine, 5-methylcytidine, and guanosine. Description and examples of non-natural nucleotides are described below.
  • pharmaceutically acceptable salt is used herein to refer to acid addition salts or base addition salts of the compounds in the present disclosure.
  • a pharmaceutically acceptable salt is any salt which retains the activity of the parent compound and does not impart any unduly deleterious or undesirable effect on a subject to whom it is administered and in the context in which it is administered.
  • Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of both inorganic and carboxylic acids.
  • Pharmaceutically acceptable salts also include metal salts such as sodium, calcium, potassium, magnesium, aluminum, iron, manganese, and complex salts.
  • salts include, but are not limited to, acid salts such as acetic, aspartic, alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic, bicarbonic, bisulfuric, bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic, citric, edetic, edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic, gluconic, glutamic, glycolic, glycolylarsanilic, hexamic, hexylresorcinoic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic, methylnitric, methylsulfur
  • a CPP-ASO that is a pharmaceutically acceptable salt may comprise a pharmaceutically acceptable salt of a CPP of the CPP-ASO, a pharmaceutically acceptable salt of an ASO of the CPP-ASO, and/or a pharmaceutically acceptable salt of any linker that may conjugate a CPP to an ASO of the CPP-ASO.
  • the phosphate groups are commonly referred to as forming the “internucleotide linkages” of the ASO.
  • the naturally occurring internucleotide linkage of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • a “phosphoram idate” group comprises phosphorus having three attached oxygen atoms and one attached nitrogen atom, while a “phosphorodiamidate” group comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms.
  • a “phosphorotriamidate” group (or a phosphoric acid triamide group) comprises phosphorus having one attached oxygen atom and three attached nitrogen atoms.
  • one nitrogen is always pendant to the linkage chain.
  • the second nitrogen, in a phosphorodiamidate linkage, is typically the ring nitrogen in a morpholino ring structure.
  • non-natural is used herein to refer to molecules that contain manmade modifications relative to their naturally occurring counterparts.
  • “non-natural” may refer to one or more nucleotide subunits having at least one modification selected from (i) a modified internucleotide linkage, e.g., an internucleotide linkage other than the standard phosphodiester linkage found in naturally-occurring oligonucleotides, (ii) modified sugar moieties, e.g., moieties other than ribose or deoxyribose moieties found in naturally occurring oligonucleotides, (iii) modified nucleobases, e.g., bases other than those found in naturally occurring oligonucleotides, or (iv) any combination of the foregoing.
  • a modified internucleotide linkage e.g., an internucleotide linkage other than the standard phosphodiester linkage found in naturally-occurring oligon
  • the ASO of a CPP-ASO is chosen from ASOs that do not have a phosphorus atom in the internucleotide linkage (backbone). In some embodiments, the ASO has a phosphorodiamidate or phosphorothioate modified internucleotide linkage (backbone).
  • morpholino is used herein to refer to a nucleotide that contains a morpholinyl ring instead of a ribose.
  • morpholino-based ASO is used herein to refer to an ASO with at least one nucleotide containing a morpholinyl ring instead of a ribose.
  • stereo-controlled is used herein to describe when a nucleotide and/or an oligonucleotide is designed or selected to have a particular stereochemistry.
  • the nucleobase portion of a nucleotide or oligonucleotide, including any and all non-natural modifications is stereo-controlled.
  • the nucleoside portion of a nucleotide or oligonucleotide, including any and all non-natural modifications is stereo-controlled.
  • the internucleotide linkage portion of a nucleotide or oligonucleotide, including any and all non-natural modifications is stereo-controlled.
  • a nucleotide may comprise one or a combination of these stereocontrolled portions.
  • an oligonucleotide may comprise a combination of nucleotides that comprise a combination of stereo-controlled nucleotides.
  • an oligonucleotide may comprise a combination of nucleotides that are stereo-controlled and not stereo-controlled.
  • the proportion of stereo-controlled nucleotides ranges from 10%- 100%, such as 15%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 50%- 90%, 50%-95%, 60%-100%, 60%-90%, 60%-95%, 70%-100%, 70%-90%, 70%- 95%, 80-100%, 80%-90%, 80%-95%, 90-100%, 90%-95%, 90%-96%, 90%-97%, 90%-98%, 90%-99%, 95%-98%, 95%-99%, 95-100%, 50%-90%, or 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of nucleotides.
  • stereopure When applied to nucleotides, the term “stereopure” is used herein to describe when at least 90% of nucleotides in an oligonucleotide are stereo-controlled.
  • the proportion of stereo-controlled nucleotides in a stereopure CPP- ASO ranges from 90-100%, 95-100%, 90%-95%, 90%-96%, 90%-97%, 90%-98%, 90%-99%, 95%-98%, 95%-99%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of nucleotides.
  • nucleotides within an oligonucleotide are stereo-controlled so that they are stereopure in the same way, i.e. , all or a portion of the nucleotides are stereo-controlled, and they are designed or selected to have the same stereochemistry.
  • all or a portion of nucleotides within an oligonucleotide are stereo-controlled so that they are not stereopure in the same way, i.e., all or a portion of the nucleotides are stereocontrolled, but they are designed or selected to have different stereochemistry.
  • stereopure When applied to the internucleotide linkage portion of an oligonucleotide, the term “stereopure” is used to describe when at least 90% of the internucleotide linkages are stereo-controlled.
  • the proportion of stereo-controlled internucleotide linkages in a stereopure CPP-ASO ranges from 90-100%, 95-100%, 90%-95%, 90%-96%, 90%-97%, 90%-98%, 90%-99%, 95%-98%, 95%-99%, or 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of internucleotide linkages.
  • all or a portion of internucleotide linkages within an oligonucleotide are stereo-controlled so that they are stereopure in the same way, i.e. , all or a portion of the internucleotide linkages are stereo-controlled, and they are designed or selected to have the same stereochemistry.
  • all or a portion of internucleotide linkages within an oligonucleotide are stereo-controlled so that they are not stereopure in the same way, i.e., all or a portion of the internucleotide linkages are stereo-controlled, but they are designed or selected to have different stereochemistry.
  • the internucleotide linkages are phosphorodiamidate linkages.
  • the internucleotide linkages are phosphorothioate linkages.
  • the term “stereorandom” is used herein to describe when the nucleotides in an oligonucleotide are not stereo-controlled.
  • the term “stereorandom” is used herein to describe when the internucleotide linkages in an oligonucleotide are not stereocontrolled.
  • the internucleotide linkages are phosphorodiamidate linkages.
  • the internucleotide linkages are phosphorothioate linkages.
  • hybridize is used herein to describe the binding of two complementary nucleotide sequences, forming one double stranded molecule. When a sufficient number of corresponding nucleotides in two sequences can hydrogen bond with each other, i.e. , they are sufficiently complementary, they may form a stable hybrid. It is understood in the art that 100% complementarity is not necessary for a CPP-ASO to hybridize with a target sequence.
  • the term “sufficient complementarity” is used herein to indicate a level of complementarity sufficient to permit an ASO of a CPP-ASO to bind to its target sequence and form a stable hybrid.
  • the complementarity of the ASO and the target sequence is at least 99%, or 98%, or 97%, or 96%, or 95%, or 94%, or 93%, or 92%, or 91 %, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81 %, or 80%, or 79%, or 78%, or 77%, or 76%, or 75%, or 74%, or 73%, or 72%, or 71 %, or 70%.
  • sequence similarity is used herein to express the similarity of two CPP-ASOs. Sequence similarity is expressed as a percentage of nucleotides shared between two CPP-ASOs. It is understood that identical sequences have 100% sequence similarity.
  • target region and “target sequence” are used interchangeably herein to designate a nucleotide sequence to which an ASO of a CPP-ASO will hybridize under physiological conditions. It is not necessary for the ASO and the target region to be 100% complementary, so long as there is sufficient complementarity for the ASO to hybridize to the target sequence and form a stable hybrid. The ASO may hybridize to all or a portion of the target sequence.
  • treat refers to ameliorating a disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • the terms also refer to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • modulating the disease or disorder either physically (e.g., through stabilization of a discernible symptom), physiologically, (e.g., through stabilization of a physical parameter), or both.
  • the terms “prevent,” “preventing,” or “prevention” are used herein to refer to inhibiting or delaying the onset of a disease or disorder.
  • the term “therapeutically effective amount” is used herein to refer to the amount of a therapeutic agent or composition effective in prevention or treatment of a disorder or disease. In some embodiments, this includes an amount of a therapeutic agent or composition effective in the prevention or treatment of a neurodegenerative disease.
  • pharmaceutically acceptable is used herein to refer to a molecular entity or composition that is pharmaceutically useful and not biologically or otherwise undesirable.
  • carrier is used herein to refer to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • excipient refers to any ingredient in a pharmaceutical composition other than the active ingredient.
  • skipping efficiency of an oligonucleotide is calculated using the following formula: and is represented on a scale of 0 to 100, wherein 100 represents 100% skipping of CD33 Exon-2. “Skipping efficiency” of an oligonucleotide as used herein is experimentally determined using one of three Standard Exon-Skipping Efficiency Assays depending on the type of antisense oligonucleotide.
  • the Standard Exon-Skipping Efficiency Assay for PMO CPP-ASOs defined below is used; for antisense oligonucleotides comprising methoxyethyl ribose oligomers, the Standard Exon-Skipping Efficiency Assay for MOE CPP-ASOs defined below is used; and for antisense oligonucleotides that do not comprise phosphorodiamidate morpholino or methoxyethyl ribose oligomers, the Standard Exon-Skipping Efficiency Assay for non-PMOs and non-MOEs described below is used.
  • the Standard Exon-Skipping Efficiency Assay for CPP-ASOs includes using mouse bone-marrow derived macrophages (mBMDM) cells that were cultured and maintained using appropriate media suggested in the vendor protocols (Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum). The Assay is performed in 96 well plate format, seeding about 50,000 cells per well and treating with the PMO CPP-ASO at a concentration of 0.5 pM without additional transfection reagents. Cells are incubated at 37°C in a cell culture incubator for 48 hours before isolating the total RNA.
  • mBMDM mouse bone-marrow derived macrophages
  • RNA transcripts Total RNA is isolated and converted to cDNA per vendor protocol, then Taqman gene expression assays are used to quantify Exon-2 skipped CD33 (Forward primer: CGCTGCTGCTACTGCTG (SEQ ID NO:207); Reverse Primer: TTCTAGAGTGCCAGGGATGA (SEQ ID NO:208); and probe: TGTGGGCAGACTTGACCCACAG (SEQ ID NQ:209)) and un-skipped CD33 (Forward primer: GGATG GAGAGAG GAAGTA (SEQ ID NQ:210) or TTCGGATGGAGAGAGGAAGTA (SEQ ID NO:291 ); Reverse Primer: GTGCCAGGGATGAGGATTT (SEQ ID NO:211); and probe: TGCATGTGACAGACTTGACCCACA (SEQ ID NO:212)) mRNA transcripts.
  • Mouse house-keeping gene HPRT1 Assay ID: Hs02800695_m1 ; ThermoFisher Scientific
  • alkyl is used herein to refer to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tertbutyl, sec-butyl, isobutyl, etc.), cyclic alkyl groups (also referred to as “cycloalkyl” groups), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups).
  • alkenyl and alkynyl refer to unsaturated aliphatic groups that are analogous to alkyls but contain at least one double or triple carbon-carbon bond, respectively.
  • alkoxy is used herein to refer to an alkyl group linked to the remainder of the molecule through an oxygen atom.
  • alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.
  • the alkoxy groups can be straight-chain or branched.
  • alkoxyalkyl is used herein to refer to an alkyl group substituted with an alkoxy group.
  • arylalkyl is used herein to refer to an alkyl group substituted with an aryl group (e.g., phenylmethyl (i.e., benzyl)).
  • alkylaryl is used herein to refer to an aryl group substituted with an alkyl group (e.g., p-methylphenyl (i.e., p-tolyl)).
  • Carbocycle and “carbocyclic” are used herein to a closed ring hydrocarbon structure (e.g., monocyclic, polycyclic ring structure).
  • Carbocyclic groups may be saturated or unsaturated. Additionally, carbocyclic groups may be aromatic or non-aromatic.
  • heterocycle and “heterocyclic” are used herein to refer to a closed ring structure (e.g., monocyclic, polycyclic ring structure) in which one or more of the atoms in the closed ring structure is a heteroatom (i.e., an atom other than carbon).
  • a heteroatom i.e., an atom other than carbon.
  • Certain exemplary heteroatoms include nitrogen, oxygen, and sulfur.
  • Heterocyclic groups may be saturated or unsaturated. Additionally, heterocyclic groups may be aromatic or non-aromatic.
  • the CPP- ASOs are directed to a target sequence in the CD33 pre-m RNA.
  • the CPP-ASOs are complementary to all or a portion of a target sequence in the CD33 pre-mRNA, represented in SEQ ID NO:1 (5 -GGGCAGGTGA GTGGCTGTGG GGAGAGGGGT TGTCGGGCTG GGCCGAGCTG ACCCTCGTTT CCCCACAGGG GCCCTGGCTA TGGATCCAAA TTTCTGGCTG CAAGTGCAGG AGTCAGTGAC GGTACAGGAG GGTTTGTGCG TCCTCGTGCC CTGCACTTTC TTCCATCCCA TACCCTACTA CGACAAGAAC TCCCCAGTTC ATGGTTACTG GTTCCGGGAA GGAGCCATTA TATCCAGGGA CTCTCCAGTG GCCACAAACA AGCTAGATCA AGAAGTACAG GAGGAGACTC AGGGCAGATT
  • SEQ ID NO:1 includes Exon-2 and portions of the bordering introns of the CD33 gene. This target sequence is involved in Exon-2 skipping, which also occurs when CD33 mRNA includes the rs3865444-A SNP. When this Exon-2 skipping occurs, pre-mRNA containing the SNP is spliced so that Exon-2 is not included in the final transcript.
  • the CPP-ASO has a CD33 Exon-2 skipping efficiency of at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% according to a Standard Exon-Skipping Efficiency Assay for the CPP-ASO.
  • the CPP- ASO has a CD33 Exon-2 skipping efficiency in a range of 25% to 99%, 30% to 99%, 35% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99% according to a Standard Exon-Skipping Efficiency Assay for the CPP- ASO.
  • the CPP-ASO has a CD33 Exon-2 skipping efficiency of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% according to a Standard Exon-Skipping Efficiency Assay for the CPP- ASO.
  • the ASO of a CPP-ASO is 16-30 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 20-30 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 25-30 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 21-30 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 21-25 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 18-21 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 18-25 nucleotides long. In some embodiments, the ASO of a CPP-ASO is 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long.
  • the ASO of a CPP-ASO comprises 16-30, such as 18- 30, nucleotides. In some embodiments, the ASO of a CPP-ASO consists of 16-30, such as 18-30, nucleotides.
  • novel CPP-ASOs complementary to all or a portion of a 10- to 16-nucleotide target sequence in the CD33 pre-mRNA, represented in SEQ ID NO:1 , which includes Exon-2 and portions of the bordering introns of the CD33 gene.
  • the ASO of the CPP-ASOs are 10-14 nucleotides long.
  • the ASO of the CPP-ASOs are 10, 11 , 12, 13, 14, 15, or 16 nucleotides long.
  • the CPP-ASOs are complementary to all or a portion of a 16- to 30-nucleotide target sequence in the CD33 pre-RNA, are sufficiently complementary to the target sequence to form a stable hybrid, and are 16-30 nucleotides in length.
  • the ASO portions of the CPP-ASOs are sufficiently complementary to all or a portion of a 25-nucleotide target sequence in the CD33 pre-RNA.
  • the ASO of the CPP-ASOs have one of the specific sequences disclosed in Table 1 or 2.
  • PMO and MOE ASOs were designed to cover CD33 Exon-2 and its surrounding introns (SEQ ID NO:1 ) in 20-25 nucleotide sections that moved down SEQ ID NO:1 5' to 3' five nucleotides at a time. Regions that exhibited increased Exon-2 skipping activity were identified where two or more consecutive PMO or MOE ASOs that are complementary to a section of SEQ ID NO:1 showed increased Exon-2 skipping activity. Those regions are identified below and in Table 3:
  • Region 1 (SEQ ID NO:213) (see, e.g., PMO-002 and PMO-003)
  • Region 2 (SEQ ID NO:214) (see, e.g., PMQ-036, PMQ-037, PMQ-004, PMO- 038, PMQ-039, and PMQ-005)
  • Region 3 (SEQ ID NO:215) (see, e.g., PMQ-082, PMQ-083, and PMQ-006)
  • Region 4 (SEQ ID NO:216) (see, e.g., PMQ-096, PMQ-007, and PMQ-097)
  • Region 5 (SEQ ID NO:217) (see, e.g., MQE-009, MOE-128, and MQE-010)
  • Region 6 (SEQ ID NO:218) (see, e.g., MOE-135, MQE-011 , and MQE-012)
  • Region 7 (SEQ ID NO:219) (see, e.g., MQE-015, MOE-183, and MOE-184)
  • Region 8 (SEQ ID NQ:220) (see, e.g., MOE-196 and MOE-197).
  • the ASO of a CPP-ASO is complementary to a region of SEQ ID NO:1 showing increased Exon-2 skipping activity, including but not limited to Regions 1 , and 2, 3, 4, 5, 6, 7, and 8.
  • the ASO of a CPP- ASO is complementary to at least a portion of SEQ ID NO:213; SEQ ID NO:214;
  • SEQ ID NO:215 SEQ ID NO:216; SEQ ID NO:217; SEQ ID NO:218; SEQ ID NO:219; and/or SEQ ID NQ:220.
  • the ASO of a CPP-ASO is complementary to at least a portion of Region 1 of SEQ ID NO: 1 .
  • the ASO of the CPP-ASO has a sequence disclosed in Table 4:
  • the ASO of a CPP-ASO is complementary to at least a portion of Region 2 of SEQ ID NO: 1 .
  • the ASO of the CPP-ASO has a sequence disclosed in Table 5:
  • the ASO of a CPP-ASO is complementary to at least a portion of Region 6 (SEQ ID NO:218) of SEQ ID NO:1.
  • the ASO of the CPP-ASO has a sequence disclosed in Table 6:
  • the CPP-ASOs may share sequence similarity with one of the CPP-ASOs disclosed in Tables 1 , 2, and 4 to 6.
  • the CPP-ASO shares at least 99%, or 98%, or 97%, or 96%, or 95%, or 94%, or 93%, or 92%, or 91 %, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81 %, or 80%, or 79%, or 78%, or 77%, or 76%, or 75%, or 74%, or 73%, or 72%, or 71 %, or 70% sequence similarity with one of the CPP-ASOs disclosed in Tables 1 , 2, and 4 to 6.
  • one or more nucleobases of the ASO of the CPP- ASOs comprise uracil. In some embodiments, one or more nucleobases of the ASO of a CPP-ASO comprise thymine. In some embodiments, one or more nucleosides of the ASO of a CPP-ASO comprise a ribose sugar moiety. In some embodiments, one or more nucleosides of the ASO of a CPP-ASO comprise a deoxyribose sugar moiety.
  • the ASO of the CPP-ASOs comprise at least one chemically modified nucleotide.
  • the at least one chemical modification of the nucleotide is chosen from chemical modification of at least one nucleobase, chemical modification of at least one sugar moiety, chemical modification of at least one phosphate, and any combination of these modifications.
  • the at least one chemical modification improves the ability of the nucleotide to resist nuclease degradation.
  • Certain exemplary chemical modifications useful in this disclosure include chemical modifications of an ASO’s phosphate backbone and non-natural internucleoside linkage(s).
  • the ASO of a CPP-ASO is chosen from ASOs having a chemically modified phosphate backbone.
  • the chemically modified phosphate backbone comprises one or more nitrogen atoms and/or one or more sulfur atoms.
  • one or more non-bridging oxygen atoms in the phosphate backbone e.g., in phosphodiester linkages
  • the ASO of a CPP-ASO has a phosphoramidate, phosphorodiamidate, phosphorodithioate, or phosphorothioate modified backbone.
  • the ASO of a CPP-ASO is chosen from ASOs that do not have a phosphorus atom in the backbone.
  • the modified backbone is stereo-controlled.
  • Certain exemplary chemical modifications useful in this disclosure include chemical modifications of at least one sugar moiety in an ASO.
  • the ASO of a CPP-ASO comprises at least one chemically modified (e.g., a non-natural) sugar moiety.
  • the ASO of a CPP-ASO comprises at least one chemically modified (e.g., a non-natural) sugar moiety that is chosen from sugar moieties substituted in at least one position.
  • the at least one chemically modified (e.g., a non-natural) sugar moiety is substituted in at least one position on the sugar chosen from the 2', 3' and 5' positions.
  • the at least one substituent on the ASO’s at least one substituted sugar moiety is chosen from hydroxyl; fluoro; alkoxy; amino; and substituted or unsubstituted, linear or branched C1-C10 alkyl groups, substituted or unsubstituted, linear or branched C2- C10 alkenyl groups, substituted or unsubstituted, linear or branched C2-Cio alkynyl groups, substituted or unsubstituted, linear or branched C7-C17 alkylaryl groups, substituted or unsubstituted, linear or branched C3-C10 allyl groups, substituted or unsubstituted, linear or branched C7-C17 arylalkyl groups, and substituted or unsubstituted, linear or branched C2-C10 alkoxyalkyl groups, each of which groups may optionally further comprise at least one heteroatom.
  • At least one substituent on at least one substituted sugar moiety of an ASO of a CPP- ASO comprises methoxy, methoxyethyl, ethoxy, propoxy, aminopropoxy, methoxyethoxy, dimethylaminoethoxy, or dimethylaminoethoxyethoxy.
  • one or more sugar moieties of the ASO of a CPP-ASO are chosen from pyranoses, derivatives of pyranoses, deoxypyranoses, derivatives of deoxypyranoses, riboses, derivatives of riboses, deoxyriboses, and derivatives of deoxyriboses.
  • the sugar moiety is stereo-controlled.
  • the ASO of a CPP-ASO comprises at least one sugar moiety that is modified in a manner that creates a bicyclic sugar moiety.
  • the bicyclic sugar moiety is formed from a bridge modification between the 4' and 2' furanose ring atoms.
  • the bridge modification comprises at least one group that forms a bridge between the 4' and 2' furanose ring atoms.
  • at least one nucleotide in a given ASO of a CPP-ASO has a bridge modification.
  • at least one nucleotide in an ASO of a CPP-ASO is a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the ASO of a CPP-ASO comprises at least one sugar moiety comprising fewer than 5 ring atoms, such as 4 ring atoms. In some embodiments, the ASO of a CPP-ASO comprises at least one sugar moiety comprising more than 5 ring atoms, such as 6 ring atoms. In some embodiments, the ASO of a CPP-ASO comprises at least one sugar moiety comprising a morpholinyl ring. In some embodiments, the ASO of a CPP-ASO is a morpholino-based ASO.
  • a morpholino-based ASO refers to an ASO comprising morpholino subunits, where morpholinyl rings replace ribose moieties.
  • Certain exemplary internucleotide linkages for such morpholino-based ASOs include, for example, phosphoram idate or phosphorodiamidate internucleotide linkages joining the morpholinyl ring nitrogen of one morpholino subunit to the 4' exocyclic carbon of an adjacent morpholino subunit.
  • Each morpholino subunit comprises a purine or pyrimidine nucleobase, which may bind by base-specific hydrogen bonding to a nucleobase in a target sequence.
  • the morpholino-based ASO may include at least one further modification.
  • the ASO of a CPP-ASO is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the ASO of a CPP-ASO has the structure of Formula I: [144]
  • B is any nucleobase described herein and n is an integer in a range from 1 to 19, 1 to 23, 1 to 28, 1 to 30, 8 to 19, 8 to 23, 8 to 28, 8 to 30, 14 to 19, 14 to 23, 14 to 28, 14 to 30, 16 to 19, 16 to 23, 16 to 28, or 16 to 30.
  • the ASO of a CPP-ASO is a methoxyethyl ribose oligomer (MOE). In some embodiments, the ASO of a CPP-ASO has the structure of Formula II:
  • B is any nucleobase described herein and m is an integer in a range from 1 to 19, 1 to 23, 1 to 28, 1 to 30, 8 to 19, 8 to 23, 8 to 28, 8 to 30, 14 to 19, 14 to 23, 14 to 28, 14 to 30, 16 to 19, 16 to 23, 16 to 28, or 16 to 30.
  • both the sugar moiety and the linkage between the nucleobase and the sugar moiety of at least one nucleotide unit in the ASO of a CPP-ASO are replaced with non-natural groups.
  • the nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound.
  • the ASO of a CPP-ASO is chosen from peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • the sugar-backbone of at least one oligonucleotide in the PNA is replaced with an am ide-containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • the ASO of a CPP-ASO comprises at least one nonnatural nucleobase (often referred to as “base”) (e.g., a nucleobase comprising one or more modifications or substitutions).
  • bases include 5-substituted pyrimidines (e.g., 5-methyl cytosine, 5-propynyl cytosine, 5- propynyl uracil), 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including but not limited to 2-aminopropyladenine.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1 ,2°C.
  • the modified nucleobase is stereo-controlled.
  • an ASO of a CPP-ASO may comprise one or more chemically modified nucleotides and one or more chemically unmodified nucleotides.
  • ASOs may contain at least one region wherein the nucleotides are modified to confer upon them increased resistance to nuclease degradation, increased cellular uptake, and/or an additional region for increased binding affinity for the target nucleic acid.
  • Certain exemplary ASOs comprising a plurality of modifications to nucleobases, sugar moieties, and/or internucleotide linkages include ASOs having a sequence shown in Table 7.
  • each nucleotide has a 2’-MOE ribose sugar moiety
  • each C represents a 5-methyl cytosine
  • each lower case letter represents a locked nucleic acid
  • each ( - ) represents a phosphodiester (PO) bond
  • each fX represents a 2’-fluoro ribonucleotide
  • each mX represents a 2’-0Me ribonucleotide.
  • nucleotides may share the same molecular formula but have a different spatial arrangement, i.e., some nucleotides may be stereoisomers.
  • modification of a phosphodiester internucleotide linkage by replacing one or more oxygens with one or more nitrogen and/or sulfur atoms may cause the phosphorus atom of the linkage to become a chiral center.
  • the ASO of a CPP-ASO comprises one or more P-chiral internucleotide linkages in an Sp or Rp configuration.
  • Sp and Rp configurations of an exemplary phosphorothioate linkage are shown below:
  • ASO of a CPP-ASO are not controlled so as to make the ASO stereorandom.
  • the nucleotides within a given ASO of a CPP-ASO are stereocontrolled.
  • one or more nucleotides within a given ASO are stereo-controlled so as to make the ASO of a CPP-ASO stereopure.
  • a given ASO of a CPP-ASO is a combination of stereo-controlled and stereorandom nucleotides.
  • the proportion of stereo-controlled nucleotides in the ASO of a CPP-ASO is in a range from 10%-100%, 15%-100%, 20%-100%, 30%- 100%, 40%-100%, 50%-100%, 50%-90%, 50%-95%, 60%-100%, 60%-90%, 60%- 95%, 70%-100%, 70%-90%, 70%-95%, 80-100%, 80%-90%, 80%-95%, 90-100%, 90%-95%, 90%-96%, 90%-97%, 90%-98%, 90%-99%, 95%-98%, 95%-99%, 95- 100%, 50%-90%, or 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the proportion of Sp internucleotide linkages in the ASO of a CPP-ASO is at least 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the proportion of Sp internucleotide linkages in the ASO of a CPP- ASO is 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the proportion of Rp internucleotide linkages in the ASO of a CPP-ASO is at least 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the proportion of Rp internucleotide linkages in the ASO of a CPP- ASO is 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. [159] In some embodiments, the ASO of a CPP-ASO is a stereopure PMO ASO having a sequence disclosed in Table 8:
  • the ASO of a CPP-ASO is a stereopure MOE ASO having a sequence disclosed in Table 9:
  • the ASO of a CPP-ASO comprises at least two regions. In some embodiments, the ASO of a CPP-ASO comprises three regions: one region near the 5' end of the ASO, one region near the 3' end of the ASO, and a gap region between the two other regions. This type of arrangement is known as a gapmer motif.
  • each motif can be equal to other motifs within the ASO of a CPP- ASO, or the length of each motif can be independent of the length of other motifs within the ASO.
  • one or more sugar moieties in an ASO of a CPP-ASO are modified so that a block of sugar moieties in one region of the ASO is different from a block of sugar moieties in a different region of the ASO.
  • an ASO of a CPP-ASO comprises modified sugar moieties arranged in a gapmer motif.
  • one or more nucleobases in an ASO of a CPP-ASO are modified so that a block of nucleobases in one region of the ASO is different from a block of nucleobases in a different region of the ASO.
  • an ASO of a CPP-ASO comprises modified nucleobases arranged in a gapmer motif.
  • one or more internucleotide linkages in an ASO of a CPP-ASO are modified so that a block of internucleotide linkages in one region of the ASO is different from a block of internucleotide linkages in a different region of the ASO.
  • a given ASO of a CPP-ASO comprises modified internucleotide linkages arranged in a gapmer motif.
  • one or more stereo-controlled nucleotides in an ASO of a CPP-ASO are modified so that a block of stereo-controlled nucleotides in one region of the ASO are different from a block of stereo-controlled nucleotides in a different region of the ASO.
  • an ASO of a CPP-ASO comprises stereo-controlled nucleotides arranged in a gapmer motif.
  • an ASO has more than one motif.
  • an ASO has more than one motif independent of each other.
  • the CPP-ASO conjugates described herein comprise a CPP conjugated to an ASO.
  • the CPP-ASO conjugate is capable of penetrating a cell membrane so that the CPP-ASO conjugate enters the cytosol of the cell.
  • more than one CPP is conjugated to an ASO.
  • a CPP-ASO conjugate comprises 2, 3, 4, or 5 CPPs conjugated to an ASO.
  • the more than one CPP includes at least two different CPPs.
  • the more than one CPP is two or more of the same CPPs.
  • conjugating more than one CPP to an ASO increases ASO activity.
  • the CPP comprises from four to 40 amino acids.
  • the CPP has 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21 , or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31 , or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39, or 40 amino acids.
  • the CPP is conjugated directly or indirectly to an ASO. In some embodiments, the CPP is conjugated at the 5' end of the ASO. In some embodiments, the CPP is conjugated at the 3' end of the ASO. In some embodiments, the CPP is conjugated at any nucleotide in the ASO. Certain methods of conjugating CPPs are known in the art. In some embodiments, the CPP is conjugated to the ASO at the C-terminus. In some embodiments, the CPP is conjugated to the N-terminus. In some embodiments, the CPP is conjugated to the ASO via a side chain of any amino acid in the CPP. In some embodiments, the CPP is covalently linked to the ASO. In some embodiments, the CPP is chemically conjugated to the ASO. In some embodiments, the CPP is non-covalently linked to the ASO.
  • the CPP comprises proteogenic and/or non- proteogenic amino acid(s).
  • Certain exemplary amino acids include alanine, betaalanine, allo-isoleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, glutamic acid, glutamine, glycine, histidine, homoproline, isoleucine, leucine, lysine, methionine, napthylalanine, norleucine, phenylalanine, phenylglycine, 4- (phosphonodifluoromethyl)phenylalanine, proline, sarcosine, selenocysteine, serine, threonine, tyrosine, tryptophan, valine, tert-butyl-alanine, penicillamine, homoarginine, nicot
  • the CPP contains at least one non-proteogenic amino acid.
  • Certain exemplary non-proteogenic amino acids include allo-isoleucine, beta-alanine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, homoproline napthylalanine, norleucine, phenylglycine, 4- (phosphonodifluoromethyl)phenylalanine, sarcosine, selenocysteine, tert-butyl- alanine, penicillamine, homoarginine, nicotinyl-lysine, triflouroacetyl-lysine, methylleucine, 3-(3-benzothienyl)-alanine, 6-aminohexanoic acid, and 5-aminopentanoic acid.
  • the at least one non-proteogenic amino acid comprises a modified proline (e.g., a substituted proline).
  • the CPP contains at least one proteogenic amino acid.
  • Certain exemplary proteogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine.
  • the CPP contains L- or D-amino acids.
  • the at least one proteogenic or non-proteogenic amino acid is substituted with one or more substituents.
  • substituents include halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, and arylthiol groups.
  • the CPP contains synthetic amino acid mimics, for example, replacement of the peptide amide bond.
  • the CPP contains a non-natural structure, for example, a non-peptide group that does not contain an amino acid.
  • the CPP comprises one or more modified or unmodified arginine residues. In some embodiments, the CPP comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 arginine residues. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amino acids of the CPP comprise a modified or unmodified arginine residue.
  • the CPP comprises one or more hydrophobic amino acids.
  • hydrophobic amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, and tryptophan.
  • at least one of the one or more hydrophobic amino acids may be substituted with one or more substituents.
  • the CPP comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 hydrophobic amino acids.
  • at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amino acids of the CPP comprise a hydrophobic amino acid.
  • the one or more hydrophobic amino acids comprise one or more aromatic hydrophobic amino acids.
  • aromatic hydrophobic amino acids include phenylalanine, tryptophan, tyrosine, naphthylalanine, 3-(3-benzothienyl)-alanine, phenylglycine, and homophenylalanine.
  • at least one of the one or more aromatic hydrophobic amino acids may be substituted with one or more substituents.
  • the CPP comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 aromatic hydrophobic amino acids.
  • the CPP comprises one or more arginine residues and one or more hydrophobic amino acids. In some embodiments, the CPP comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 arginine residues and 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 hydrophobic amino acids. In some embodiments, the CPP comprises one or more arginine residues and one or more aromatic hydrophobic amino acids. In some embodiments, the CPP comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 arginine residues and 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 aromatic hydrophobic amino acids.
  • the CPP comprises one or more modified or unmodified lysine residues. In some embodiments, the CPP comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 lysine residues.
  • the CPP is linear.
  • Certain exemplary linear CPPs include Pip6a peptide (Wood M. JA et al, Mol Therapy - Nucleic Acids, 2012, 1 , e38), ApoE peptide (Gait, M. J. et al, Nucleic Acid Therapeutics, 2017, 27, 130), neurotensin-based peptides (Prakash, T. P. et al, J. Med. Chem.
  • the CPP is a linear peptide having a sequence disclosed in Table 10.
  • B is beta-alanine and X is 6-aminohexanoic acid:
  • the CPP is a cyclic cell penetrating peptide (“cCPP”).
  • cCPPs include CPP9, CPP12 (Pei, D. et al. Biochemistry, 2016, 55, 2601) and others as outlined in Tiwari, K. et al. Mol. Pharmaceutics 2019, 16, 9, 3727.
  • a cyclic CPP of a CPP-ASO has one of the following structures:
  • the CPP is synthesized using a solid-phase approach and/or a solution phase approach. In some embodiments, the CPP is synthesized using both a solid phase and solution phase approach where part of the synthesis occurs using a solid phase and another part of the synthesis occurs using a solution phase. Further information about synthesis of certain CPPs according to some embodiments is included in the Examples below.
  • the CPP comprises a lipoic acid group.
  • the lipoic acid moiety is an (R)-lipoic acid group.
  • the lipoic acid moiety is an (S)-lipoic acid group.
  • the lipoic acid group is conjugated to a lysine residue of the CPP.
  • the lipoic acid group is conjugated to a non-lysine residue (e.g., a 5-aminopentanoic acid) of the CPP.
  • the CPP comprises one to five lipoic acid groups.
  • the CPP comprises one, two, three, four, or five lipoic acid groups.
  • a cyclic CPP of a CPP-ASO has one of the following structures:
  • a CPP comprising a lipoic acid group may be synthesized according to an exemplary synthesis scheme described in the Examples below.
  • the CPP comprises one or more lactam amino acids. In some embodiments, the CPP comprises 1 , 2, 3, 4, or 5 lactam amino acids. In some embodiments, each lactam amino acid is independently an 8, 9, or 10- membered ring.
  • the one or more lactam amino acids each independently have a structure according to Formula III:
  • R 1 and R 2 are each independently selected from the group consisting of H, an aryl group, a heteroaryl group, an alkylaryl group, an arylalkyl group, a linear or branched alkyl group, and a guanidine-comprising group, each of which may be independently substituted with one or more substituents, and wherein n is an integer from 1 to 3.
  • R 1 and/or R 2 comprise an aryl group. In some embodiments, R 1 and R 2 each independently comprise an aryl group.
  • the aryl group may be monocyclic or polycyclic. Certain exemplary monocyclic aryl groups include phenyl and benzyl groups. An example of a polycyclic aryl group is a naphthyl group.
  • R 1 and/or R 2 comprise a guanidine-comprising group. In some embodiments, R 1 and R 2 each independently comprise a guanidine-comprising group.
  • An example of a guanidine-comprising group is (CH2)2CNsH4.
  • R 1 and R 2 are H. In some embodiments, R 1 is H and R 2 is phenyl. In some embodiments, R 1 is phenyl and R 2 is H. In some embodiments, R 1 and R 2 are phenyl. In some embodiments, R 1 is 2-naphthyl and R 2 is H. In some embodiments, R 1 is H and R 2 is 2-naphthyl. In some embodiments, R 1 and R 2 are 2- naphthyl. In some embodiments, R 1 is 2-naphthyl and R 2 is phenyl. In some embodiments, R 1 is (CH2)2CNsH4 and R 2 is H.
  • R 1 is H and R 2 is (CH2)2CNSH4. In some embodiments, R 1 is (CH2)2CNsH4 and R 2 is 2-naphthyl. In some embodiments, R 1 and R 2 are (CH2)2CNsH4.
  • n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
  • a CPP comprising a lactam amino acid has a structure shown below:
  • a CPP comprising a lactam amino acid may be synthesized according to an exemplary synthesis scheme shown in Figs. 22-27 and the Examples below.
  • the CPP comprises one or more modified proline residues.
  • the one or more modified proline residues comprise one or more substituents.
  • Certain exemplary suitable substituents include an aryl group and a guanidine-comprising group. Examples of suitable aryl groups include phenyl, benzyl, and naphthyl groups. An example of a suitable guanidine-comprising group includes, but is not limited to, -(CH2)2-CNsH4.
  • the one or more modified proline residues of the CPP each independently have a structure according to Formula IV:
  • R 1 is an aryl group or a guanidine-comprising group.
  • R 1 is a guanidine-comprising group.
  • R 1 is -(CH2)2-CNsH4.
  • R 1 is an aryl group.
  • R 1 is benzyl.
  • R 1 is phenyl.
  • R 1 is naphthyl.
  • a CPP comprising a modified proline residue has a structure shown below:
  • a CPP comprising a modified proline residue may be synthesized according to an exemplary synthesis scheme shown in the Examples below.
  • the CPP comprises one or more oxadiazole linkages.
  • the one or more oxadiazole linkages have a structure according to Formula V:
  • R is a substituted or unsubstituted aryl group.
  • aryl groups include phenyl, benzyl, naphthyl, and methyl naphthyl.
  • a CPP comprising an oxadiazole linkage has a structure according to Formula VI:
  • R is a substituted or unsubstituted aryl group.
  • R is phenyl, benzyl, naphthyl, or methyl naphthyl.
  • a CPP comprising an oxadiazole linkage may be synthesized according to an exemplary synthesis scheme shown in the Examples below.
  • a CPP-ASO conjugate further comprises a nuclear localization sequence (NLS).
  • a nuclear localization sequence generally refers to an amino acid sequence that facilitates transport of molecules comprising the sequence into the nucleus of eukaryotic cells.
  • the nuclear localization sequence may be a monopartite or bipartite nuclear localization sequence.
  • Suitable nuclear localization sequences include sequences comprising all or a portion of one or more of the following sequences: PKKKRKV (SEQ ID NO: 273) from simian virus 40 (SV40), PKLKRQ (SEQ ID NO: 274), RPRK (SEQ ID NO: 275), RRARRPRG (SEQ ID NO: 276), KRPAATKKAGQAKKKK (SEQ ID NO: 277) from nucleoplasmin, PAAKRVKLD (SEQ ID NO: 278) and RQRRNELKRSP (SEQ ID NO: 279) from c- myc, RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 280) from the IBB domain of importin-alpha, VSRKRPRP (SEQ ID NO: 281 ) and PPKKARED (SEQ ID NO: 282) from polyomavirus large T, PQPKKKPL (SEQ ID NO: 283) from human p53
  • the NLS is covalently or non-covalently coupled to the CPP, ASO, and/or linker of a CPP-ASO. In some embodiments, the NLS is covalently or non-covalently coupled to the CPP of a CPP-ASO. In some instances, the CPP is a linear peptide. In some instances, the CPP is a cyclic peptide. In some embodiments, the NLS is covalently or non-covalently coupled to the ASO of a CPP- ASO. In some embodiments, the NLS is covalently or non-covalently coupled to a linker coupling a CPP and an ASO.
  • the antisense molecules used in accordance with this disclosure may be made through well-known techniques of solid phase synthesis. Equipment for such synthesis is available from several sources including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.
  • oligonucleotides such as phosphorothioates and alkylated derivatives.
  • diethyl-phosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., Tetrahedron Letters, 22:1859- 1862 (1981 ).
  • the ASOs are synthesized in a way so that all nucleotides of the ASO are stereopure.
  • the ASOs are synthesized in vitro and do not include antisense compositions of biological origin.
  • the ASOs may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures, or mixtures of compounds, as for example, liposomes, lipids, receptor targeted molecules for assisting in uptake, distribution and/or absorption.
  • the antisense oligonucleotides are conjugated to cell penetrating peptides using known chemical reactions. Examples are found in: Gait. M. J. et al. Curr. Pharm. Des. 2005, 11, 3639; Prescher, J. A. et al. Nat. Rev. Chem.
  • the CPP is conjugated to the ASO via strain-promoted azide-alkyne cycloaddition reaction (“click chemistry”). In some embodiments, the CPP is conjugated to the ASO via strained alkene-tetrazine cycloaddition reaction. In some embodiments, the CPP is conjugated to the ASO by an amide bond. In some embodiments, the CPP is conjugated to the ASO using one of the bonds in the image below:
  • the CPP is conjugated directly to the ASO. In some embodiments, the CPP is indirectly conjugated to the ASO with a linker between the CPP and ASO.
  • the linker comprises an alkyl group, a carbocyclic group, a heterocyclic group, a polyethylene glycol, or one or more of these groups.
  • the linker comprises one or more proteogenic or non-proteogenic amino acids. In some embodiments, the one or more proteogenic or non-proteogenic amino acids comprise sarcosine. In some embodiments, the linker is a cleavable linker.
  • Certain exemplary suitable cleavable linkers include linkers comprising valine-citrulline (“Val-Cit”), valine-alanine (“Val-Ala”), glutamic acid-valine-citrulline (“Glu-Val-Cit”), and/or alanine-alanine-asparagine (“Ala-Ala- Asn”).
  • the CPP-ASOs are used to induce Exon-2 skipping during processing of CD33 pre-mRNA.
  • at least one CPP- ASO disclosed herein is used to induce Exon-2 skipping in CD33 pre-mRNA during pre-mRNA splicing.
  • the at least one CPP-ASO is introduced into a cell, wherein the at least one CPP-ASO is complementary to all or a portion of SEQ ID NO:1 , wherein the CPP-ASO hybridizes to a target region of the CD33 gene, and wherein the CPP-ASO induces Exon-2 skipping during pre-mRNA splicing of the CD33 gene.
  • the CPP-ASO administered to induce Exon-2 skipping during pre-mRNA splicing comprises one of SEQ ID NOS: 2-10. In some embodiments, the CPP-ASO administered to induce Exon-2 skipping during pre- mRNA splicing comprises one of SEQ ID NOS:2-15, 36-39, 82, 83, 96, 97, 128, 132, 135, 136, 183, 184, 190, 196, 197, 202, 224, or 252. In some embodiments, the CPP-ASO administered to induce Exon-2 skipping during pre-mRNA splicing comprises one of SEQ ID NOS: 2, 12, 224, or 252.
  • a CPP-ASO can be introduced by transfection along with one or more transfection agents.
  • excipients or transfection agents are capable of forming complexes, nanoparticles, micelles, vesicles, and/or liposomes that help deliver each CPP-ASO complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art.
  • Suitable excipients or transfection agents include LipofectAMINETM 2000 (Invitrogen), Endo-Porter peptide, polyethylenimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), LipofectinTM, DOTAP and/or viral capsid proteins that are capable of self-assembly into particles that can be used when delivering a CPP-ASO to a cell.
  • Their high transfection potential is combined with an expected low to moderate toxicity in terms of overall cell survival.
  • the ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
  • the methods comprise administering a therapeutically effective amount of at least one CPP-ASO disclosed herein. In some embodiments, the methods comprise administering a therapeutically effective amount of at least one CPP-ASO that hybridizes to all or a portion of SEQ ID NO:1 . In some embodiments, the methods comprise administering a therapeutically effective amount of at least one CPP-ASO comprising one of SEQ ID NOS:2-10.
  • the methods comprise administering a therapeutically effective amount of at least one CPP-ASO comprising one of SEQ ID NOS:2-15, 36-39, 82, 83, 96, 97, 128, 132, 135, 136, 183, 184, 190, 196, 197, 202, 224, or 252.
  • the methods comprise administering a therapeutically effective amount of at least one CPP-ASO comprising one of SEQ ID NOS: 2, 12, 224, or 252.
  • the neurodegenerative disease is characterized by a mutation in the CD33 gene.
  • the neurodegenerative disease is characterized by an aberrant microglial phenotype.
  • the neurodegenerative disease is Alzheimer’s Disease, microfibromialgia, or multiple sclerosis.
  • the CPP-ASO administered to a subject having a neurodegenerative disease may be administered in a pharmaceutical composition.
  • the amount of CPP-ASO administered in a pharmaceutical composition may be dependent on the subject being treated, the subject’s weight, the manner of administration, and the judgment of the prescribing physician.
  • a dosing schedule may involve the daily or semidaily administration of the pharmaceutical composition at a perceived dosage of about 1 pg to about 1000 mg.
  • intermittent administration such as on a weekly, monthly, quarterly, or yearly basis, of a dose of the pharmaceutical composition may be employed.
  • physicians will readily determine optimum dosages and will be able to readily modify administration to achieve such dosages.
  • a therapeutically effective amount of a compound or composition disclosed herein can be measured by the therapeutic effectiveness of the compound.
  • the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being used.
  • the therapeutically effective amount of a disclosed compound is sufficient to establish a maximal plasma concentration.
  • preliminary doses as, for example, determined according to animal tests, and the scaling of dosages for human administration is performed according to artaccepted practices.
  • toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • compositions that exhibit large therapeutic indices are desirable.
  • data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans.
  • therapeutically effective dosages achieved in one animal model may be converted for use in another animal, including humans, using conversion factors known in the art (see, e.g., Freireich et al., Cancer Chemother. Reports 50(4):219244 (1966).
  • the CPP-ASOs herein may be administered in a pharmaceutical composition comprising therapeutically effective amounts of an CPP-ASO together with pharmaceutically acceptable excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH, and ionic strength, and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol).
  • the material may be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
  • Hyaluronic acid may also be used.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and/or rate of in vivo clearance of the present CPP-ASOs and derivatives.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • a pharmaceutical composition comprising a CPP-ASO and a pharmaceutically acceptable carrier or excipient may be prepared for administration according to techniques well known in the pharmaceutical industry. In some embodiments, such techniques include combining the CPP-ASO with the carrier and/or excipient(s) into association in a unit dosage form.
  • compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a compound of the present disclosure as powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • such formulations may be prepared by any suitable method which includes the step of bringing into association at least one embodiment of the present disclosure as the active compound and at least one carrier or excipient (which may constitute one or more accessory ingredients).
  • the at least one carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and is not deleterious to the recipient.
  • the carrier may be a solid or a liquid, or both, and may be formulated with at least one compound described herein as the active compound in a unit-dose formulation, for example, a tablet, which may contain from about 0.05% to about 95% by weight of the at least one active compound.
  • other pharmacologically active substances may also be present including other compounds.
  • the formulations of the present disclosure may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • liquid pharmacologically administrable compositions can, for example, be prepared by, for example, dissolving or dispersing, at least one active compound of the present disclosure as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • suitable formulations may be prepared by uniformly and intimately admixing the at least one active compound of the present disclosure with a liquid or finely divided solid carrier, or both, and then, if desired, shaping the product.
  • a tablet may be prepared by compressing or molding a powder or granules of at least one embodiment of the present disclosure, which may be optionally combined with one or more accessory ingredients.
  • compressed tablets may be prepared by compressing, in a suitable machine, at least one embodiment of the present disclosure in a free-flowing form, such as a powder or granules, which may be optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent(s).
  • molded tablets may be made by molding, in a suitable machine, where the powdered form of at least one embodiment of the present disclosure is moistened with an inert liquid diluent.
  • formulations suitable for buccal (sub-lingual) administration include lozenges comprising at least one embodiment of the present disclosure in a flavored base, for example, sucrose and acacia or tragacanth, and pastilles comprising the at least one compound in an inert base such as gelatin and glycerin or sucrose and acacia.
  • formulations suitable for parenteral administration comprise sterile aqueous preparations of at least one embodiment of the present disclosure, which are approximately isotonic with the blood of the intended recipient.
  • these preparations are administered intravenously, although administration may also be affected by subcutaneous, intramuscular, intraperitoneal, intracerebroventricular, or intradermal injection.
  • these preparations are administered via osmotic pump.
  • such preparations may conveniently be prepared by admixing at least one embodiment described herein with water and rendering the resulting solution sterile and isotonic with the blood.
  • injectable compositions according to the present disclosure may contain from about 0.1 to about 5% w/w of the active compound.
  • formulations suitable for rectal administration are presented as unit-dose suppositories.
  • these may be prepared by admixing at least one embodiment as described herein with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • formulations suitable for topical application to the skin may take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • carriers and excipients which may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof.
  • the CPP-ASO is generally present at a concentration of from about 0.1 % to about 15% w/w of the composition, for example, from about 0.5 to about 2%.
  • ASO antisense oligonucleotide
  • CPP-ASO cell penetrating peptide conjugated to an antisense oligonucleotide
  • RNA ribonucleic acid
  • mRNA messenger ribonucleic acid
  • SNP single nucleotide polymorphism
  • PNA peptide nucleic acid
  • DOTAP 1 ,2 dioleoyl 3 trimethylammoniopropane
  • PEI polyethylenimine
  • HATLI Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium
  • DIPEA N,N-diisopropylethylamine
  • HPRT1 hypoxanthine phosphoribosyltransferase 1
  • GAPDH1 glyceraldehyde 3 phosphate dehydrogenase 1
  • NTC non-targeting control
  • Trt trityl
  • SNP rs3865444 was reported to be associated with an increased skipping of Exon-2 of CD33 and with reduced levels of full length CD33 on the surface of monocytes.
  • the allele was found to be associated with decreased levels of full length CD33 in human cerebrospinal fluid (CSF) and plasma when measured using Somascan technology (Fig. 1).
  • CSF human cerebrospinal fluid
  • Fig. 2 the Alzheimer’s Disease Neuroimaging Initiative
  • the allele was found to be associated with decreased ventricle volume and increased midtemporal volume, which are both consistent with protection against Alzheimer’s Disease (Fig. 2).
  • the allele was associated with improved slope for Alzheimer’s Disease Assessment Scale (ADAS) 11 , mini-mental state examination (MMSE), Rey Auditory Verbal Learning Test (RAVLT) immediate, Trial Making TestB (TRABSCOR), Functional Activities Questionnaire (FAQ), 18 F-fluorodeoxyglucose-positron emission tomography (FDG PET), ventricle volume, fusiform gyrus, and midtemporal volume (Fig. 3), indicating protection against the disease.
  • ADAS Alzheimer’s Disease Assessment Scale
  • MMSE mini-mental state examination
  • RAVLT Rey Auditory Verbal Learning Test
  • TABSCOR Trial Making TestB
  • FAQ Functional Activities Questionnaire
  • FDG PET F-fluorodeoxyglucose-positron emission tomography
  • ventricle volume ventricle volume
  • fusiform gyrus fusiform gyrus
  • midtemporal volume Fig. 3
  • rs201074739 is a 4-base pair deletion in exon3 of the CD33 gene. This causes a frameshift in the open reading frame and a premature translation termination.
  • the indel was associated with decreased levels of full length CD33 in human CSF and plasma when measured using SomaScan technology (Fig. 4). However, this indel has not been associated with a reduced risk of the disease so far. Moreover, it was associated with increased ventricle volume and a worse functional activities questionnaire (FAQ) score, suggesting a deleterious effect (Fig. 2).
  • FAQ functional activities questionnaire
  • PMO oligonucleotides were designed for screening.
  • the designed oligonucleotides listed in Tables 11 and 12 below were made by GeneTools LLC (www ⁇ Table 11 lists the top PMO oligonucleotides with their deconvoluted MS data.
  • Table 1 includes the top PMO oligonucleotides in Table 11 , as well as other PMO oligonucleotides. All PMO oligonucleotides listed in Tables 11 and 1 contain a phosphorodiamidate-attached sarcosine linker (Sar) at the 5’ end. All PMO oligonucleotides in Tables 11 and 1 were synthesized with unmodified cytosine PMO nucleotide.
  • Sar phosphorodiamidate-attached sarcosine linker
  • PMO oligonucleotides listed in Tables 11 and 1 have stereorandom internucleotide linkages, and thus are called stereorandom PMO oligonucleotides.
  • the general formula of the PMO oligonucleotides listed in Tables 11 and 1 is:
  • MOE oligonucleotides were designed for screening.
  • the designed oligonucleotides listed in Tables 12 and 2 were made by either Integrated DNA Technologies (www. idtdna. com ) or GeneDesign (Ajinomoto Bio Pharma, https://aiibio-pharma.com/).
  • Table 12 lists the top MOE sequences with their deconvoluted MS data. All MOE oligonucleotide listed in Tables 12 and 2 contain a hydroxyl at the 5’ end. All MOE oligonucleotides listed in Tables 12 and 2 contain 2’- O-MOE-modified ribonucleotides with phosphorothioate backbone except when noted. All MOE oligonucleotides listed in Tables 12 and 2 were synthesized with 5- methylcytosine 2’-O-MOE ribonucleotide. All MOE oligonucleotides listed in Tables
  • MOE oligonucleotides 12 and 2 have stereorandom internucleotide linkages, and thus are called stereorandom MOE oligonucleotides.
  • the general formula of the MOE oligonucleotides listed in Tables 12 and 2 depicted as free form is:
  • the reaction mixture was stirred for 30 min and monitored by UPLC-MS. Upon completion, MTBE (14 mL) was added over 1 minute with a syringe. The suspension was stirred for 10 min and then sonicated. The suspension was filtered over a sintered filter funnel and rinsed with MTBE 10mL (2x5mL). The solids were dried, transferred to a new flask and then dissolved by addition of DCM (3.5mL). 1 ,2,2,6, 6-pentamethylpiperidine (292 pL) was added via syringe. After 10 min at rt, MTBE (15.8mL) was added over 1 minute. White solids were formed.
  • reaction mixture was stirred for 2 hours at rt. Upon completion, MTBE (15 mL) was added. The solids were filtered and rinsed with MTBE (10mL). The solids were dried and then transferred to a flask and dissolved by addition of DCM (2.7 mL).
  • Trityl deblock solution was prepared as follows: To a flask were added DCM (8 mL), 2,2,2-trifluoroethanol (2 mL), 4-cyanopyridine (100 mg), ethanol (100 pL) and trifluoroacetic acid (105 mg) in that order. The solution was mixed until all components are dissolved and then used in deprotection as is.
  • Step 1 - trityl deprotection To a flask with “trityl-protected PMO oligonucleotide” (1 wt, 1 equiv.) was added trityl deblock solution (8 volumes compared to trityl-protected PMO oligonucleotide mass). The reaction mixture was stirred for 5-30 minutes and monitored by UPLC MS. Upon completion (>99.5% target), added EtOAc (10-40 vols) and MTBE (10-40 volumes) to form a white precipitate. The solids were filtered on a sintered funnel, rinsed with EtOAc/MTBE 1 :1 , dried under vacuum and collected to afford “TFA salt PMO oligonucleotide” for the next step.
  • Step 2 free basing: To a flask with “TFA salt PMO oligonucleotide” (1 wt, 1 equiv.) was added DCM (7-10 vols compared to TFA salt PMO oligonucleotide mass) and EtOH (0.3-0.5 vol). The solution was treated with 1 , 2, 2,6,6- pentamethylpiperidine (5 equiv.). The reaction mixture was stirred for 5-10 minutes and then treated with EtOAc (10-40 vols) and MTBE (10-40 volumes) to form a white precipitate. The solids were rinsed with EtOAc/MTBE 1 :1 , dried under vacuum and collected for the next step.
  • PMO oligonucleotides were designed for screening. The designed oligonucleotides were made by GeneTools LLC (webs ite : w : flene tools : co ) by solid-phase method. Table 13 below lists synthesized PMO oligonucleotides with their deconvoluted MS data. These PMO oligonucleotides are complementary to a section of SEQ ID NO:1 showing increased Exon-2 skipping activity. In particular, PMO-221 through PMO 240, PMO-324, PMO-424, PMO-402 and PMO-502 are complementary to Region 1 ; and PMO-241 through PMO-244 are complementary to Region 2.
  • PMO oligonucleotides listed in Table 13 below contain a phosphorodiamidate-attached sarcosine (Sar) linker at the 5’ end. All PMO oligonucleotides listed in Table 13 below were synthesized with unmodified cytosine PMO nucleotide. All PMO oligonucleotides listed in Table 13 below have stereorandom internucleotide linkages, and thus are called stereorandom PMO oligonucleotides.
  • the structure of PMO-224 is as follows:
  • the synthesis includes iterative steps of deprotection/free basing/coupling as depicted here for all Rp internucleotide linkages):
  • Tm The Melting temperature (Tm) of PMO oligonucleotides:
  • Tm measurement device Shimadzu UV-2700 UV-Vis Spectrophotometer
  • ASO samples were prepared by dissolving ⁇ 0.6-0.8 mg of solid to ⁇ 3.2 ug/mL using nuclease free water.
  • Reverse complementary RNA obtained from IDT Technologies Inc.
  • 10 pL aliquots of each stock solution were diluted to 1 mL using nuclease free water to determine their concentrations by UV-Vis Spectrophotomer.
  • Test Samples 500 pL were prepared containing 4.0 pM PMO with 4.0 pM reverse complimentary RNA in buffer (100 mM NaCI, 10 mM Na Phosphate pH 7.0 with 0.1 mM EDTA).
  • Test samples were incubated in a 1 mL cuvette and heated from 15 °C to 105 °C at 0.5 °C/min. UV absorbance increase due to strand melting was monitored at 260 nm. Prior to the experiment, the samples were melted and reannealed by heating from 25 °C to 95 °C at 5 °C/min and cooling to starting temperatures to ensure complete annealing. Shimadzu Tm Analysis software was used to calculate the Tm (curve inflection point: 50% melting) using the derivative function.
  • Fmoc-SAR-Wang resin purchased from Aapptec, RWG103, Lot#9953380, 0.65 mmol/g, 110-200 mesh
  • DMF 8 m L
  • the resin was treated with 20% piperidine in DMF (6 mL), shaked for 3 minutes, removed solvent, and dried for 1 minute under N2 gas (repeated the same sequence for 4 times).
  • the resin was washed with DMF (5 mL x 5 times), washed with CH2CI2 (5 mL x 5 times), and dried under vacuum using N2 gas for overnight to give 0.8 g of resin.
  • Fig. 11 shows the UV chromatogram of trityl-protected 21-mer (all-Sp-Sar-CCTCACCTGTCACATGCACAG-Tr) after cleavage from resin.
  • the synthesized PMO- loaded resin was dried, transferred to centrifugal bottle, and charged with 7N NHs/MeOH ( ⁇ 0.5 mL/1 pmol). The mixture was stirred at 50 - 55 °C for 60 hours. The reaction was cooled to room temperature, filtered the solids, and washed with methanol. The resulting filtrate was concentrated under reduced pressure to approximate final volume of ⁇ 20 mL, then, filtered any solids over 0.4 micron membrane filter. The filtrate was concentrated to dryness and weighed. The obtained crude residue was dissolved with 60 mL of solvent mixture of aq. 50 mM EtsNHOAc (used cell culture water)/MeCN (1/1) with EtsN (0.1 %). The filtrate was purified by reversed phase HPLC conditions as shown in Table 19.
  • oligonucleotides listed in Table 22 contain a 2’-O-MOE modified ribonucleotides and a hydroxyl group at the 5’ end. Oligonucleotides in Table 22 contain stereopure phosphorothioate internucleotide linkages, and thus are called stereopure MOE oligonucleotides. All oligonucleotides listed in Table 22 are complementary to Region 6: (SEQ ID NO:218).
  • Step 1 To 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(2- methoxyethoxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1 H,3H)-dione (14.2 g, 44.893 mmol) in pyridine (99 mL, 1228.96 mmol) was added 1-[chloro-(4- methoxyphenyl)-phenylmethyl]-4-methoxybenzene (18.25 g, 53.871 mmol) at room temperature.
  • Step 2 To an aqueous solution of Na2COs (242 mL, 121 .225 mmol) were added 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1 H,3H)- dione (25 g, 40.408 mmol) in DCM (250 mL, 3885.69 mmol), Tetrabutylammoniumhydrogensulfate (5.49 g, 16.163 mmol), and chloromethyl pivalate (7.30 g, 48.49 mmol) at room temperature.
  • the resulting precipitate was filtered, and washed subsequently with water (100 mL) and n- heptane (125 mL).
  • the filter cake was dissolved in CH2CI2 (200 mL) and the aqueous layer was removed.
  • the organic layer was concentrated in vacuo to ca. 50 mL and treated with n-heptane (75 mL).
  • the mixture was stirred at room temperature for 20 min and concentrated in vacuo to ca. 50 mL.
  • the resulting precipitate was filtered, washed with n-heptane (20 mL), and dried over N2 purge for 2 hours to give the title compound (30.1 g, 91 %).
  • Sp phosphorothioate linkage was obtained using Rp-PSI-monomers that were prepared from (-)-PSI reagent; Rp phosphorothioate linkage was obtained using Sp-PSI-monomers that were synthesized from (+)-PSI, and PO internucleotide linkages were obtained using PO-PSI monomers 1 .
  • the cartridge was washed with 2 N NaCI/MeCN (5/1 , v/v) to elute truncated sequences, and 3% TFA in water (150 mL), then water (50 mL).
  • the crude DMTr-off PS-oligonucleotide was eluted with 50 mL of aceton itrile-water (1 :1 , v/v) containing 0.5% of 28% NH4OH.
  • the solution containing crude DMTr-off oligonucleotide was dried under vacuum. The weight was measured by Nanodrop (RNA-40) and 31 P NMR was taken. It was analyzed by RP-HPLC, IEX-HPLC and UPLC/MS.
  • the absorbance of the diluted solution was measured at 260 nm on a Nanodrop UV-Vis spectrophotometer to give a yield (7 ⁇ 15% yield) and endotoxin level was confirmed to be less than 0.06 EU/mg by a kinetic chromogenic LAL method (Charles River, Endosafe® nexgen-PTS).
  • Tm measurement device Shimadzu UV-2700 UV-Vis Spectrophotometer
  • Protocol 1 ASO samples were prepared at a concentration of 400 pM using deionized water. IDT’s reverse complementary RNA (rcRNA) was dissolved to 400 pM using UltraPure Distilled water. 10 pL aliquots of each stock solutions were diluted to 1 mL using ultra pure distilled water and their actual concentrations were measured by UV-Vis Spectrophotomer. Test samples (500 pL) were prepared containing 4.0 pM ASO with 4.0 pM rcRNA in buffer (100 mM NaCI, 10 mM Na phosphate pH 7.0 with 0.1 mM EDTA).
  • Test samples were incubated in a 1 mL cuvette and heated from 15 °C to 105 °C at 0.5 °C/minute. UV absorbance increase due to strand melting was monitored at 260 nm. Prior to the experiment, the samples were melted and reannealed by heating from 25 °C to 95 °C at 5 °C/m inute and cooling to starting temperatures to ensure complete annealing. Shimadzu Tm Analysis software was used to calculate the Tm (curve inflection point: 50% melting) using the derivative function.
  • Protocol 2 ASO samples were prepared at a concentration of 200 pM using PBS and then followed the same procedure as protocol 1 with adjusted amount.
  • Fig. 12 shows the Tms of MOE-012, MOE-277, and MOE-278.
  • Fig. 13 shows an example of overlay HPLC chromatogram (MOE-252 and
  • Example 12 BCN-functionalized-PMO-002 (Compound 10)
  • R 1 and R 2 are the options shown for (Compound 13) in the box above: Ac-RXRRBRRXRYQFLIRXRBRXRB-OH.
  • Example 14 General Procedure C for attachment of (/?)-lipoic acid (LA) to Compounds 18, 19 and 20.
  • Example 16 General procedure E for peptide-PMO conjugate by amide bond
  • PMO-002 (12 mg, 1 .43 pmol) and DMSO (100 pL) were added to a first vial and the suspension was warmed to 37 °C until a clear solution was formed.
  • 13 (7.5 mg, 2.57 pmol, 1.8 equiv) and N-methylpyrroldinone (50 pL) were added to a second vial.
  • HOBT 0.5 mg, 2.8 pmol, 2.0 equiv.
  • Hunig’s base (1 pL, 5 pmol, 3.5 equiv) and N-methylpyrroldinone (50 pL) were added to the second vial and mixed to ensure uniformity.
  • Example 17 Exon-Skipping Efficiency Assay in mouse bone-marrow derived macrophages (mBMDM) cells in vitro
  • Mouse house-keeping gene HPRT1 was used to normalize the target transcript expressions.
  • mice were necropsied 1 week after the injection, or longer in the case of a duration study. At necropsy, mice were transcardially perfused with PBS under avertin anesthesia. Brains were rapidly removed from the skull, and the cortex and hippocampus were dissected from the injected hemisphere for use in exon skipping evaluation. For RNA isolation, frozen tissue was added with 9X volume of Trizol and homogenized for 3 minutes. 500 pL of the Trizol lysate was transferred to a 1 mL deep well plate. 100 pL of chloroform was added to each sample, shaken vigorously, and centrifuged at 4000xg for 5 minutes.
  • Compounds 30, 31 and 33 were quantified in mouse cortex and hippocampus using a hybridization-based immunoassay method (HELISA). Tissues were lysed in TRIzol, 1 :10 (Thermo Fisher Scientific, Waltham, MA), and were diluted in hybridization buffer (1 :100, 1 M NaCI in TE-Buffer and 0.1 % Tween®20). Compound 30 was spiked in diluted tissue homogenate to prepare standard curves and quality controls (QC). 35 pL of diluted samples, standards, and QCs were transferred to a 96-well PCR plate.
  • HELISA hybridization-based immunoassay method
  • 35 pl of detection probe solution (5’- GTGACAGGTGAGG/3Bio/-3’ (for compound 30, 33, Integrated DNA Technologies, Inc, Coralville, IA), 5’-/5DigN/CTGTGCATGT-3’ (for compound 31, Integrated DNA Technologies, Inc, Coralville, IA), 100 nM in hybridization buffer), was added to the PCR plate containing standards and samples. Sample and detection probe were hybridized on a thermal cycler under the following conditions: 95 °C for 10 minutes, 37 °C for 60 minutes, and a final hold at 4 °C.
  • MSD Gold 96-well Streptavidin SECTOR plate (Meso Scale Diagnostics, LLC., Rockville, MD) was blocked with 150 pL of Casein in TBS blocker (Thermo Fisher Scientific, Waltham, MA) at room temperature for 1 .5 hours. After washing with the wash buffer (Tris buffered saline with Tween®20, Sigma-Aldrich, St.
  • MSD GOLD SULFO-TAG label Anti-Digoxigenin, Fab fragment (made in-house from conjugation of MSD GOLD SULFO-TAG NHS-Ester (Meso Scale Diagnostics, LLC., Rockville, MD) with Anti-Digoxigenin, Fab fragments (Sigma-Aldrich, St. Louis) (in Casein-TBS Blocking Buffer and 0.05% Tween20.
  • Compound 30 The duration of Compound 30 was also evaluated.
  • a single 30 pg dose of Compound 30 maintained exon skipping up to 60 days in the mouse brain (cortex and hippocampus, Fig. 16).
  • PMO-002 showed peak activity at 7 days and declined in activity after 14 days.
  • Analysis of the brain concentration of Compound 30 compared to PMO-002 showed a dramatically improved PK profile by higher exposure.
  • Compound 30 had a 10-fold improvement in brain exposure relative to PMO-002 (Fig. 17).
  • Lipoic acid contains a 5-membered disulfide ring which can increase peptide interaction with proteins and improve cellular uptake. Lipoic acid was incorporated in conjugates Compounds 32, 33, and 34 by attachment to a lysine residue.
  • Compounds 32, 33, and 34 were tested in vivo at 10 pg doses (Fig. 19).
  • Compound 33 contains a lysine-N-lipoic acid conjugate in the macrocyclic ring instead of the phenylalanine present in Compound 30.
  • Compound 33 had improved skipping efficacy relative to Compounds 32 and 34, and when compared to Compound 30.
  • Example 19 Additional examples of cell-penetrating peptides
  • cell-penetrating peptides include peptides containing a cyclic lactam.
  • the cyclic lactam may contain an eight-, nine- or ten-membered ring.
  • the cyclic lactam may be constructed such that it contains side chains (R 1 , R 2 ) with aromatic, linear or branched alkyl groups and functionalized alkyl groups.
  • the side chain may contain guanidine group such as the one found in arginine which promotes cell-penetrating activity.
  • the stereochemistry of each center may be varied accordingly to achieve the best cell-penetrating potency.
  • lactam amino acids are listed in Fig. 20.
  • Examples of cell-penetrating peptides with lactam amino-acids AA1-AA10 are listed in Figure 21.
  • the synthesis of the lactam amino acids AA1-AA10 for use in peptide synthesis follows synthetic routes as illustrated in Figs. 22-27.
  • Additional examples of cell-penetrating peptides include peptides containing chemically modified proline residues (Fig. 28). Proline residues provide conformational bias which may not be achieved by acyclic amino acids.
  • the modified proline is constructed such that it contains side chain functional groups with aromatic, linear, and/or branched alkyl groups.
  • the side chain contains one or more guanidine group such as the one found in arginine.
  • the stereochemistry of each center may be varied accordingly to achieve the best cell-penetrating potency. For Examples 19-47, if a compound does not depict a specific stereochemical configuration, it includes every possible stereochemical configuration.
  • synthesis of proline modified with a guanidine side chain is conducted according to a previously reported method (Ishiguro et al. J. Med. Chem. 2004, 47, 489-492).
  • Another example includes novel peptide containing peptide-bond isosteres such as 1 ,3,4-oxadiazole as illustrated in Fig. 29.
  • the peptide is constructed by reported methodology (Yudin A. K. et al. Nature Chem. 8, 2016, 1104.)
  • the synthesis of the cyclic peptide portion follows general solid-phase synthesis protocols.
  • the linker between the peptide and the PMO is selected from linkers illustrated in compounds in Examples 15 and 16.
  • attachment of the peptide to the PMO is achieved as described in Examples 15 and 16 to construct the Peptide-PMO conjugate (e.g. amide bond formation, azide-alkyne click chemistry etc.).
  • Compound 41 was made by adding DMSO (22.4 ml, 315 mmol) dropwise to a solution of oxalyl chloride (13.8 ml, 158 mmol) in DCM (400 mL) at -78 °C under nitrogen. The solution was stirred at -78 °C for 10 minutes. A solution of 41-1 (28.1 ml, 131 mmol) in DCM (7.50 mL) was added slowly, and the reaction was stirred at -78 °C for 1 hour. Triethylamine (92 ml, 656 mmol) was then added, and the reaction mixture was maintained at -78 °C for 10 minutes before being warmed to room temperature and stirred for 2 hours.
  • Compound 115 is prepared from Compound 109 using General Procedure K.
  • Compound 120 is prepared from the Ethyl ester of Compound 115 using
  • Compound 121 is prepared from Compound 117 using General Procedure M.
  • Compound 122 is prepared from Compound 118 using General Procedure M.

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Abstract

L'invention concerne de nouveaux conjugués d'oligonucléotides antisens et de peptides de pénétration cellulaire qui induisent un saut d'Exon-2 dans le gène CD33 pendant l'épissage de pré-ARNm, et leur utilisation dans le traitement d'une maladie neurodégénérative, telle que la maladie d'Alzheimer.
PCT/US2023/078121 2022-10-27 2023-10-27 Oligonucléotides antisens-peptides et leur utilisation pour le traitement de troubles neurodégénératifs WO2024092256A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118707003A (zh) * 2024-08-28 2024-09-27 北京悦康科创医药科技股份有限公司 靶向pcsk9基因的寡核苷酸药物的中间体杂质检测方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
WO2017024264A2 (fr) 2015-08-05 2017-02-09 Eisai R&D Management Co., Ltd. Réactifs chiraux pour la préparation d'oligomères homogènes

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011060832A1 (fr) * 2009-11-20 2011-05-26 Polyphor Ag Composés peptidomimétiques fixés sur une matrice et présentant une activité antagoniste du ccr10
EP4058032A4 (fr) * 2019-12-19 2024-01-10 Entrada Therapeutics, Inc. Compositions pour administrer des composés antisens
JP2024518780A (ja) * 2021-04-28 2024-05-02 エーザイ・アール・アンド・ディー・マネジメント株式会社 神経変性障害の治療用アンチセンスオリゴヌクレオチド及びその使用
AU2022271873A1 (en) * 2021-05-10 2024-01-04 Entrada Therapeutics, Inc. Compositions and methods for intracellular therapeutics

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
WO2017024264A2 (fr) 2015-08-05 2017-02-09 Eisai R&D Management Co., Ltd. Réactifs chiraux pour la préparation d'oligomères homogènes

Non-Patent Citations (33)

* Cited by examiner, † Cited by third party
Title
ACS CHEM. BIOL., vol. 8, 2013, pages 423 - 431
AUGUSTO-OLIVEIRA ET AL.: "What Do Microglia Really Do in Healthy Adult Brain?", CELLS, vol. 8, 2019, pages 1293
AUSUBEL, F ET AL.: "Current Protocols in Molecular Biology", 2002, GREENE PUBLISHING ASSOCIATES/WILEY INTERSCIENCES
BEAUCAGE ET AL., TETRAHEDRON LETTERS, vol. 22, 1981, pages 1859 - 1862
BUSCHAUER, A, J. MED. CHEM., vol. 13, no. 59, 2016, pages 6045
CREIGHTON, C. J, BIOORG. MED. CHEM, vol. 12, 2004, pages 4375
DOWLING: "Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line", NATURE RΕV. NEUROLOGY, vol. 12, 2016, pages 675, XP055727395, DOI: 10.1038/nrneurol.2016.180
ECHIGOYA ET AL.: "Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges", J. PERS. MED, vol. 8, 2018, pages 41
EVSTIGNEEV ET AL., BIOPOLYMERS, vol. 93, no. 12, 2010, pages 1023 - 1038
FREIREICH ET AL., CANCER CHEMOTHER. REPORTS, vol. 50, no. 4, 1966, pages 219244
GAIT, M. J ET AL., NUCLEIC ACID THERAPEUTICS, vol. 27, 2017, pages 130
GLOVER: "DNA Cloning: A Practical Approach", vol. I-II, 1985, MRL PRESS, LTD
GRICIUC ET AL.: "Alzheimer's Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta", NEURON, vol. 78, 2013, pages 631
HUANG ET AL.: "A P(V) platform for oligonucleotide synthesis", SCIENCE, vol. 373, no. 6560, 2021, pages 1265 - 1270, XP055974798, DOI: 10.1126/science.abi9727
ISHIGURO ET AL., J. MED. CHEM., vol. 47, 2004, pages 489 - 492
J. SAMBROOKE. F. FRITSCHT MANIATIS: "Books", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS, article "Molecular Cloning: A Laboratory Manual"
JEON, J ET AL., MOLECULES, vol. 24, 2019, pages 3567
KNOUSE ET AL.: "Unlocking P(V): Reagents for chiral phosphorothioate synthesis", SCIENCE, vol. 361, no. 6408, 2018, pages 1234 - 1238, XP055593726, DOI: 10.1126/science.aau3369
LU, J. ET AL.: "Types of nuclear localization signals and mechanisms of protein import into the nucleus", CELL COMMUN SIGNAL, vol. 19, 2021, pages 60, XP093073531, DOI: 10.1186/s12964-021-00741-y
M. J. ET AL., CURR. PHARM. DES, vol. 11, 2005, pages 3639
MALIK ET AL.: "CD33 Alzheimer's Risk-Altering Polymorphism, CD33 Expression, and Exon 2 Splicing", J. NEUROSCIENCE, vol. 33, 2013, pages 13320, XP055283071, DOI: 10.1523/JNEUROSCI.1224-13.2013
MARTIN: "Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING CO
PEI, D ET AL., BIOCHEMISTRY, vol. 55, 2016, pages 2601
PRAKASH, T. P ET AL., J. MED. CHEM., vol. 63, 2020, pages 8471
PRESCHER, J.A. ET AL., NAT. REV. CHEM, vol. 4, 2020, pages 476
R. BEHRENDT ET AL., J. PEPT. SCI, vol. 22, 2016, pages 4 - 27
SONYOKOTA: "Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy", EXON SKIPPING & INCLUSION THERAPIES, 2018, pages 57 - 68
STETSENKO, D, MOLECULES, vol. 26, 2021, pages 5420
TIWARI, K. ET AL., MOL. PHARMACEUTICS, vol. 16, no. 9, 2019, pages 3727
WILLLUHRMANN: "Spliceosome Structure and Function", COLD SPRING HARB. PERSPECT. BIOL, vol. 3, 2011, pages 1
WOJTERA ET AL.: "Microglial cells in neurodegenerative disorders", FOLIA NEUROPATHOLOGY, vol. 43, 2005, pages 311
WOOD M. JA ET AL., MOL THERAPY - NUCLEIC ACIDS, vol. 1, 2012, pages e38
YUDIN A ET AL., NATURE CHEM, vol. 8, 2016, pages 1104

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