WO2024251725A1 - Oligonucléotides ciblant gal3st1 - Google Patents

Oligonucléotides ciblant gal3st1 Download PDF

Info

Publication number
WO2024251725A1
WO2024251725A1 PCT/EP2024/065312 EP2024065312W WO2024251725A1 WO 2024251725 A1 WO2024251725 A1 WO 2024251725A1 EP 2024065312 W EP2024065312 W EP 2024065312W WO 2024251725 A1 WO2024251725 A1 WO 2024251725A1
Authority
WO
WIPO (PCT)
Prior art keywords
antisense oligonucleotide
gal3st1
nucleotides
seq
modified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2024/065312
Other languages
English (en)
Inventor
Søren Vestergaard Rasmussen
Peter Hagedorn
Marianne LERBECH JENSEN
Tue FRYLAND
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Contera Pharma AS
Original Assignee
Contera Pharma AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Contera Pharma AS filed Critical Contera Pharma AS
Priority to EP24729318.6A priority Critical patent/EP4720283A1/fr
Publication of WO2024251725A1 publication Critical patent/WO2024251725A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/1137—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 enzymes
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • 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
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl
    • 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
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3525—MOE, methoxyethoxy
    • 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
    • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon
    • C12N2310/3533—Halogen
    • 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

Definitions

  • the present invention relates to antisense oligonucleotides (oligomers) complementary to GAL3ST1 pre-mRNA sequences, which are capable of inhibiting the expression of GAL3ST1. Inhibition of GAL3ST1 expression is expected to be beneficial for the treatment of metachromatic leukodystrophy.
  • Metachromatic leukodystrophy is a rare autosomal recessive lysosomal storage disorder caused by a deficiency in the arylsulfatase A (ARSA) enzyme. Mutations in the ARSA gene result in the accumulation of toxic sulfatides in the nervous system (e.g. oligodendrocytes, microglia, some CNS neurons) leading to progressive demyelination and neurological dysfunction.
  • MLD Clinical manifestation of MLD can vary depending on the age of onset, which can range from infancy to adulthood.
  • the most common form is the late-infantile form, which usually presents between 6 months and 2 years of age. Children with this form typically have developmental delay, followed by regression of skills, muscle weakness, spasticity, seizures, and vision and hearing loss.
  • onset occurs between 2 and 16 years of age, with symptoms including gait abnormalities, cognitive decline, and behavioral changes.
  • the adult form of MLD is the least common and usually presents after age 16, with symptoms such as cognitive decline, psychiatric symptoms, and motor dysfunction (Polten et al., 1991, Shaimardanova et al., 2020). In all forms of MLD, the disease is progressive, leading to severe disability and for most patients’ death.
  • MLD The prognosis for MLD is poor, with a life expectancy of 5-10 years for the late-infantile form, 10-20 years for the juvenile form, and variable in the adult form.
  • the severity and progression of the disease can be influenced by the age of onset, genotype, and residual ARSA enzyme activity.
  • MLD a progressive disease 2019
  • Bone marrow or hematopoietic stem cell transplantation has shown some success in slowing disease progression, particularly in patients with the late-infantile form.
  • this treatment is associated with significant risks and is only effective if done early in the disease course (Van Rappards et al., 2014, Boucher et al., 2015).
  • GAL3ST1 galactose-3-O-sulfotransferase-l
  • PAPS 3-phosphoadenosine-5-phosphosulfate
  • GalC galactosylceramide
  • GAL3ST1 As for the biosynthesis of sulfatides GAL3ST1 appears to be the sole responsible enzyme as Gal3stl homozygous knock out mice (GAL3ST1 completely lack sulfatides in brain (Honke et al., 2001). Moreover, it has been shown that overexpression of Gal3stl in Arsa (-/-) mice leads to increased sulfatide storage in the nervous system and augmentation of the MLD like pathology (Ramakrishnan et al., 2007). Moreover, Eckhardt et al. discloses that ASA-deficient mice have been used for more than a decade as an animal model of MLD.
  • Sulfatide storage pattern as detected by blue staining, in ASA-deficient mice closely resembles the sulfolipid storage pattern observed in MLD patients.
  • Jones E et al. discloses that cerebrosides in vertebrates may be sulphated by the cerebroside sulfotransferase enzyme (encoded by the GAL3ST1 gene) to make sulfatide. Altogether this indicates that inhibition or reduction of GAL3ST1 expression could be an effective therapeutic concept for treating MLD.
  • target sequences within the human GAL3ST1 pre-mRNA and potent antisense oligonucleotides targeting the human GAL3ST1 gene for treatment of MLD.
  • the present invention provides an antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in a GAL3ST1 (Galactosylceramide sulfotransferase) gene.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 522 to SEQ ID NO: 632.
  • the sequences are shown in Table Bl in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 522, 523, 525, 532, 537, 540, 546, 547, 550, 551, 562, 563, 565, 569, 570, 571, 572, 573, 594, 598, 600, 616, 617, 618, 623, 624, 627, and 629.
  • the sequences are shown in Table B2 in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 522, 532, 537, 540, 546, 547, 562, 565, 569, 570, 573, 600, 617, 624, 627, 629.
  • the sequences are shown in Table B3 in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 522.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 527.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 540.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 546.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 547.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 562.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 565.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 569. In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 570.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 600.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 617.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 624.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 627.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 629.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 627.
  • the antisense oligonucleotide comprises a stretch of at least 12, or at least 14 nucleotides which is at least 90% complementary to a target sequence as set forth herein.
  • said stretch is 95%, in particular 100% complementary to the target sequence.
  • the stretch of at least 10 nucleotides is fully complementary to a target sequence comprised by SEQ ID NO: 522.
  • the stretch of at least 10 nucleotides is fully complementary to a target sequence comprised by SEQ ID NO: 624.
  • the stretch of at least 10 nucleotides is fully complementary to a target sequence comprised by SEQ ID NO: 627.
  • the stretch of at least 10 nucleotides is fully complementary to a target sequence comprised by SEQ ID NO: 629.
  • the antisense oligonucleotide has a length of 12 to 30 nucleotides, more preferably, a length of 14 to 22 nucleotides, and most preferably a length of 16 to 20 nucleotides.
  • the antisense oligonucleotide comprises or consists of a nucleic acid sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 521. In some embodiments, the antisense oligonucleotide consists of a nucleic acid sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 521.
  • the antisense oligonucleotide is an antisense compound as shown in Table Al in Fig. 1, wherein
  • Adx represents 2'deoxyadenosine-3'-phosphorothioate
  • Aox represents 2'-O-methyladenosine-3'-phosphorothioate
  • Amx represents 2'-O-Methoxyethyladenosine-3'-phosphorothioate
  • Alx represents 2'-O-beta-D-oxy LNA adenosine-3'-phosphorothioate
  • Cdx represents 2'deoxycytidine-3'-phosphorothioate
  • Cox represents 2'-O-methylcytidine-3'-phosphorothioate
  • Edx represents 2'deoxy-5-methylcytidine-3'-phosphorothioate
  • Emx represents 2'-O-Methoxyethyl-5-methylcytidine-3 '-phosphorothioate
  • Elx represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3 '-phosphorothioate
  • Gdx represents 2'deoxy guanosine-3 '-phosphorothioate
  • Gox represents 2'-O-methylguanosine-3 '-phosphorothioate
  • Gmx represents 2'-O-Methoxyethylguanosine-3 '-phosphorothioate
  • Glx represents 2'-O-beta-D-oxy LNA guanosine-3 '-phosphorothioate
  • Tdx represents 2'deoxythymidine-3 '-phosphorothioate
  • Tmx represents 2'-O-Methoxyethylthymidine-3'-phosphorothioate
  • Tlx represents 2'-O-beta-D-oxy LNA thymidine-3 '-phosphorothioate
  • Ado represents 2'deoxyadenosine-3 '-phosphodiester
  • Aoo represents 2'-O-methyladenosine-3 '-phosphodiester
  • Amo represents 2'-O-Methoxyethyladenosine-3 '-phosphodiester
  • Alo represents 2'-O-beta-D-oxy LNA adenosine-3 '-phosphodiester
  • Cdo represents 2'deoxy cytidine-3 '-phosphodiester
  • Coo represents 2'-O-methyl cytidine-3 '-phosphodiester
  • Edo represents 2'deoxy-5-methylcytidine-3 '-phosphodiester
  • Emo represents 2'-O-methoxyethyl-5-methylcytidine-3 '-phosphodiester
  • Elo represents 2'-O-beta-D-oxy LNA-5-methylcytidine-3 '-phosphodiester
  • Gdo represents 2'deoxy guanosine-3 '-phosphodiester
  • Goo represents 2'-O-methylguanosine-3 '-phosphodiester
  • Gmo represents 2'-O-Methoxyethylguanosine-3 '-phosphodiester
  • Gio represents 2'-O-beta-D-oxy LNA guanosine-3 '-phosphodiester
  • Tdo represents 2'deoxythymidine-3 '-phosphodiester
  • Tmo represents 2'-O-Methoxyethylthymidine-3'-phosphodiester
  • Tlo represents 2'-O-beta-D-oxy LNA thymidine-3 '-phosphodiester
  • Ad represents 2'deoxyadenosine
  • Ao represents 2'-O-methyladenosine
  • Al represents 2'-O-beta-D-oxy LNA adenosine
  • Cd represents 2'deoxycytidine
  • Co 2'-O-methylcytidine
  • Em represents 2'-O-Methoxyethyl-5-methylcytidine
  • El represents 2'-O-beta-D-oxy LNA -5-methylcytidine
  • Gd represents 2'deoxyguanosine
  • Gm represents 2'-O-Methoxy ethylguanosine
  • G1 represents 2'-O-beta-D-oxy LNA guanosine
  • Td represents 2'deoxythymidine
  • Tm represents 2'-O-Methoxy ethylthymidine
  • T1 represents 2'-O-beta-D-oxy LNA thymidine
  • Uoo represents 2'-O-methyluridine-3 '-phosphodiester
  • Uox represents 2'-O-methyluridine-3'-phosphorothioate.
  • the antisense oligonucleotide is an antisense compound as shown in Table C in Fig. 2.
  • Table C for the annotation, please see the previous paragraph.
  • the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 1_41, 1 103, 1 131, 1 129, 1 116, 1 164, 1 193, 16 17, 19_23, 25_85, 25_16, 108 15, 108 16, 108 12, 108_6, 102_4, 108_20, 108_7, 108 17, 106_7, 103 10, 96_21, 96_12, 48_2, 48_7, 48_3, 79_3, 95 _7, and 95_9, as shown in Table Al in Fig. 1.
  • the ASO compounds with these ASO IDs had a strong effect on the down-regulation of the target gene (see Examples).
  • the antisense oligonucleotide of the present invention is capable of reducing the amount of GAL3ST1 (Galactosylceramide sulfotransferase) mRNA (typically pre-mRNA) in a host cell expressing said GAL3ST1 mRNA.
  • the host cell is a mammalian cell, such as a primate cell. In a preferred embodiment, said host cell is human host cell.
  • the antisense oligonucleotide of the present invention is capable of reducing the amount of GAL3ST1 the antisense oligonucleotide is capable of reducing galactosylceramide sulfotransferase activity in a host cell, and/or Alternatively or additionally, the antisense oligonucleotide of the present invention is capable of reducing the amount of sulfatide in a host cell.
  • the antisense oligonucleotide is a chemically modified antisense oligonucleotide.
  • a chemically modified antisense oligonucleotide typically comprises modifications of the phosphodiester backbone chemistry, nucleobase modifications and sugar modifications.
  • the chemically modified antisense oligonucleotide comprises at least one 2’ modified sugar or bicyclic sugar.
  • the chemically modified antisense oligonucleotide contains at least one modified nucleobase.
  • at least one modified nucleobase is 5- methylcytosine.
  • the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2'-O-Methoxyethyl- RNA, 2’-O-Methyl-RNA, 2’-Fluoro-RNA.
  • the chemically modified antisense oligonucleotide may comprise at least one modified internucleoside linkage. In an embodiment, at least five, such as at least 10 internucleoside linkages are modified internucleoside linkages. In an embodiment, all internucleoside linkages are modified internucleoside linkages.
  • the antisense oligonucleotide may comprise unmodified internucleoside linkages (i.e. phosphodiester linkages), modified internucleoside linkages, or a combination thereof.
  • the modified linkage(s) is (are) selected from: a Phosphorothioate internucleoside linkage, a Phosphorodithioate internucleoside linkage, a Phophoroamidate internucleoside linkage, a methyl phosphonate intemucleoside linkage, a phosphotriester internucleoside linkage, a boranophosphate internucleoside linkage and a phosphoryl guanidine internucleoside linkage.
  • internucleoside linkage can be stereodefined versions of said linkages
  • the at least one modified linkage is a phosphorothioate linkage.
  • at least 50% of the internucloside linkages, such as all internucleoside linkages, are phosphorothioate internucleoside linkages.
  • the antisense oligonucleotide comprises at least one nucleoside with a modified sugar moiety, typically at least four nucleosides with a modified sugar moiety (herein also referred to as sugar modified nucleosides).
  • the antisense oligonucleotide comprises at least one, such as one, two, three, four or more LNA (locked nucleic acid) or MOE (2’-O-Methoxyethyl) nucleosides.
  • LNA locked nucleic acid
  • MOE 2’-O-Methoxyethyl
  • the LNA nucleoside is a beta-D-oxy LNA nucleoside.
  • the antisense oligonucleotide has a gapmer structure, i.e. is a gapmer.
  • the present invention further relates to a conjugate comprising the antisense oligonucleotide according to the present invention, wherein the said antisense oligonucleotide is covalently attached to a conjugate moiety.
  • the present invention further relates to pharmaceutical composition comprising an inhibitor of the human GAL3ST1 protein.
  • the present invention relates to pharmaceutical composition comprising the antisense oligonucleotide according to the present invention or the conjugate according to the present invention.
  • the composition further comprises diluents and carriers.
  • the present invention further relates to the inhibitor of human GAL3ST1 or the pharmaceutical composition according to the present invention for use in treating metachromatic leukodystrophy.
  • the inhibitor in particular, is the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating metachromatic leukodystrophy.
  • the present invention further relates to a method for treating metachromatic leukodystrophy, comprising administering to a subject suffering from metachromatic leukodystrophy a pharmaceutically effective amount of inhibitor of human GAL3ST1, in particular the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating metachromatic leukodystrophy.
  • the inhibitor is a siRNA or short-hairpin RNA targeting the GAL3ST1 gene.
  • the inhibitor is an antibody, or antigen binding fragment thereof that specifically binds to GAL3ST1.
  • the present invention further relates to a method for identifying a candidate compound for the treatment of metachromatic leukodystrophy, comprising a) providing an antisense oligonucleotide according to the present invention, b) contacting a host cell expressing GAL3ST1 mRNA with said antisense oligonucleotide, c) determining the amount of GAL3ST1 mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
  • GAL3ST1 Galactosylceramide sulfotransferase
  • GAL3ST1 Galactosylceramide sulfotransferase
  • target regions within the GAL3ST1 pre-mRNA were identified which - when targeted by antisense oligonucleotides - allow for efficient downregulation of the human GAL3ST1 pre-mRNA (or mRNA) in a host cell expressing said pre-mRNA or mRNA.
  • the sequences of the target regions are shown in Tables Bl, B2 and B3.
  • the invention provides antisense oligonucleotides, which are capable of downregulating GAL3ST1.
  • the antisense oligonucleotides comprise a stretch of at least 10 nucleotides which is preferably 90%, more preferably, 95% and most preferably fully complementary (i.e. 100% complementary) to the target region (herein also referred to as target sequence).
  • target sequence the target region
  • the antisense oligonucleotides of the present invention are candidates for the treatment of metachromatic leukodystrophy.
  • the present invention relates to an antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in a GAL3ST1 (Galactosylceramide sulfotransferase) gene.
  • GAL3ST1 Galactosylceramide sulfotransferase
  • oligonucleotide as used herein is well known in the art. As used herein, the term refers to a molecule of at least ten covalently linked nucleotides. Typically, the oligonucleotides as referred to herein are chemically synthesized, for example by solidphase chemical synthesis. The oligonucleotides as referred to herein shall contain various chemical modifications which typically do not occur in nature. For example, the antisense oligonucleotide may contain at least one 2’ modified sugar. In a preferred embodiment, the antisense oligonucleotides are gapmers.
  • the oligonucleotides of the present invention are antisense oligonucleotides, and in particular single-stranded oligonucleotides. Accordingly, they shall be capable of binding the GAL3ST1 gene, in particular to the GAL3ST1 pre- mRNA, when expressed in a cell, thereby down- regulating the expression of GAL3ST1 gene in the cell.
  • the cell is a human cell is a cell of the central nervous system (CNS).
  • the cell is a brain cell.
  • the GAL3ST1 (Galactosylceramide sulfotransferase- 1 or Galactose-3-O-sulfotransferase- 1, CST) gene is well known the art. Alternative names are 3'-phosphoadenosine-5'- phosphosulfate:GalCer sulfotransferase gene, 3'- phosphoadenylylsulfate:galactosylceramide 3 '-sulfotransferase 1 gene, or cerebroside sulfotransferase or galactose-3-O-sulfotransferase-l gene.
  • the GAL3ST1 gene is typically the human GAL3ST1 gene. Information on the gene, such as on the nucleic acid sequence, can be found in the known databases, for example, under NCBI Gene ID: 9514).
  • the human GAL3ST1 gene encodes an enzyme, i.e. protein having galactosylceramide sulfotransferase activity (EC 2.8.2.11). Typically, the enzyme catalyzes the transfer of a sulfate group to position 3 of non-reducing beta-galactosyl residues in glycerolipids and sphingolipids. Typically, the enzyme catalyzes the synthesis of galactosylceramide sulfate (also known as sulfatide). Typically, sulfatides are a major lipid component of the myelin sheath and of monogalactosylalkylacylglycerol sulfate
  • the catalyzed reaction is as follows:
  • the protein sequence can be assessed in the Uniprot database under the accession number Q99999 (G3 STI HUMAN).
  • the human GAL3ST1 protein has an amino acid sequence as shown in SEQ ID NO: 636.
  • the GAL3ST1 protein is typically referred to as “Galactosylceramide sulfotransferase”.
  • the ASO of the present invention targets the human GAL3ST1 pre-mRNA, i.e. downregulates expression of said pre-mRNA.
  • the sequence of the human GAL3ST1 pre- mRNA can be e.g. assessed in the Ensembl database under accession number in ENST00000406361. Typically, it is encoded by a region on human Chromosome 22: position 30554635-30574665 on the reverse strand (Assembly GRCh38).
  • the sequence of the human pre-mRNA has a sequence as shown in SEQ ID NO: 633.
  • SEQ ID NO: 633 shows the sequence of the sequence of the refseq transcript, NM_001318108 (20.031 bases).
  • SEQ ID NO: 634 and 635 include the pre-mRNA sequence of the other two brain expressed transcripts, i.e. of SEQ ID NO: 634 and 635.
  • the pre- mRNA is further processed, i.e., by splicing, thereby generating a protein coding mRNA (herein also referred to as transcript).
  • the antisense oligonucleotide of the present invention may also target the processed human GAL3ST1 mRNA (if the target region is located within an exon, either coding or in the 3’- or 5’UTR).
  • SEQ ID NO: 633. 634 and 635 are RNA sequences. In the sequence listing, they are provided as DNA sequences. It is understood by the skilled person that the target RNA sequences have uracil (U) bases instead of thymine bases (T).
  • the antisense oligonucleotides of the present invention shall be capable of down-regulating, i.e. reducing expression of the GAL3ST1 pre-mRNA in a cell that expresses said pre-mRNA.
  • the expression is reduced in a call by antisense oligonucleotides of the present invention by least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a control cell (i.e. an untreated control cell).
  • a control cell i.e. an untreated control cell.
  • How to assess whether the expression is reduced can be assessed by well-known methods, i.e. by measuring the expression level (i.e. the amount of the target mRNA) in ASO treated cells.
  • the down-regulation of the target gene is assessed as described in the Examples section.
  • As control for down regulation untreated cells can be used.
  • Down-regulating the expression of the GAL3ST1 mRNA typically, leads down-regulation of the GAL3ST1 protein and thus to reduced levels of sulfatide as compared to a control.
  • Down-regulation of the GAL3ST1 protein can be assessed by e.g. assessing the Galactosylceramide sulfotransferase activity in cells treated with the ASO of the present invention by using well known enzymatic assays or by or quantifying the protein expression, such as by Western Blotting, mass spectrometry or ELISA.
  • the Galactosylceramide sulfotransferase activity is reduced in a cell by antisense oligonucleotides of the present invention by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a control cell (i.e. an untreated control cell).
  • the above assessments can be done in vivo or in vitro. If they are done in vitro, they are typically done in human cells, such as in human cells used in the Examples section. In vivo, it is e.g. envisaged that a down-regulation of the target mRNA or protein, of at least 30%, such as at least 40% is achieved. For example, the down-regulation of the target mRNA or protein may be between 40% to 60%, or between 50% to 60% as compared to a control.
  • target sequences within the GAL3ST1 pre-mRNA were identified which can be efficiently targeted with ASOs.
  • 111 of such target regions/ sequences were identified. These regions are shown in the following table.
  • each identified target region was assigned a so called “Target ID” (Target ID 1 to 111). These IDs are used throughout the application.
  • the terms “target region”, “target sequence” and “target nucleic acid” are used interchangeably herein.
  • Table Bl Information on the identified target regions can be found in Table Bl in the Examples section (e.g. the sequence of the target region and the SEQ ID NO).
  • Table B2 lists more preferred target regions.
  • Table B3 lists the most preferred target regions.
  • the target regions in Tables B2 and B3 may be present in the target regions shown in Table Bl, but may be shorter.
  • the antisense oligonucleotide of the present invention is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in the above table or in Table Bl.
  • the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 522 to SEQ ID NO: 632.
  • the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table B2.
  • the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 522, 523, 525, 532, 537, 540, 546, 547, 550, 551, 562, 563, 565, 569, 570, 571, 572, 573, 594, 598, 600, 616, 617, 618, 623, 624, 627, and 629.
  • the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table B3.
  • the target sequence has a sequence selected from the sequences consisting of 522, 532, 537, 540, 546, 547, 562, 565, 569, 570, 573, 600, 617, 624, 627, 629.
  • the antisense oligonucleotide typically comprises stretch of at least 10 nucleotides which is at least 90% complementary (such as 95% or 100%) to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 522 to SEQ ID NO: 632.
  • the target sequence has a sequence as shown in SEQ ID NO: 522. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 532. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 537. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 540. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 546. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 547. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 562. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 565.
  • the target sequence has a sequence as shown in SEQ ID NO: 569. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 570. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 573. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 600. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 617. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 624. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 627. In an embodiment, the target sequence has a sequence as shown in SEQ ID NO: 629.
  • the antisense oligonucleotides of the present invention are preferably single-stranded antisense oligonucleotides.
  • the antisense oligonucleotides of the invention shall comprise a “stretch of nucleotides” which is sufficient complementary to a target sequence as referred to herein.
  • the stretch of nucleotides is at least 90% complementary to a target sequence.
  • the stretch of nucleotides is at least 95% complementary to a target sequence.
  • the stretch of nucleotides is fully complementary (i.e. 100% complementary to the target sequence).
  • the term “complementary” is well known in the art.
  • the percentage of complementary is typically calculated by calculating the proportion of nucleotides (in %) within the stretch of oligonucleotides of the ASO of the present invention which are complementary to the target sequence within the GAL3ST1 gene.
  • a nucleotide present in the ASO of the present invention are considered as complementary if it forms a Watson-Crick base pair with the nucleotide present in the target RNA sequence.
  • Watson Crick base pairs are guanine-cytosine; adenine-uracil, and adenine-thymine, i.e. G-C, A-U or A-T.
  • modified nucleotides have also the capacity to form such base pairs. For more information, see e.g. Table A2.
  • the “stretch of nucleotides” as referred to herein needs to have a certain length in order to allow for the binding of the oligonucleotide of the present invention to the target region.
  • the stretch of nucleotides has a length of at least 10 nucleotides, more preferably of at least 12 nucleotides and most preferably of at least 14 nucleotides.
  • the antisense oligonucleotide of the present invention may comprise further nucleotides - i.e. in addition to the stretch of nucleotides as referred to above, such as linker nucleotides. These further nucleotides may be complementary to the target sequence, or not.
  • the antisense oligonucleotide of the present invention preferably, has a length of 12 to 30 nucleotides, more preferably, of 14 to 22 nucleotides, and most preferably of 16 to 20 nucleotides. Accordingly, it is envisaged that the antisense oligonucleotide in not longer than 30 nucleotides. In some embodiments, the antisense oligonucleotide in not longer than 22 nucleotides or 20 nucleotides.
  • the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 521. In a preferred embodiment, the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 521.
  • the antisense oligonucleotide of the present invention is a chemically modified antisense oligonucleotide. Accordingly, it does not occur in nature.
  • a wide range of chemical modification can be incorporated into an oligonucleotide, such modification are e.g. reviewed in Crooke et al. which herewith is incorporated by reference in its entirety (Stanley T Crooke, Xue-Hai Liang, Brenda F Baker, Rosanne M Crooke. Review J Biol Chem. 2021 Jan-Jun;296, antisense technology: A review).
  • At least one of the nucleotides (herein also referred to a monomer) present in the oligonucleotide comprises a chemical modification. More preferably, at least 30%, such as at least 50% of the nucleotides present in the oligonucleotide comprise a chemical modification. In some embodiments, all of the nucleotides comprise chemical modification. Modifications include modifications of the phosphodiester backbone chemistry (“backbone modifications”), nucleobase modifications and sugar modifications. Preferred chemical modifications are shown in Table A2, see in particular the column “Nucleotide”. The ASOs of the present invention may comprise such nucleotides.
  • the ASO of the present preferably comprises one or more internucleoside linkages other than a phosphodiester linkage.
  • the antisense oligonucleotide comprises at least five, such as at least ten modified internucleoside linkages. Most preferably, all internucleoside linkages are modified linkages. However, some linkages may be phosphodiester linkages, such as one, up to two, up to three or up to four, up to six, or up to eight phosphodiester linkages.
  • the at least one modified internucleoside linkage is selected from the group consisting of at least one Phosphorothioate internucleoside linkage, at least one Phosphorodithioate internucleoside linkage, at least one Phophoroamidate internucleoside linkage, at least one methyl phosphonate internucleoside linkage, at least one phosphotriester internucleoside linkage, at least one boranophosphate internucleoside linkage and at least one phosphoryl guanidine intemucleoside linkage.
  • the at least one modified internucleoside linkage can be a stereodefined versions of said linkages.
  • the oligonucleotide comprises at least one phosphorodithioate internucleoside linkage.
  • the oligonucleotide comprises at least one phosphoryl guanidine internucleoside linkage. Most preferably, all internucleoside linkage are phosphoryl guanidine internucleoside linkages.
  • the oligonucleotide comprises at least one phosphorothioate internucleoside linkage. Most preferably, at least 40%, such as at least 60% are internucleoside linkage are phosphorothioate linkages. Most preferably, all internucleoside linkage are phosphorothioate linkages.
  • the backbone may be modified with Morpholino Phosphorodiamidate (PMO) and Peptide Nucleic Acid (PNA).
  • PMO Morpholino Phosphorodiamidate
  • PNA Peptide Nucleic Acid
  • Modification applied to the sugar group could be acyclic modifications such as UNA (unlocked nucleic acid), FNA (Flexible nucleic acid), (S)- and (R)-GNA (glycol nucleic acid), D- and L-aTNA (threofuranosyl nucleic acids), SNA (Serinol nucleic acids), as described in further details in Bege & Borbas 2021 (Miklos Bege & Aniko Borbas Review Pharmaceuticals (Basel) . 2022 Jul 22;15(8):909. doi: 10.3390/phl5080909. The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic).
  • the chemically modified antisense oligonucleotide comprises one or more modified nucleosides.
  • the one or more modified nucleosides are sugar modified nucleosides, such as one, two, three, four or more sugar modified nucleosides. Typically, it comprises four sugar modified nucleosides.
  • a sugar modified nucleoside is nucleoside with a modified sugar.
  • the one or more sugar modified nucleosides are 2’ sugar modified nucleosides, such as 2’0 modified sugar nucleosides.
  • the 2’0 modified sugar is, selected from the group consisting of 2’-0-Me, 2’MOE (2’-O-Methoxyethyl)), 2’-N-propyl, 2’-O-allyl, 2’F RNA, 2’-O-ethylamine.
  • the 2’0 modified sugar is 2’MOE (2’-O-Methoxyethyl).
  • the modified nucleosides are 2’MOE nucleosides.
  • the one or more modified nucleotides could be locked nucleic acids such as, beta-D-oxy-LNA, 2 ',4 '-constrained 2'-0-ethyl (cEt), such as R-cET and S-cEt, Beta-D- amino LNA, Beta-D-thio LNA, alpha-L-oxy LNA, ENA and other modifications as described in Wan & Seth 2016 (W Brad Wan, Punit P Seth, Review J Med Chem. 2016 Nov 10;59(21):9645-9667. The Medicinal Chemistry of Therapeutic Oligonucleotides).
  • the oligonucleotide of the invention preferably comprises one more Locked Nucleic Acid Nucleosides (LNA nucleosides) which are well known 2’- modified nucleosides.
  • LNA nucleosides Locked Nucleic Acid Nucleosides
  • the one or more modified nucleosides are (S)-6’-methyl-beta-D-oxy-LNA (ScET) LNA nucleosides. More preferably, the one or more modified nucleosides are beta- D-oxy-LNA nucleosides, Nucleobase modification include, but are not limited to, 5-methyl-cytosine, pseudouridine, 5-Methyluridine, 8-Oxoguanine, 2-thio-thymine, Diaminopurine, abasic nucleosides and others as also described in Brad&Seth 2016 and Robert et al., 2020 (Thomas C Roberts, Robert Langer, Matthew J A Wood. Review Nat Rev Drug Discov. 2020 Oct;19(10):673-694. Advances in oligonucleotide drug delivery)
  • the chemically modified antisense oligonucleotide contains at least one modified nucleobase.
  • the at least one modified nucleobase is 5- methylcytosine.
  • the ASO may comprise at least pseudouridine, or at least one 8- oxoguanine as modified nucleobase.
  • the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of 2-O-Methoxyethyl- RNA, 2’-O-Methyl-RNA, 2’-Fluoro-RNA.
  • the antisense oligonucleotide of the present invention has a gapmer structure, i.e. is a gapmer.
  • Gapmers are well known in the art. The term refers to (single stranded) DNA antisense oligonucleotide structures with RNA-like segments on both sides (flanking regions). Gapmers bind to the target sequence and down-regulate target gene expression through the induction of RNase H cleavage.
  • Suitable gapmer designs are well known in the art and are e.g. reviewed in Crooke et al., which herewith is incorporated by reference in its entirety (Stanley T Crooke, Xue-Hai Liang, Brenda F Baker, Rosanne M Crooke. Review J Biol Chem. 2021 Jan-Jun;296. Antisense technology: A review).
  • the gapmer is a LNA gapmer in which the flanking regions comprise LNA nucleosides, such as D-oxy LNA nucleosides.
  • the gapmer may also comprise 2’O-Methoxyethyl (MOE) nucleosides in the flanking regions.
  • MOE gapmers are frequently referred to as MOE gapmers.
  • the antisense oligonucleotide of the present invention has a gapmer structure and at least one modified internucleoside linkage.
  • the S oligonucleotide of the present invention has a gapmer structure and at least 10 modified intemucleoside linkages.
  • the antisense oligonucleotide of the present invention has a gapmer structure and, at least 40%, such as at least 60%, in particular all linkages are modified internucleoside linkages.
  • the modified linkages are described herein above.
  • the modified linkages are phosphorothioate internucleoside linkages.
  • the linkages are phosphorodithioates linkages.
  • the antisense oligonucleotide of the present invention is a compound selected from the compounds shown in Table Al in Table Al in Fig. 1 (see column “Compound”), wherein
  • Adx represents 2'deoxyadenosine-3'-phosphorothioate
  • Aox represents 2'-O-methyladenosine-3'-phosphorothioate
  • Alx represents 2'-O-beta-D-oxy LNA adenosine-3'-phosphorothioate
  • Cox represents 2'-O-methylcytidine-3'-phosphorothioate
  • Emx represents 2'-O-Methoxyethyl-5-methylcytidine-3'-phosphorothioate
  • Gdx represents 2'deoxy guanosine-3 '-phosphorothioate
  • Gox represents 2'-O-methylguanosine-3 '-phosphorothioate
  • Gmx represents 2'-O-Methoxyethylguanosine-3 '-phosphorothioate
  • Glx represents 2'-O-beta-D-oxy LNA guanosine-3 '-phosphorothioate
  • Tdx represents 2'deoxythymidine-3 '-phosphorothioate
  • Tmx represents 2'-O-Methoxyethylthymidine-3'-phosphorothioate
  • Tlx represents 2'-O-beta-D-oxy LNA thymidine-3 '-phosphorothioate
  • Ado 2'deoxyadenosine-3 '-phosphodiester
  • Aoo represents 2'-O-methyladenosine-3 '-phosphodiester
  • Alo represents 2'-O-beta-D-oxy LNA adenosine-3 '-phosphodiester
  • Emo represents 2'-O-Methoxyethyl-5-methyl cytidine-3 '-phosphodiester
  • Gio represents 2'-O-beta-D-oxy LNA guanosine-3 '-phosphodiester
  • Tdo represents 2'deoxythymidine-3' -phosphodiester
  • Tmo 2'-O-Methoxyethylthymidine-3'-phosphodiester
  • Tlo represents 2'-O-beta-D-oxy LNA thymidine-3 '-phosphodiester
  • Ad 2'deoxyadenosine
  • Al represents 2'-O-beta-D-oxy LNA adenosine
  • Em represents 2'-O-Methoxyethyl-5-methylcytidine
  • G1 represents 2'-O-beta-D-oxy LNA guanosine
  • Tm represents 2'-O-Methoxyethylthymidine
  • T1 represents 2'-O-beta-D-oxy LNA thymidine
  • internucleoside linkages present in the compounds shown in Table Al in Fig. 1 and in Table C in Fig. 2 are phosphorothioate linkages.
  • the presence of an “o” at the third position of the three-letter code indicates the presence of a phosphodiester internucleoside linkage.
  • the presence of an “x” at the third position of the three-letter code indicates the presence of a Phosphorothioate internucleoside linkage.
  • the compound selected from Table Al in Fig. 1 is a compound, which resulted in an efficient down-regulation of the target gene in the studies described in the Examples, such as more than 50% or more than 34%.
  • the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 1_41, 1 103, 1 131, 1 129, 1 116, 1 164, 1 193, 16 17, 19_23, 25_85, 25_16, 108 15, 108 16, 108 12, 108_6, 102_4, 108_20, 108_7, 108 17, 106_7, 103 10, 96_21, 96_12, 48_2, 48_7, 48_3, 79_3, 95_7, and 95_9, as shown in Table Al in Fig. 1.
  • the compound is a compound selected from Table C in Fig. 2. More preferably, the compound is a compound from Table C, which resulted in an efficient down-regulation of the target gene in the studies described in the Examples, such as more than 50% or more than 34%.
  • the antisense oligonucleotide (or composition) of the present invention shall be administered to the CNS, in particular to the brain.
  • the ASO is delivered to CNS through intrathecal injection - as it is e.g. the current state of art for similar ASO e.g. Nusinersen/Spinraza.
  • the ASO of the present invention or the pharmaceutical composition is, preferably, administered intrathecally.
  • the ASO can be administered by subcutaneous or intravenous administration either with or with or without a conjugate in order to reach the peripheral nervous system.
  • the present invention further relates to a conjugate comprising the antisense oligonucleotide of the present invention and a conjugate moiety.
  • the conjugate moiety is covalently bound to the antisense oligonucleotide, e.g. via one or more linker nucleotides, such as one, two, three or four linker nucleotides (or more).
  • the linker may be cleaved after administration to the patient.
  • the antisense oligonucleotide of the present invention shall be delivered or administered to the CNS, in particular to the brain.
  • the conjugate moiety is a moiety that allows the crossing of the conjugate of the blood brain barrier.
  • the moiety can be and antibody or antigen-binding fragment thereof targeting the transferrin receptor.
  • the antisense oligonucleotides of the present invention can be administered/delivered ‘unassisted’ in saline solution. However, distribution to certain tissues and uptake in cells can be enhanced by conjugates and formulation techniques. Conjugation to ASOs could be, peptides, antibodies and aptamers binding to receptors on target cells or proteins mediating transcytosis e.g. the transferrin receptor. Antisense oligos can also be conjugated to naturally occurring ligands or modifications hereof as exemplified by GalNac conjugation binding with high affinity to asialoglycoprotein receptor 1 (ASGR1, ASPGR) and Alphatocopherol conjugation and interaction with transfer protein Alfa-TTP.
  • ASOs conjugation to ASOs
  • ASOs could be, peptides, antibodies and aptamers binding to receptors on target cells or proteins mediating transcytosis e.g. the transferrin receptor.
  • Antisense oligos can also be conjugated to naturally occurring
  • tissues delivery and cellular uptake of ASOs of the present invention can be enhanced through formulation with nanocarriers that facilitates crossing of biological barriers such as cellular membranes.
  • nanocarriers Various types of nanocarriers have been described with favorable properties for delivery of nucleic acids e.g. lipid nanoparticles (LNPs) as used for BioNTech mRNA vaccines, LNPs functionalized with peptides, PEGylated lipids, cationic lipids, exomes (lipid bilayers) both artificial and natural exosomes such as milk exosomes and spherical nucleic acids and others as described in further details in Roberts et al., 2020 (Thomas C Roberts, Robert Langer, Matthew J A Wood. Review Nat Rev Drug Discov. 2020 Oct;19(10):673-694. Advances in oligonucleotide drug delivery).
  • the present invention further relates to a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or the conjugate of the present invention.
  • a pharmaceutical composition comprises the antisense oligonucleotide or the conjugate of the present invention together with a pharmaceutically acceptable carrier and/or, in particular, a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable refers to the non-toxicity of a material which, in certain exemplary embodiments, does not interact with the action of the oligonucleotide or the conjugate present in the pharmaceutical composition.
  • carrier refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application.
  • excipient is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surface active agents, preservatives, emulsifiers or buffer substances.
  • binders e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol
  • fillers e.g., lubricants, thickeners, surface active agents, preservatives, emulsifiers or buffer substances.
  • the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc.
  • the pharmaceutical composition can be formulated for intrathecal administration.
  • the antisense oligonucleotide or conjugate of the present invention is preferably administered by intrathecal administration a route of administration for drugs via an injection into the spinal canal. Thereby, it reaches the cerebrospinal fluid and the brain.
  • the present invention further relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in medicine.
  • the present invention relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating metachromatic leukodystrophy (MLD).
  • MLD metachromatic leukodystrophy
  • the present invention relates to the use of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament for treating metachromatic leukodystrophy.
  • Metachromatic leukodystrophy is a lysosomal storage disease.
  • MLD is typically listed in the family of leukodystrophies as well as among the sphingolipidoses as it affects the metabolism of sphingolipids.
  • Leukodystrophies affect the growth and/or development of myelin, the fatty covering which acts as an insulator around nerve fibers throughout the central and peripheral nervous systems.
  • MLD involves cerebroside sulfate accumulation.
  • MLD is degenerative disease that is associated with a progressive damage to brain cells. The disease is inherited in an autosomal recessive manner.
  • the present invention relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use treating metachromatic leukodystrophy.
  • the present invention relates to the use of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament for treating metachromatic leukodystrophy.
  • the present invention relates to a method of treating metachromatic leukodystrophy, comprising administering pharmaceutically effective amount of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention to a subject suffering from metachromatic leukodystrophy.
  • treating refers to the administration of a compound or composition or a combination of compounds or compositions to a subject in order to ameliorate metachromatic leukodystrophy.
  • the term encompasses both the amelioration of one or more symptoms of the metachromatic leukodystrophy or prevention of the worsening of one or more symptoms, i.e. prophylaxis.
  • the amelioration of symptoms also includes the reduction of one or more symptoms.
  • the term preferably, refers to the reduction of one or more symptoms of the disease.
  • the treatment is typically a disease modifying treatment that reduces one or more symptoms of metachromatic leukodystrophy.
  • the development of disease pathology is inhibited. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic or disease modifying. It is to be understood that the treatment does not allow a complete cure of metachromatic leukodystrophy.
  • treatment also includes increasing the life expectancy of a subject (as compared to an untreated subject).
  • the subject to be treated is a subject suffering from metachromatic leukodystrophy.
  • the patient might not yet show symptoms of metachromatic leukodystrophy at the time of the treatment.
  • the subject shows symptoms of metachromatic leukodystrophy.
  • Clinical manifestation of MLD can vary depending on the age of onset, which can range from infancy to adulthood.
  • Symptoms of metachromatic leukodystrophy are well known in the art and include (but are not limited to) one or more of include muscle wasting and weakness, muscle rigidity, developmental delays, progressive loss of vision leading to blindness, convulsions, impaired swallowing, paralysis, and dementia.
  • the subject has been diagnosed through genetic testing to suffer from metachromatic leukodystrophy. The most common and severe form of MLD is diagnosed in the infant age. Also typically, the diagnosis involves genetic testing.
  • MLD low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low density low
  • the terms “subject” and “patient” are used interchangeably herein.
  • the “subject” or “patient” may be a vertebrate.
  • the term includes both humans and other animals, particularly mammals, and other organisms.
  • the subject is a mammal.
  • the subject is a primate.
  • the subject is a human subject suffering from metachromatic leukodystrophy.
  • the present invention further relates to a method for identifying a candidate compound for the treatment of metachromatic leukodystrophy, comprising a) providing an antisense oligonucleotide according to the present invention, b) contacting a host cell expressing GAL3ST1 mRNA with said antisense oligonucleotide, c) determining the amount of GAL3ST1 mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
  • the antisense oligonucleotide to be provided in step a) is preferably the antisense oligonucleotide of the present invention. Accordingly, it shall comprise a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in the human GAL3ST1 gene (i.e. mRNA or premRNA).
  • mRNA or premRNA a target sequence in the human GAL3ST1 gene
  • the provided antisense oligonucleotide targets a sequence disclosed in Table Bl, more preferably, a sequence disclosed in Table B2 and most preferably a sequence disclosed in Table B3.
  • said method is an in vitro method.
  • an antisense oligonucleotide of the present invention is provided.
  • the antisense oligonucleotides are complementary to a target region as set forth herein elsewhere.
  • the antisense oligonucleotide shall be contacted with a host cell.
  • Said host cell shall express the GAL3ST1 gene.
  • an antisense olignonucleotide which leads to a reduction of the amount of GAL3ST1 mRNA (such as GAL3ST1 mRNA) is considered as candidate compound.
  • the present invention relates to an inhibitor for GAL3ST1, or the pharmaceutical composition comprising said inhibitor for use in treating metachromatic leukodystrophy.
  • the inhibitor is an inhibitory RNA, such as a siRNA or short-hairpin RNA, which is capable of binding to GAL3ST1 mRNA, such as the pre-mRNA and which is capable of downregulating the GAL3ST1 mRNA in a cell.
  • an inhibitory RNA such as a siRNA or short-hairpin RNA
  • the inhibitor is an antisense oligonucleotide, which is capable of binding to GAL3ST1 mRNA, such as the pre-mRNA and which is capable of downregulating the GAL3ST1 mRNA in a cell, such as the antisense oligonucleotide of the present invention.
  • the inhibitor is an antibody, or antigen binding fragment thereof which specifically binds to the GAL3ST1 protein, and which is capable of downregulating the GAL3ST1 protein in a cell.
  • the inhibitor such as the antisense oligonucleotide can be used in combination with Libmeldy stem cell therapy.
  • Libmeldy stem cell therapy is an autologous haematopoietic stem cell (HSC) gene therapy product currently used for late infantile or early juvenile forms of the MLD.
  • HSC autologous haematopoietic stem cell
  • Figure 1 Table Al, Compounds tested in the Examples section (e.g. in Example 1)
  • Figure 2 Table C, Compounds tested in the Examples section (e.g. in Example 5)
  • Oligonucleotide synthesis a well-known technique in the field, was utilized for the experiments described herein.
  • the oligonucleotides were procured from Biosearch Technologies (Lystrup, Denmark). Following solid support cleavage, the oligonucleotides were subjected to cartridge purification utilizing ammonium acetate. Subsequently, the oligonucleotides were dissolved to a concentration of 750 pM in PBS, and purity was assessed by LC/MS with a minimum threshold of 80% purity.
  • Fig. 1 showcases the compounds produced by the aforementioned methodology, all of which underwent testing in either A549 cells (Example 1) or CaCo2 cells (Example 2). Subsequently, select ASO compounds that displayed efficient downregulation of GAL3ST1 underwent further assessment, as detailed in Example 3, where EC50 values were determined.
  • Example 1 Testing in vitro efficacy of antisense oligonucleotides targeting GAL3ST1 in A549 cells at single test concentration.
  • a cell-based screening assay was developed in cells showing endogenous expression of GAL3ST1 premRNA.
  • Various cell densities, compound incubation periods, and concentrations were optimized prior to establishing the assay conditions, as delineated below.
  • the A549 cells were cultured and expanded as per the supplier's instructions (ECACC, acquired from Merck, 86012804-1VL). The cells were grown to 70-80% confluency, trypsinized, and suspended in growth media. Viable cells were counted using trypan blue and a Countess 3 automatic cell counter. The requisite number of cells were diluted in complete growth media, mixed via gentle pipetting, added to reagent reservoirs, and dispensed into 96-well plates using a multichannel pipette in a total volume of 195 pl/well.
  • sterile PBS was added to the moats of the 96-well culture plates (NuncTM EdgeTM 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplates).
  • GAL3ST1 ASOs (refer to Table 3, columns "ASO ID"; the compounds are listed in Fig. 1) were added directly to the growth media from a 20-fold stock dilution in PBS, resulting in a final ASO concentration of either 10 or 25 pM.
  • Table 1 provides a summary of the crucial parameters pertaining to the cellular work.
  • RNA was used as input template for qPCR, using qScriptTM XLT One-Step RT-qPCR ToughMix® (cat# 95134-500) from QuantaBio and duplexed PCR reaction with probe based qPCR assays from Integrated DNA technologies (IDT) listed in Table 2.
  • Table 2 qPCR assay used for screening GAL3ST1 targeting ASOs.
  • the qPCR reaction was performed in 384 wells using a QuantStudio 7 Flex instrument (Applied Biosystems by Thermo Fisher Scientific). Quantification of GAL3ST1 mRNA was carried out using the ddCT method, with the median value of all the PBS-treated wells within the same plate serving as the untreated control.
  • Table 3B Expression level of GAL3ST1 in relation to PBS treated control cells (in %) a549 10 pM
  • Example 2 Testing in vitro efficacy of antisense oligonucleotides targeting GAL3ST1 in CACO2 cells at single test concentration.
  • ASOs antisense oligonucleotides
  • a cell-based screening assay was developed using Caco2 cells and in SK-N-AS cells.
  • the cells were cultured and expanded according to the supplier's recommendations. When reaching 70-80% confhiency the cells are trypsinized and resuspended in growth media. Viable cells are counted using trypan blue and a Vi-CELL automatic cell counter (Beckman Coulter). A predetermined number of cells were diluted in growth media, added to 96-well plates, and treated with the ASOs. To minimize plate effect and evaporation, sterile PBS was added to the moats of the culture plates.
  • Table 4 Information on Caco2 and SK-N-AS cells
  • Each plate contained antisense oligonucleotides (ASOs) designed to target GALST1, as well as 10 PBS controls (10 pL), 4 GAL3ST1 positive controls, and 2 controls targeting the ATXN3 gene.
  • ASOs antisense oligonucleotides
  • the Caco2 cells were harvested by gently aspirating the growth media, and RNA was extracted using the Macher ey-Nagel NucleoSpin 96 RNA Kit according to the manufacturer's instructions. The RNA was eluted in 75 pl of water.
  • Example 3 Determination of EC50 values of GAL3ST1 targeting ASOs in Caco2 and SK-N-AS cells
  • concentration response experiments were performed for compounds showing high levels of knockdown.
  • the cells were incubated with varying concentrations of ASOs (0.01, 0.0316, 0.1, 0.316, 1.0, 3.16, 10, and 31.6 pM), and concentration response curves and ECso values were generated using the same method as described in Example 2.
  • concentration response curves were generated using GraphPad Prism software version 9 with the "log(inhibitor) vs. response— Variable slope (four parameters)" fit, with the bottom constrained to >0 and top set to 100.
  • the ECso values are presented in Table 7.
  • Example 4 Overview on identified target regions within the GAL3ST1 pre-mRNA sequence (SEQ ID NO: 1) which allow for efficiently downregulating GAL3ST1
  • Table B2 Targeting sequences as shown in Table B2 allowed for a very efficient down-regulation of the target gene.
  • Table B3 Targeting sequences as shown in Table B3 allowed for the most efficient down-regulation of the target gene.
  • Example 5 Testing in vitro efficacy of additional antisense oligonucleotides targeting GAL3ST1 in CACO2 cells at single test concentration.
  • GAL3ST1 target regions identified in Example 1 to 4 above further ASOs were designed with different sugar and back-bone modifications.
  • An overview on the compounds is provided in Table C in Figure 2.
  • Table Al the annotation for the individual nucleotides is provided in Table A2.
  • the new ASOs were tested for activity in Caco2 cells at a single concentration of 10 pM.
  • some compounds that were tested in Examples 1 to 4 were included in the tests.
  • the protocol described in Example 2 was used. The results are shown in Table 8. The results for compounds that have been already tested in Examples 1 to 4 (such as the compound with ASO ID 1 41) are given in bold.
  • Table 8 Expression level of GAL3ST1 in relation to PBS treated control cells (% UTC) Caco2 10 pM
  • Example 6 Determination of EC50 values of GAL3ST1 targeting ASOs in Caco2 cells
  • Example 7 Testing in vitro efficacy and potency of antisense oligonucleotides targeting GAL3ST1 in iPSC derived human neurons at multiple test concentrations.
  • the activity of preferred ASOs were tested in iPSC derived human neurons to confirm activity and potency in a relevant cellular model.
  • the iPSC derived neurons were maintained as recommended by the supplier (Fuji film, 01279) and RNA were purified and analyzed by microarray as described in example 8. Table 10. Effect on GAL3ST1 as evaluated by microarray ASO knockdown expressed as %PBS. Evaluation of ASO in vitro activity in Caco2 cells and iPSC derived neurons shows good activity for all ASOs leading to significant reduction of GAL3ST1 mRNA.
  • Example 8 Off-target effects of selected gapmers in vitro evaluated by microarrays
  • Modified oligonucleotide gapmers complementary to human GAL3ST1 pre-mRNA were designed and tested for their transcriptome-wide off-target effects in human neurons by unassisted uptake.
  • the gapmers were tested at four different concentrations (0.2, 1, 5, and 25 pM) and compared to PBS-treated controls in a series of experiments under similar conditions. For each concentration typically three replicates were done.
  • Human glutamatergic-enriched cortical neurons derived from induced pluripotent stem cells (iCell GlutaNeurons; FUJIFILM Cellular Dynamics, Inc.) were plated at a density of 80000 cells per well. Media and supplements were according to manufacturer’s specifications and half media change was performed on day 1 and 4 after plating the cells. Gapmers were added 4 days after plating and incubated with the cells for 4 days. After 4 days of incubation, total RNA was isolated from the cells and DNase-treated using RNeasy kit (QIAGEN) according to the manufacturer’s instructions.
  • RNeasy kit QIAGEN
  • first strand cDNA was synthesized from total RNA with a combination of a Poly-dT and random primers containing a 5 '-adaptor sequence.
  • a 3 ’-adaptor was added to the single stranded cDNA followed by low-cycle PCR amplification.
  • the amplified cDNA was used as template for in vitro transcription to produce amplified amounts of complementary mRNA (cRNA).
  • cRNA complementary mRNA
  • the cRNA was then used as input for a second round of cDNA synthesis, producing double stranded cDNA.
  • the cDNA was hybridized to Human Clariom GO Screen 384-array plates, stained, and imaged on a GeneTitan Multi-Channel Instrument.
  • Probe intensities from the imager were summarized and corrected for technical variation between arrays by Robust Multichip Average (RMA) normalization.
  • RMA Robust Multichip Average
  • concentration-response curves of RNA levels (as percent of PBS) after treatment with gapmer at four different concentrations were analyzed by nonlinear least-squares fitting of a two-parameter logistic function (fitting parameters were slope and half-maximal effective concentration, EC50).
  • Genes were selected for CRC analysis if they (1) showed significant and at least 25% reduction in RNA levels between PBS-treated samples and samples treated with 25 pM gapmer (by empirical Bayes-moderated Estatistics with a false discovery rate, FDR, below 5%), (2) showed general concentration-dependent reductions by gapmer treatment (by onesided multiple regression analysis, treating the concentrations of gapmer at 0 (PBS), 0.2, 1, 5 or 25 pM as the ordinal independent variable, with p-value ⁇ 0.005 from the resulting t- statistic for the linear trend), and (3) were not identified in 1 and 2 across multiple different gapmers with different nucleobase sequence.
  • genes for which CRCs were fitted and EC50s estimated were exclusively seen for individual gapmers or for groups of gapmers with identical or near-identical nucleobase sequence. Such genes could therefore be hybridization-based off-targets or genes affected downstream of perturbing such off-targets.
  • EC50 ratios between potential off-targets and the on-target were calculated, grouped, and counted as shown in the table below. The weaker the effect on the off-target compared to the on-target, the larger the EC50 ratio.
  • Table 11 Number of potential off-targets stratified by their EC50 relative to GAL3ST1 as evaluated by microarray. Potential off-targets with EC50 values no more than 10-fold weaker than the GAL3ST1 EC50 are shown in the ‘ ⁇ 10’ column, those with EC50 values between 10- and 20-fold weaker in the ’ 10 to 20’ column, and so forth.
  • Example 9 Single dose in vivo efficacy test in heterozygous GAL3ST1 humanized mice of ASOs.
  • mice In vivo acute tolerability of the antisense oligonucleotides were tested in mice, with 5-6 mice per ASO group. Mice at 8-10 weeks of age were housed in European IVC cages type IIL with TAPVEI aspen bedding (Tapvei Eatonis Oil, Estonia). The cages were enriched with nesting material, wooden sticks and hiding material. The light cycle was 12-hour dark and 12-hour light. Diet was pelleted complete diet (Altromin 1324, Brogaarden), and municipal drinking water. Diet and water were administered ad libitum.
  • the G23 needle was mounted on a stand so it precisely penetrated 3.9 mm through the mouse's skull.
  • the dose volume of 5 pL was injected over 30 seconds and the animal was then placed back in its cage.
  • the most preferred ASOs were dosed ICV to a mouse model containing the human version of GAL3ST1, a humanized mice model. In vivo efficacy in the CNS was evaluated 4 weeks after dosing by necropsy and harvest of brain tissues.
  • RNA samples were sacrificed by cervical dislocation and brain tissue was sampled from striatum, hippocampus, cortex right, cortex left and cerebellum and dissected tissue was snap frozen in Precellys tube (CK14, 2 mL, Cat.no. P000912-LYSK0-A) by submerging in liquid nitrogen. The tissue was stored at -80°C until RNA purification. At the day of RNA extraction, the tissue sample (30-80 mg) was homogenized in 280 pL RLT buffer (Qiagen No.: 79216) using a Precellys 24 tissue homogenizer. The RNA was then extracted using TRIzol (ThermoFisher, Cat.no. 15596023) in combination with a Qiacube (Qiagen) for automated RNA extraction. After extraction RNA concentrations were normalized to 2 ng/pl.
  • the diluted RNA was used as input template for the qPCR, using TaqPathTM 1-Step RT- qPCR Master Mix, CG (ThermoFisher cat. No. Al 5299) and qPCR assays from Integrated DNA technologies (IDT) custom made GAL3ST1 assay as seen in Table 13 for human GAL3ST1 and Ppia-Mm.PT.39a.2gs as endogenous normalizer.
  • IDTT Integrated DNA technologies
  • Table 13 GAL3ST1 qPCR assay used for RNA quantification of the GAL3ST1 humanized mice.
  • the expression level of human GAL3ST1 was calculated using the ddCt approach and normalized to the average level of GAL3ST1 expression in samples from saline treated mice.
  • a single lOOpg dose of ASO administered intracerebroventricular show significant reduction of human GAL3ST1 relative to mouse ppia as endogenous control in the brain regions striatum, hippocampus, cortex right, cortex left and cerebellum of the humanized mice as seen in Table 14, as well as having an acute tox score below 4.
  • Table 14 Levels of human GAL3ST1 mRNA (normalized to mouse ppia) after ASO treatment (4-weeks) expressed as % of saline treated mice (%UTC).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des oligonucléotides (oligomères) antisens complémentaires de séquences de pré-ARNm GAL3ST1, qui sont aptes à inhiber l'expression de GAL3ST1. L'inhibition de l'expression de GAL3ST1 devrait être bénéfique pour le traitement de la leucodystrophie métachromatique.
PCT/EP2024/065312 2023-06-05 2024-06-04 Oligonucléotides ciblant gal3st1 Ceased WO2024251725A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP24729318.6A EP4720283A1 (fr) 2023-06-05 2024-06-04 Oligonucléotides ciblant gal3st1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23177340.9 2023-06-05
EP23177340 2023-06-05

Publications (1)

Publication Number Publication Date
WO2024251725A1 true WO2024251725A1 (fr) 2024-12-12

Family

ID=86692660

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/065312 Ceased WO2024251725A1 (fr) 2023-06-05 2024-06-04 Oligonucléotides ciblant gal3st1

Country Status (2)

Country Link
EP (1) EP4720283A1 (fr)
WO (1) WO2024251725A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3360549A1 (fr) * 2017-02-14 2018-08-15 Rheinische Friedrich-Wilhelms-Universität Bonn Thérapeutiques par petites molécules pour le traitement de la leucodystrophie métachromatique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3360549A1 (fr) * 2017-02-14 2018-08-15 Rheinische Friedrich-Wilhelms-Universität Bonn Thérapeutiques par petites molécules pour le traitement de la leucodystrophie métachromatique

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BEGEBORBAS, MIKLÓS BEGE & ANIKÓ BORBAS REVIEW PHARMACEUTICALS (BASEL, vol. 15, no. 8, 22 July 2022 (2022-07-22), pages 909
BOUCHER AAMILLER WSHANLEY RZIEGLER RLUND TRAYMOND GORCHARD PJ., ORPHANET J RARE DIS., vol. 10, 7 August 2015 (2015-08-07), pages 94
HONKE KHIRAHARA YDUPREE JSUZUKI KPOPKO BFUKUSHIMA KFUKUSHIMA JNAGASAWA TYOSHIDA NWADA Y, PROC NATL ACAD SCI U S A., vol. 99, no. 7, 26 March 2002 (2002-03-26), pages 4227 - 32
JONES EMEAD S.: "Genetic risk factors for Creutzfeldt-Jakob disease", NEUROBIOL DIS., vol. 142, 18 June 2020 (2020-06-18), pages 104973, XP086210328, DOI: 10.1016/j.nbd.2020.104973
MOL NEUROBIOL., vol. 37, no. 2-3, 9 May 2008 (2008-05-09), pages 93 - 103
POLTEN AFLUHARTY ALFLUHARTY CBKAPPLER JVON FIGURA K, GIESELMANN V. N ENGL J MED, vol. 324, no. 1, 3 January 1991 (1991-01-03), pages 18 - 22
RAMAKRISHNAN HHEDAYATI KKLILLMANN-RAUCH RWESSIG CFEWOU CN, MAIER H, GOEBEL HGIESELMANN VECKHARDT M., J NEUROSCI., vol. 27, no. 35, 29 August 2007 (2007-08-29), pages 9482 - 90
ROBERTS THOMAS C ET AL: "Advances in oligonucleotide drug delivery", NATURE REVIEWS DRUG DISCOVERY, NATURE PUBLISHING GROUP, GB, vol. 19, no. 10, 11 August 2020 (2020-08-11), pages 673 - 694, XP037256878, ISSN: 1474-1776, [retrieved on 20200811], DOI: 10.1038/S41573-020-0075-7 *
SHAIMARDANOVA ACHULPANOVA SVSOLOVYEVA VVMULLAGULOVA AIKITAEVA KVALLEGRUCCI C, RIZVANOV AA. REVIEW FRONT MED (LAUSANNE, vol. 7, 20 October 2020 (2020-10-20), pages 576221
STANLEY T CROOKEXUE-HAI LIANGBRENDA F BAKERROSANNE M CROOKE, REVIEW J BIOL CHEM., January 2021 (2021-01-01), pages 296
THOMAS C ROBERTSROBERT LANGERMATTHEW J A WOOD, REVIEW NAT REV DRUG DISCOV., vol. 19, no. 10, October 2020 (2020-10-01), pages 673 - 694
THURAIRATNAM SUKANTHINI ET AL: "Brain Penetrable Inhibitors of Ceramide Galactosyltransferase for the Treatment of Lysosomal Storage Disorders", ACS MEDICINAL CHEMISTRY LETTERS, vol. 11, no. 10, 16 June 2020 (2020-06-16), US, pages 2010 - 2016, XP093103346, ISSN: 1948-5875, DOI: 10.1021/acsmedchemlett.0c00120 *
VAN RAPPARD DFBOELENS JJWOLF NI, BEST PRACT RES CLIN ENDOCRINOL METAB, vol. 29, no. 2, 16 October 2014 (2014-10-16), pages 261 - 73
W BRAD WANPUNIT P SETH, REVIEW J MED CHEM., vol. 59, no. 21, 10 November 2016 (2016-11-10), pages 9645 - 9667

Also Published As

Publication number Publication date
EP4720283A1 (fr) 2026-04-08

Similar Documents

Publication Publication Date Title
US11781135B2 (en) Methods for treating Alzheimer's disease
RU2566724C9 (ru) Композиции и способы модуляции smn2 сплайсинга у субъекта
US10829761B2 (en) Treatment of idiopathic pulmonary fibrosis using RNA complexes that target connective tissue growth factor
CN101980726A (zh) 治疗与dna重复不稳定性相关的遗传病症的方法和装置
KR20180104075A (ko) IL4Rα, TRPA1, 또는 F2RL1을 표적화하는 RNA 복합체를 사용한 아토피 피부염 및 천식의 치료
US20240043843A1 (en) Treatment for lysine degradation-associated disorders
EP4720283A1 (fr) Oligonucléotides ciblant gal3st1
JP2001510336A (ja) 合成アンチセンスオリゴデオキシヌクレオチド類およびそれらを含む医薬組成物
US20210214727A1 (en) Enhanced oligonucleotides for inhibiting scn9a expression
US20260103714A1 (en) Antisense oligonucleotides for the treatment of canavan disease
WO2025068557A1 (fr) Oligonucléotides antisens pour le traitement de la maladie de canavan
WO2025238004A2 (fr) Oligonucléotide antisens
WO2024068997A2 (fr) Oligonucléotides antisens pour le traitement d'une maladie de canavan
RU2793459C2 (ru) Композиции и способы модуляции smn2 сплайсинга у субъекта
CN122003502A (zh) 用于治疗卡纳万病的反义寡核苷酸
EP4677088A2 (fr) Compositions et procédés de modulation de gls1
EP4677091A2 (fr) Compositions et procédés de modulation de grin2a
RU2858547C2 (ru) Композиции для модулирования экспрессии sod-1
AU2024231822A1 (en) Compositions and methods for modulating sptlc1
WO2026087529A1 (fr) Arn d'inhibition
EP4728075A2 (fr) Compositions et procédés de modulation de l'apolipoprotéine e
WO2025136934A1 (fr) Compositions et procédés de modulation de sptlc2
WO2025111653A1 (fr) Compositions et méthodes améliorées pour le traitement de troubles neurodéveloppementaux monogéniques
AU2024390205A1 (en) Improved compositions and methods for treatment of monogenic neurodevelopmental disorders
WO2025190276A1 (fr) Molécule polynucléotidique pour la suppression de l'expression de dmpk et son utilisation

Legal Events

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

Ref document number: 24729318

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2024729318

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024729318

Country of ref document: EP

Effective date: 20260105

ENP Entry into the national phase

Ref document number: 2024729318

Country of ref document: EP

Effective date: 20260105

ENP Entry into the national phase

Ref document number: 2024729318

Country of ref document: EP

Effective date: 20260105

ENP Entry into the national phase

Ref document number: 2024729318

Country of ref document: EP

Effective date: 20260105

WWP Wipo information: published in national office

Ref document number: 2024729318

Country of ref document: EP