WO2025262002A2 - Oligonucléotides antisens pour le traitement de la maladie d'alzheimer - Google Patents

Oligonucléotides antisens pour le traitement de la maladie d'alzheimer

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Publication number
WO2025262002A2
WO2025262002A2 PCT/EP2025/066815 EP2025066815W WO2025262002A2 WO 2025262002 A2 WO2025262002 A2 WO 2025262002A2 EP 2025066815 W EP2025066815 W EP 2025066815W WO 2025262002 A2 WO2025262002 A2 WO 2025262002A2
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seq
aso
exon
antisense oligonucleotide
nucleotides
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WO2025262002A3 (fr
Inventor
Olav Michael Andersen
Emilie DAM ROSENBERG
Mads FUGLSANG KJØLBY
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Aarhus Universitet
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Aarhus Universitet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • ASOs antisense oligonucleotides
  • SORLA is important for Amyloid Precursor Protein (APP) transport out of the endosomes where, if not counteracted by SORLA, amyloidogenic processing of APP into pathogenic fragments (i.e. the Amyloid p-peptide (A )) occurs.
  • This SORLA- assisted transport of APP ensures a decreased cleavage of APP by the p-secretase, thereby reducing the production of the p-C-terminal fragment (CTF) that can subsequently be further processed to generate amyloid beta (Ap) peptides.
  • AD Alzheimer’s disease
  • Ap accumulates in amyloid plaques within the brain, and is the most important pathological hallmark of the disease.
  • the etiology of the disease is rather linked to the level of P-CTF, and other cargo proteins, that in AD cannot be recycled out of the endosome, leading to endosomal swelling and dysfunctional endosomal activity (i.e. the Endosomal Traffic Jam hypothesis for Alzheimer’s disease).
  • the SORL1 gene - encoding the endosomal sorting receptor SORLA - has been associated with the development of Alzheimer’s disease during the last 15 years. More recently, large whole-exome sequencing studies have identified how SORL1 is the gene harbouring the most genetic variation across the human genome in groups of AD patients.
  • CR-domains represent the main ligand-binding site in all known receptors that contain clusters of CR-domains. Also the cluster of CR-domains of SORLA is involved in binding to ligands, incl. APP. Consequently, mutations in CR-domains can have grave consequences on the functionality of SORLA, both with regard to ligand binding but also with regard to misfolding and ER retention of the protein.
  • AD While knowledge has been gained regarding genetic markers predicting a risk or causal connection for developing AD, no approved treatment for AD is available to date and AD remains to be an immense burden to patients and the health care system.
  • CR-domains represent the main ligand-binding site in most receptors that contain clusters of CR-domains. Also the cluster of CR-domains of SORLA is involved in binding to ligands, incl. APP. However, the typical binding of any ligand does not depend on any isolated CR-domain, but many studies have rather shown how binding is achieved by combined interaction of a number of CR-domains with several sites on their ligand.
  • a functional SORLA protein lacking the specific CR-domain encoded by exon 23, can be produced.
  • This approach has several advantages. Firstly, ASO’s delivered to the brain of AD patients can restore functional SORLA protein in vivo. AD patients carrying genetic variants that lead to mutations in CR-domains are producing SORLA protein, however, due to the mutation this protein is not functional. This mutated SORLA protein misfolds and gets pathologically retained in the endoplasmatic reticulum (ER).
  • ER endoplasmatic reticulum
  • mutated SORLA protein can have a dominant negative effect on non-mutated SORLA (produced from a non-affected allele) due to SORLA dimer formation.
  • the mutated SORLA will lead to misfolding and retention of the non-mutated SORLA in the dimer.
  • the level of functional SORLA will be even more reduced, and as a consequence amyloidogenic processing of APP into pathogenic fragments cannot be counteracted any longer.
  • ASO mediated exon-skipping of mutated a SORLA CR-domain SORLA protein lacking a CR-domain will be produced in the cell, and this variant will not induce misfolding.
  • the present invention concerns an antisense oligonucleotide (ASO) comprising or consisting of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity, wherein binding of the antisense oligonucleotide (ASO) comprising or consist
  • the present disclosure is directed to an antisense oligonucleotide (ASO) binding or capable of binding to a target site comprising or consisting of a target polynucleotide sequence having at least 80% sequence identity to a target polynucleotide sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:
  • SEQ ID NO: 46 SEQ ID NO: 47 and SEQ ID NO: 48, such as at least 85%, 90%, 95%, 98%,
  • the present disclosure is directed to a composition comprising the antisense oligonucleotide, such as a pharmaceutical composition.
  • the present disclosure is directed to an antisense oligonucleotide (ASO) and/or the composition, for use in the prevention, treatment and/or alleviation of Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies, or a disease or disorder associated with Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies.
  • ASO antisense oligonucleotide
  • the present disclosure is directed to a method for treatment of Alzheimer’s Disease, Parkinson’s Disease, and/or Dementia with Lewy bodies comprising administration of a therapeutically effective amount of an antisense oligonucleotide to a subject in need thereof.
  • the present disclosure is directed to a method for mediating exon skipping in SORL1 transcripts in a cell, tissue or organ, wherein the method comprises the step of contacting said cell, tissue or organ with the antisense oligonucleotide and/or the composition, wherein the exon is exon 23 of SORL1.
  • the present disclosure is directed to a method of determining the efficiency of ASO mediated SORL1 exon skipping in a subject, the method comprising the following steps: a) analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in a first sample comprising cerebrospinal fluid derived from a patient, obtained before treatment with an ASO, such as any ASO as defined herein, b) analyzing the levels of SORLA lacking the complement-type repeat encoded by exon 23 in a second sample comprising cerebrospinal fluid derived from the same patient as in a), obtained after treatment with an ASO, c) comparing the level of SORLA lacking the complement-type repeat encoded by exon 23 in the samples of a) and b), and d) determining exon skipping if the level of SORLA lacking the complement-type repeat encoded by exon 23 in b) is higher than in a).
  • the present disclosure is directed to a method for testing if a patient identified with a SORL1 mutation will benefit from treatment with an ASO mediating exon skipping, the method comprising the steps of: a. determining if the mutation is in exon 23 of SORL1 , b. if yes, obtaining a cell comprising the SORL1 mutation identified in the patient, c. selecting an ASO that targets the exon 23, such as any ASO as defined herein, d. contacting a first aliquot of cells with medium comprising the selected ASO, and contacting a second aliquot of cells with medium not comprising an ASO, e.
  • Panels A), C) and E) display agarose gels with PCR-products from reactions using samples from HEK293 cells transfected with ASO 23.17, ASO 23.54, and ASO 23.63, respectively, at concentrations at 100, 200, and 400nM. All three ASOs were tested using MOE and O-Me backbone chemistry as indicated.
  • the amplified product for transcripts with exon 23 were 382 nt in length, while transcripts without exon 23 were 268 nt in length.
  • the ladder shows DNA bands of the indicated lengths.
  • TD.63/ASO23.63 (SEQ ID NO: 26)).
  • E-F display representative images of agarose gel analysis from one biological replicate (out of three) demonstrating the effect on exon 23 skipping by the 30 ASOs designed for the Exon-Walk (EW) strategy.
  • G displays a bar graph showing the percentage of SORL1 transcript that have been deleted of exon 23 by the three biological replicate experiments that measured the effect on exon 23 skipping by the 30 ASOs designed for the Exon-Walk (EW) strategy.
  • the agarose gel image show products for RT-PCR analysis of transcripts isolated from HEK293 cells treated with the indicated ASOs at 12 nM.
  • the migration of the amplicons generated from transcript including (FL) or excluding SORL1 exon 23 (Aex23) are indicated with arrows.
  • the amplified product for transcripts with exon 23 were 377 nt in length, while transcripts without exon 23 were 267 nt in length.
  • the upper panel show SORL1 protein as detected with the LR11 antibody that bind to both full-length SORL1 as well as the truncated receptor form deleted of CR1 (as the epitope for the monoclonal LR11 resides in CR7).
  • the bottom panel represent a loading control with detection of beta-Actin.
  • Cells transfected with Lipofectamine without any ASO content (Lipo; Onm) is set as negative control.
  • the migration of the amplicons generated from transcript including (FL) or excluding SORL1 exon 23 (Aex23) are indicated with arrows.
  • ASO23.63 (SEQ ID NO: 2), ASO23.63.10 (SEQ ID NO: 1) or ASO EW.09 (SEQ ID NO: 3) as also tested by RT-PCR.
  • the qPCR results depicted in the graph were normalized to the expression of housekeeping gene HPRT and the total level of SORL1 (determined with a taq-man assay specific for the exon3-exon4 boundary) and are displayed as relative percentage to non-treated cells corresponding to 0 nM ASO.
  • the amplified product for transcripts with exon 24 were 351 nt in length, while transcripts without exon 24 were 228 nt in length
  • the amplified product for transcripts with exon 30 were 326 nt in length, while transcripts without exon 30 were 198 nt in length
  • the amplified product for transcripts with exon 25 were 342 nt in length, while transcripts without exon 25 were 222 nt in length
  • the amplified product for transcripts with exon 26 were 324 nt in length, while transcripts without exon 26 were 198 nt in length
  • the amplified product for transcripts with exon 33 were 360 nt in length, while transcripts without exon 33 were 220 nt in length
  • the amplified product for transcripts with exon 31 were 358 nt in length, while transcripts without exon 31 were 202 nt in length.
  • FL indicate the full- length transcripts a) the amplified product of full-length transcript for exon 25 should be 342 nt in length and b) the amplified product of full-length transcript for exon 30 should 326 nt in length.
  • A) A25 indicate the amplified products of transcripts without exon 25 were 222 nt in length, while B) A30 indicate the amplified products of transcripts without exon 30 were 198 nt in length
  • FL indicate the full- length transcripts a) the amplified product of full-length transcript for exon 31 should be 358 nt in length and b) the amplified product of full-length transcript for exon 24 should be 351 nt in length.
  • A) A31 indicate the amplified products of transcripts without exon 31 were 202 nt in length, while B) A24 indicate the amplified products of transcripts without exon 24 were 228 nt in length Figure 13 Testing for off-target effects of most promising ASOs for effects on splicing of exon 26
  • sequence identity refers to the extent to which two optimally aligned polynucleotide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e. , the entire reference sequence or a smaller defined part of the reference sequence.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference polynucleotide molecule (or its complementary strand) as compared to a test polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totalling less than 20 percent of the reference sequence over the window of comparison).
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction times 100.
  • the comparison of one or more polynucleotide sequences refer to a full-length polynucleotide sequence.
  • ASO includes a plurality of such ASOs, such as one or more ASOs, at least one ASOs, or two or more ASOs.
  • exon skipping is herein defined as inducing, producing or increasing production within a cell of a mature mRNA that does not contain a particular exon that would be present in the mature mRNA without exon skipping.
  • exons are coding sections of an RNA transcript, or the DNA encoding it, that may be translated into protein. Exons can be separated by intervening sections of DNA that do not code for proteins, known as introns. Following transcription, new, immature strands of messenger RNA, called pre-mRNA, may contain both introns and exons. These pre-mRNA molecules may undergo a modification process in the nucleus called splicing during which the noncoding introns are cut out and only the coding exons remain. Splicing produces a mature messenger RNA molecule that may be translated into a protein.
  • GC content refers generally to the cytosine and guanine content of a nucleic acid molecule.
  • SORLA as used herein is synonymous to the terms SORLA, Sortilin-related receptor, sortilin related receptor 1 , SORL1 , Low-density lipoprotein receptor relative with 11 ligand-binding repeats, LDLR relative with 11 ligand-binding repeats, LR11 , SorLA-1 , Sorting protein-related receptor containing LDLR class A repeats and gp250.
  • Human sorLA is annotated in UniProt under the accession number Q92673.
  • TD tool-designed
  • TD refers to ASOs designed using the eSkip-finder online tool.
  • the TD ASOs are interchangeable referred to with the prefix “23.”.
  • TD.63 and “23.63” are used interchangeably.
  • TD.17 and “23.17” are used interchangeably.
  • TD.54 and “23.54” are used interchangeably.
  • CA is interchangeable referred to with the prefix “23.63”.
  • CA.1 and 23.63.1 are used interchangeably herein.
  • CA.2 and 23.63.2 are used interchangeably herein.
  • CA.3 and 23.63.3 are used interchangeably herein.
  • CA.4 and 23.63.4 are used interchangeably herein.
  • CA.5 and 23.63.5 are used interchangeably herein.
  • CA.6 and 23.63.6 are used interchangeably herein.
  • CA.7 and 23.63.7 are used interchangeably herein.
  • CA.8 and 23.63.8 are used interchangeably herein.
  • CA.9 and 23.63.9 are used interchangeably herein.
  • CA.10 and 23.63.10 are used interchangeably herein.
  • modified nucleotide or “nucleotide modification” or “modification” refers to a nucleotide the basic structural unit of nucleic acids, RNA or DNA that has been chemically modified, but still functions as a nucleotide.
  • modification refers modifications of the nucleic acid backbone, the nucleobase, the ribose sugar and/or 2'-ribose substitutions, wherein the polynucleotide sequence remains unaltered.
  • antisense oligonucleotide encompasses nucleic acids-based molecules complementary to a target mRNA, particularly a seed sequence of the target mRNA to form duplex with the target mRNA.
  • PCR Real-time polymerase chain reaction
  • qPCR qRT-PCR
  • the present invention takes advantage of antisense oligonucleotides for inducing exonskipping in the pre-mRNA transcripts (also referred to as precursor mRNA).
  • This type of antisense-mediated splicing modulation uses antisense oligonucleotides (ASOs) to manipulate the splicing.
  • ASOs may induce exon skipping by sterically blocking the binding of splicing factors to pre-mRNA transcripts (also referred to as precursor mRNA).
  • the Antisense Oligonucleotides described herein may, in some embodiments, be further characterized by the modifications, and/or properties described in the sections “Modifications of the ASOs” and section “Properties of the ASOs” of the present disclosure.
  • the present disclosure concerns an antisense oligonucleotide (ASO) comprising or consisting of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity, wherein binding of the antisense oligonucleotide to
  • the present disclosure concerns an antisense oligonucleotide (ASO) comprising or consisting of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity, wherein binding of the antisense oligonucleotide to
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, and SEQ ID NO: 13
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 14 or SEQ ID NO: 17
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 5.
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 10.
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 11. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 15.
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 17. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 20.
  • the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 22. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 23. In some embodiments, the ASO comprises or consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 24.
  • the ASOs of the present invention may be defined by the polynucleotide sequence to which the ASOs bind or are capable of binding.
  • the polynucleotide sequence to which the ASOs bind or are capable of binding may be referred to as target sites or target polynucleotide sequences.
  • the present disclosure concerns an antisense oligonucleotide (ASO) binding or capable of binding to a target site comprising or consisting of a target polynucleotide sequence having at least 80% sequence identity to a target polynucleotide sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33,
  • SEQ ID NO: 34 SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,
  • SEQ ID NO: 39 SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43,
  • the antisense oligonucleotide is targeted to a 5’ splice site, a 3’ splice site and/or an exonic splice enhancer site (ESE).
  • the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 25. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 27.
  • the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 30.
  • the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 31. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 32. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 33.
  • the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 34. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 35. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 36.
  • the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 40. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 41. In some embodiments, the (ASO) binding or capable of binding to a target site comprises or consists of a target polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42.
  • binding to a target site causes exon skipping of exon 23 encoding a complement-type repeat (CR) domain of SORLA, wherein exon 23 comprises or consists of a polynucleotide sequence of SEQ ID NO: 49, or a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 49, such as at least 85%, 90%, 95%, 98% or 99% sequence identity.
  • exon 23 comprises or consists of a polynucleotide sequence of SEQ ID NO: 49, or a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 49, such as at least 85%, 90%, 95%, 98% or 99% sequence identity.
  • binding to a target site causes exon skipping of exon 23 encoding a complement-type repeat (CR) domain of SORLA
  • exon 23 comprises or consists of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107 such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity.
  • binding to a target site causes exon skipping of exon 23 encoding a complement-type repeat (OR) domain of SORLA, wherein exon 23 comprises a mutation selected from the list consisting of: p.C1078R (T>C), p.R1080C (C>T), p.R1084C (C>T), p.W1095C (G>C), P.W1096C (G>C), p.D1102N (G>A), p.C1103Y (G>A), p.D1105H (G>C), p.D1108N (G>A), and p.C1112Y (G>A).
  • exon 23 comprises a mutation selected from the list consisting of: p.C1078R (T>C), p.R1080C (C>T), p.R1084C (C>T), p.W1095C (G>C), P.W1096C (G>C), p.D1102N (
  • oligonucleotides of the present invention may comprise one or more modifications, for example to increase stability of the antisense oligonucleotides.
  • the antisense oligonucleotide further comprises one or more modifications at at least one nucleotide position, or at each nucleotide position.
  • the modifications are modifications of the nucleic acid backbone, the nucleobase, the ribose sugar and/or 2'-ribose substitutions.
  • the antisense oligonucleotide comprises one or more modifications, such as two or more, such as three or more modifications, selected from the group consisting of: Peptide nucleic acid (PNA), Serinol nucleic acid (SNA), Phosphorodiamidate morpholino oligomer (PMO), Thiomorpholino oligonucleotide (TMO), and Morpholino nucleic acid (MNA).
  • PNA Peptide nucleic acid
  • SNA Serinol nucleic acid
  • PMO Phosphorodiamidate morpholino oligomer
  • TMO Thiomorpholino oligonucleotide
  • MNA Morpholino nucleic acid
  • the antisense oligonucleotide comprises a Peptide nucleic acid (PNA) modification.
  • the antisense oligonucleotide comprises a Serinol nucleic acid (SNA) modification.
  • the antisense oligonucleotide comprises a Phosphorodiamidate morpholino oligomer (PMO) modification. In some embodiments, the antisense oligonucleotide comprises a Thiomorpholino oligonucleotide (TMO) modification. In some embodiments, the antisense oligonucleotide comprises a Morpholino nucleic acid (MNA) modification.
  • PMO Phosphorodiamidate morpholino oligomer
  • TMO Thiomorpholino oligonucleotide
  • MNA Morpholino nucleic acid
  • the antisense oligonucleotide comprises a 2’-O-methyl (2’-OMe) modification of the ribose sugar. In some embodiments, the antisense oligonucleotide comprises 2’-O-methoxyethyl (2’MOE) modification of the ribose sugar. In some embodiments, the antisense oligonucleotide comprises a 2’-fluoro (2’-F) modification of the ribose sugar. In some embodiments, the antisense oligonucleotide comprises a 2’- 0,4’C-ethylene-bridged nucleic acid (ENA) modification of the ribose sugar.
  • ENA nucleic acid
  • the antisense oligonucleotide comprises a 2’,4’-constrained 2’-0-ethyl (cEt) modification of the ribose sugar.
  • the antisense oligonucleotide comprises an Amido-bridged nucleic acid (AmNA) modification of the ribose sugar.
  • the antisense oligonucleotide comprises a Guanidine-bridged nucleic acid (GuNA) modification of the ribose sugar. Cyclohexenyl nucleic acid (CeNA) modification of the ribose sugar.
  • the antisense oligonucleotide comprises an Anhydrohexitol nucleic acid (HNA) modification of the ribose sugar. In some embodiments, the antisense oligonucleotide comprises an Altritol nucleic acid (ANA) modification of the ribose sugar. In some embodiments, the antisense oligonucleotide comprises a Tricyclo-DNA (tc-DNA) modification of the ribose sugar. In some embodiments, the antisense oligonucleotide comprises a 7’, 5’-alpha- bicyclo-DNA (7’5’-a-bc-DNA) modification of the ribose sugar.
  • the antisense oligonucleotide comprises one or more 2’, 4’- constrained 2'-0-Ethyl (cEt) modifications. In some embodiments, the antisense oligonucleotide comprises one or more 2'-O-Methylation (2’-0me) modifications. In some embodiments, the antisense oligonucleotide comprises one or more phosphorothioate modifications. In some embodiments, the antisense oligonucleotide comprises a 2’-O-methoxyethyl (2’MOE) sugar modification. In some embodiments, the antisense oligonucleotide comprises locked nucleic acid.
  • the ASOs may further be conjugated to moieties which may attribute properties to the ASO, non-limiting examples of such properties may be the cell-penetrance or celltargeting.
  • the antisense oligonucleotide is conjugated to a moiety or to a nanoparticle formulation.
  • the moiety is a celltargeting moiety and/or a cell-penetrating moiety.
  • the antisense oligonucleotide is conjugated to Triantennary N-acetylgalactosamine (GalNAc) moiety, TAT (SEQ ID NO: 108), and/or a peptide.
  • GalNAc Triantennary N-acetylgalactosamine
  • the antisense oligonucleotide is conjugated to a Triantennary N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the antisense oligonucleotide is conjugated to TAT (SEQ ID NO: 108). In some embodiments, the antisense oligonucleotide is conjugated to a peptide.
  • GalNAc Triantennary N-acetylgalactosamine
  • TAT SEQ ID NO: 108
  • the antisense oligonucleotide is conjugated to a peptide.
  • oligonucleotides of the present invention may be characterized, in some embodiments, by any of the following properties.
  • the length and GC content of the antisense oligonucleotides of the present invention may be varied.
  • the antisense oligonucleotide is between 12 to 25 nucleotides in length, such as between 12 and 23 nucleotides, between 12 and 21 nucleotides, between 12 and 20 nucleotides, between 12 and 19 nucleotides, between 12 and 18 nucleotides, between 12 and 16 nucleotides, between 12 and 14 nucleotides, between 14 and 25 nucleotides, between 14 and 23 nucleotides, between 14 and 21 nucleotides, between 14 and 20 nucleotides, between 14 and 19 nucleotides, between 14 and 18 nucleotides, between 14 and 16 nucleotides, between 16 and 25 nucleotides, between 16 and 23 nucleotides, between 16 and 21 nucleotides, between 16 and 20 nucleotides, between 16 and 19 nucleotides, between 16 and 18 nucle
  • the antisense oligonucleotide is at least 12 nucleotides long, such as at least 14 nucleotides, and/or at least 16 nucleotides and/or at least 18 nucleotides, and/or at least 20, and/or at least 22 nucleotides, and/or at least 24 nucleotides, and/or at least 26 nucleotides, and/or at least 28 nucleotides, and/or at least 30 nucleotides long.
  • the antisense oligonucleotide is 23 nucleotides long. In some embodiments, the antisense oligonucleotide has a GC-content of 40 to 60%, such as 45 to 55%.
  • the antisense oligonucleotides of the present invention may e.g. be formulated in a composition such as a pharmaceutical composition.
  • the present disclosure concerns an composition comprising the antisense oligonucleotide as described herein, such as a pharmaceutical composition.
  • Such compositions may comprise more than one of the antisense oligonucleotides of the present disclosure.
  • the composition comprises one or more of said antisense oligonucleotides.
  • the ASOs of the present invention may facilitate skipping of exon 23.
  • mutations in exon 23 may for example cause or promote the development of e.g. Alzheimer’s disease.
  • the present disclosure concerns an antisense oligonucleotide (ASO) as described herein and/or the composition as described herein, for use as a medicament.
  • ASO antisense oligonucleotide
  • the present disclosure concerns an antisense oligonucleotide (ASO) as described herein and/or the composition as described herein, for use in the prevention, treatment and/or alleviation of Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies, or a disease or disorder associated with Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies.
  • ASO antisense oligonucleotide
  • the present disclosure concerns use of an antisense oligonucleotide as described herein in the manufacture of a medicament for treatment of Alzheimer’s Disease, Parkinson’s Disease, and/or Dementia with Lewy bodies.
  • the present disclosure concerns a method for treatment of Alzheimer’s Disease, Parkinson’s Disease, and/or Dementia with Lewy bodies comprising administration of a therapeutically effective amount of an antisense oligonucleotide as described herein to an subject in need thereof.
  • an effective amount of the antisense oligonucleotide is administered to the eye, to the spinal cord, to the nose, to the cerebrospinal fluid, to the brain and/or to the liver, such as wherein the antisense oligonucleotide is administered intrathecally or intranasally.
  • the present invention takes advantage of antisense oligonucleotides for inducing exon-skipping in the pre-mRNA transcripts (also referred to as precursor mRNA).
  • This type of antisense-mediated splicing modulation uses antisense oligonucleotides (ASOs) to manipulate the splicing.
  • ASOs may induce exon skipping by sterically blocking the binding of splicing factors to pre-mRNA transcripts (also referred to as precursor mRNA).
  • the present disclosure concerns a method for mediating exon skipping in SORL1 transcripts in a cell, tissue or organ, wherein the method comprises the step of contacting said cell, tissue or organ with the antisense oligonucleotide as described herein and/or the composition as described herein, wherein the exon is exon 23 of SORL1.
  • the method for mediating exon skipping may be facilitated by one ASO or more than one ASO.
  • one ASO is used.
  • more than one ASO is used, such as 2 ASOs, such as 3 ASOs, such as 4 ASOs, such as 5 ASOs, such as 6 ASOs, such as 7 ASOs, such as 8 ASOs, such as 9 ASOs, such as 10 ASOs, such as 11 ASOs, such as 12 ASOs, such as 13 ASOs, such as 14 ASOs, such as 15 ASOs, such as 16 ASOs, such as 17 ASOs, such as 18 ASOs, such as 19 ASOs, such as 20 ASOs, such as 21 ASOs, such as 22 ASOs, such as 23 ASOs, or 24 ASOs.
  • the skipping of exon 23 may for example be analyzed by analyzing the inhibition of inclusion of exon 23 in the spliced transcripts or by analyzing the promotion of skipping of exon 23 in the spliced transcripts and the results of these analyses may for example be presented as IC50 values or EC50 values, respectively.
  • IC50 half maximal inhibitory concentration
  • EC50 half maximal effective concentration
  • IC50 half maximal inhibitory concentration
  • EC50 half maximal effective concentration
  • exon skipping of one exon is mediated.
  • the ASO inhibits the inclusion of exon 23 of SORL1 with an IC50 of 20 nM or less, such as 19 nM or less, such as 18 nM or less, such as 17 nM or less, such as 16 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less, such as 9 nM or less, such as 8 nM or less, such as 7 nM or less, such as 6 nM or less, such as 5 nM or less, such as 4 nM or less, or such as 3 nM or less.
  • the ASO inhibits the inclusion of exon 23 of SORL1 with an IC50 of 2.6 nM or less. In some embodiments, the ASO promotes skipping of exon 23 of SORL1 with an EC50 of 20 nM or less, such as 19 nM or less, such as 18 nM or less, such as 17 nM or less, such as 16 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less, such as 9 nM or less, such as 8 nM or less.
  • 20 nM or less such as 19 nM or less, such as 18 nM or less, such as 17 nM or less, such as 16 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11
  • the amount of SORL1 transcripts in said cell, tissue or organ is at least 80% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition, such as at least 81%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition.
  • the amount of SORL1 transcripts in said cell, tissue or organ is determined by qPCR.
  • the amount of SORLA protein in said cell, tissue or organ is at least 80% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition, such as at least 81%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition.
  • the amount of SORLA protein in said cell, tissue or organ is determined by western blotting.
  • the efficiency of the ASO in mediating SORL1 exon skipping in a subject may for example be analyzed by analyzing the inclusion of exon 23 before and after treatment with said ASO.
  • the present disclosure concerns a method of determining the efficiency of ASO mediated SORL1 exon skipping in a subject, the method comprising the following steps: a) analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in a first sample comprising cerebrospinal fluid derived from a patient, obtained before treatment with an ASO, such as any ASO as defined herein, b) analyzing the levels of SORLA lacking the complement-type repeat encoded by exon 23 in a second sample comprising cerebrospinal fluid derived from the same patient as in a), obtained after treatment with an ASO, c) comparing the level of SORLA lacking the complement-type repeat encoded by exon 23 in the samples of a) and b), and d) determining exon skipping if the level of SORLA lacking the complement-type repeat encoded by exon 23 in b) is higher than in a).
  • the method optionally comprising the step of obtaining sample b) at several time points after treatment with an ASO, thus monitoring the efficiency of ASO mediated exon skipping over time.
  • a method to test whether a patient may benefit from treatment with an ASO mediating exon skipping may for example involve contacting a cell comprising the mutation with an ASO and comparing exon skipping in said cell to the exon skipping in a non-treated counterpart.
  • the present disclosure concerns a method for testing if a patient identified with a SORL1 mutation will benefit from treatment with an ASO mediating exon skipping, the method comprising the steps of: a. determining if the mutation is in exon 23 of SORL1 , b. if yes, obtaining a cell comprising the SORL1 mutation identified in the patient, c. selecting an ASO that targets the exon 23, such as any ASO as defined herein, d.
  • contacting a first aliquot of cells with medium comprising the selected ASO, and contacting a second aliquot of cells with medium not comprising an ASO e. analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in the first and the second aliquot, f. comparing the level of SORLA lacking the complement-type repeat encoded by exon 23 in the first and the second aliquot, and g. concluding that the patient will benefit from treatment with the ASO if the level of SORLA lacking the complement-type repeat encoded by exon 23 in the first aliquot is higher than in the second aliquot.
  • the present disclosure concerns a method for testing if a patient identified with a SORL1 mutation will benefit from treatment with an ASO mediating exon skipping, the method comprising the steps of: a. determining if the mutation is in exon 23 of SORL1 , b. if yes, obtaining a cell comprising the SORL1 mutation identified in the patient, c. selecting an ASO that targets the exon 23, such as any ASO as defined herein, d. contacting a first aliquot of cells with medium comprising the selected ASO, and contacting a second aliquot of cells with medium not comprising an ASO, e. analyzing the levels of endoplasmic reticulum-resident SORLA in the first and the second aliquot, f.
  • the method is an in-vitro method.
  • said ASO is a ASO as described herein, such as e.g. in the sections “Antisense Oligonucleotides”, “Modifications of the ASOs”, and/or “Properties of the ASOs”.
  • a method of identifying an ASO suitable for treatment of a patient with Alzheimer’s Disease may for example involve the evaluation of whether the target site of the ASO comprise the mutation.
  • the present disclosure concerns a method of producing an ASO suitable for treatment of a patient with Alzheimer’s Disease, wherein the patient carries a mutation in an exon encoding a complement-type repeat (OR) domain of SORLA, the method comprising the following steps: a. identifying an ASO as described herein, b. determining if the target site of the ASO comprises the mutation, or if the target site of the ASO does not comprise the mutation, and c. determining that the ASO that binds to a target site not comprising the mutation is suitable for treatment of a patient with Alzheimer’s Disease.
  • the mutation is a calcium-cage-mutation, an asx-turn mutation or an odd-numbered cysteines-mutation. In some embodiments, the mutation is a mutation in exon 23. In some embodiments, the mutation is a substitution of cysteine to arginine at position 1078 (C1078R) of human SORL1 , wherein the mutation is a substitution of arginine to cysteine at position 1080 (R1080C) of human SORL1 , wherein the mutation is a substitution of arginine to cysteine at position 1084 (R1084C) of human SORL1 , wherein the mutation is a substitution of tryptophan to cysteine at position 1095 (W1095C) of human SORL1 , wherein the mutation is a substitution of tryptophan to cysteine at position 1096 (W1096C) of human SORL1 , wherein the mutation is a substitution of aspartic acid to asparagine at position 1102 (D1102N) of human
  • the mutation is a substitution of cysteine to arginine at position 1078 (C1078R) of human SORL1. In some embodiments, the mutation is a substitution of arginine to cysteine at position 1080 (R1080C) of human SORL1. In some embodiments, the mutation is a substitution of arginine to cysteine at position 1084 (R1084C) of human SORL1. In some embodiments, the mutation is a substitution of tryptophan to cysteine at position 1095 (W1095C) of human SORL1. In some embodiments, the mutation is a substitution of tryptophan to cysteine at position 1096 (W1096C) of human SORL1.
  • the mutation is a substitution of aspartic acid to asparagine at position 1102 (D1102N) of human SORL1. In some embodiments, the mutation is a substitution of cysteine to tyrosine at position 1103 (C1103Y) of human SORL1 . In some embodiments, the mutation is a substitution of aspartic acid to histidine at position 1105 (D1105H) of human SORL1. In some embodiments, the mutation is a substitution of aspartic acid to asparagine at position 1108 (D1108N) of human SORL1 . In some embodiments, the mutation is a substitution of cysteine to tyrosine at position 1112 (C1112Y) of human SORL1 .
  • the mutation is selected from the list of mutations recited in the table below:
  • the mutation is a substitution of thymine (T) to cytosine (C) at position chr11:121570165. In some embodiments the mutation is a substitution of cytosine (C) to thymine (T) at position chr11:121570171.
  • the mutation is a substitution of cytosine (C) to thymine (T) chr11:121570183.
  • the mutation is a deletion a deletion of cytosine (C) at position chr11:121570194
  • the mutation is an insertion of guanine (G) after thymine (T) at position chr11 :121570219.
  • the mutation is: a. a substitution of guanine (G) to cytosine (C) at position chr11:121570221, b. a substitution of guanine (G) to adenine (A) at position chr11 :121570221, c. a substitution of guanine (G) to adenine (A) at position chr11:121570237, d. a substitution of thymine (T) to cytosine (C) at position chr11:121570240, e. a substitution of guanine (G) to adenine (A) at position chr11:121570241, f.
  • guanine (G) to cytosine (T) at position chr11:121570241 g. a substitution of guanine (G) to cytosine (C) at position chr11:121570246, h. a deletion of adenine (A) and cytosine (C) at position chr11:121570247, i. a substitution of guanine (G) to adenine (A) at position chr11:121570255, j. a substitution of guanine (G) to cytosine (C) at position chr11:121570255, k.
  • a substitution of thymine (T) to cytosine (C) at position chr11:121570165 q. a substitution of cytosine (C) to thymine (T) at position chr11 : 121570171 , r. a substitution of cytosine (C) to thymine (T) chr11:121570183, s. a deletion a deletion of cytosine (C) at position chr11:121570194, or t. an insertion of guanine (G) after thymine (T) at position chr11:121570219.
  • the mutation is a substitution of guanine (G) to cytosine (C) at position chr11:121570221.
  • the mutation is a substitution of guanine (G) to adenine (A) at position chr11:121570221.
  • the mutation is a substitution of guanine (G) to adenine (A) at position chr11:121570237.
  • the mutation is a substitution of thymine (T) to cytosine (C) at position chr11:121570240.
  • the mutation is a substitution of guanine (G) to adenine (A) at position chr11:121570241.
  • the mutation is a substitution of guanine (G) to cytosine (T) at position chr11:121570241. In some embodiments the mutation is a substitution of guanine (G) to cytosine (C) at position chr11:121570246.
  • the mutation is the mutation is a deletion of adenine (A) and cytosine (C) at position chr11:121570247.
  • the mutation is a substitution of guanine (G) to adenine (A) at position chr11:121570255.
  • the mutation is a substitution of guanine (G) to cytosine (C) at position chr11:121570255
  • the mutation is a substitution of adenine (A) to thymine (T) at position chr11:121570256.
  • the mutation is a substitution of guanine (G) to adenine (A) at position chr11:121570258.
  • the mutation is a substitution of adenine (A) to guanine (G) at position chr11:121570259.
  • the mutation is a substitution of guanine (G) to adenine (A) at position chr11:121570268.
  • the mutation is a substitution of guanine (G) to thymine (T) at position chr11:121570268.
  • Example 001 Establishing RT-PCR detection method for the skipping of exon 23 from the SORL1 transcripts
  • the aim of this example was to establish an RT-PCR based method to monitor SORL1 transcripts with and without exon 23.
  • Primers were designed within the flanking regions, to cover exon-exon boundaries. Temperature optimization was done by PCR.
  • HEK293 cells were transfected using plasmids with cDNA that encoded either the full- length SORL1-FL or exon 23 deleted SORL1 (SORL1-Aex23) with FuGENE.
  • RNA was harvested by standard methods (PureLinkTM RNA from Invitrogen), preparation of cDNA from 1 pg of purified RNA, and PCR was performed using 1 pg of cDNA as template, and taq-man polymerase and PCR instrument to scan temperatures for the amplification step ranging from 42 to 71 Celsius.
  • Gel Loading Dye, Purple (6X) was added to PCR products before they were run on an agarose gel (2%) with gelred, and gel bands were visualized with i Bright Imaging Systems.
  • the primers used in the present example were: Forward primer: CTGTGTGCCCAGGCCAT (17 nt) (SEQ ID NO: 52) Reverse Primer: GTTGTCTCCACAGTCATCCTCAAG (24 nt) (SEQ ID NO: 53)
  • the two DNA sequences (in bold) in Table 1 indicate where the forward and reverse primers anneals to the SORL1 transcript.
  • the amplified product for transcripts with exon 23 were 382 nt in length, while transcripts without exon 23 were 268 nt in length
  • Example 002 Identification and validation of ASOs for skipping of SORL1 exon 23 using the eSkip-Finder online tool
  • the aim of this example was to identify antisense oligonucleotides (ASOs) that can lead to skipping of exon 23 of SORL1 transcripts using a set of ASOs predicted by eSkip-Finder to have a strong ability to lead to skipping of exon 23.
  • ASOs antisense oligonucleotides
  • the eSkip-finder online tool was applied with the SORL1 exon 23 sequence (114 nt) including flanking intronic sequences (200 nt on both upstream and downstream intron).
  • TD.63 SEQ ID NO: 2
  • TD.54 SEQ ID NO: 14
  • TD.17 SEQ ID NO: 15
  • HEK293 cells were transfected with lipofectamine and 100, 200 or 400 nM of the identified ASOs.
  • Time for monitoring ASO effects were based on a publication testing skipping of exons from the APP gene (PMID 29628304), and 48 hrs post-transfection for harvesting cells for RNA extraction and cDNA synthesis was used. RT-PCR and agarose gels.
  • ASO were produced with either MOE (for DNA) or OMe (for RNA) backbone chemistry.
  • the eSkip-Finder was used to identify ASO sequences that could be used for skipping the human SORL1 exon 23.
  • the inventors identified the ASO 23.63 target sequence, which can lead to skipping of >80% of exon 23 of SORL1 transcripts from transfected HEK293 cells.
  • the backbone chemistry of OMe was the superior chemistry for screening of the skipping of exon 23 from SORL1 transcripts in HEK293 cells. 200nM was a preferred concentration for screening purposes.
  • Example 003 Sanger sequencing of exon 23 skipped S0RL1 transcript
  • PCR product from HEK293 cells transfected with 200 nM of ASO 23.63 (O’Me) was analyzed by agarose gel analysis, and the band migrating corresponding to 268 nt was excised from the gel, purified following standard protocols and sequenced by Sanger sequencing.
  • the inventors extracted the shorter PCR product that migrated in agarose gel corresponding to 268 nt and send for Sanger sequencing.
  • the inventors analyzed the sequence and confirmed the presence of the expected new exon-exon boundary corresponding to exclusion of the 114 nt of exon 23, a novel exon-exon boundary corresponding to joining exons 22 and 24 ( Figure 3 and Table 2).
  • the underlined sequences corresponds to the primer sequences (SEQ ID NOs: 52-53).
  • the bold sequences corresponds to the junction between exons 22 and 24 (SEQ ID NOs: 90-91).
  • Example 004 Identification and validation of ASOs for skipping of SORL1 exon 23 using Exon-Walk
  • the aim of this example was to identify antisense oligonucleotides (ASOs) that could lead to skipping of exon 23 of SORL1 transcripts using a set of tiling ASOs spanning the entire exonic sequence based on an experimental approach.
  • ASOs antisense oligonucleotides
  • ASO were produced with OMe backbone chemistry
  • the inventors did an exon-walk experiment for the SORL1 exon 23 with overlapping ASOs tested at 200 nM in transfected HEK293 cells.
  • the inventors designed a set of overlapping ⁇ 25-mer ASO sequences that spanned from the upstream intron, across the entire exon 23, and into the downstream intron with an overlap of 4-6 nucleotides in order to achieve the best GO content and least ability for self-annealing/secondary structure prediction (Figure 4A-D).
  • WT untreated cells
  • Lipo lipofectamine
  • App cells transfected with an ASO generated for a target unrelated in sequence to SORL1 (App).
  • the inventors identified several ASO sequences that could induce skipping of SORL1 exon 23, with EW.08 (SEQ ID NO: 7) and EW.09 (SEQ ID NO: 3) as the most efficient and that led to >80% skipping at the applied conditions.
  • Example 005 ASO shortening according to avoid overlap with the nucleotide substituted by p.Arg1080Cys
  • the aim of the present example was to shorten the ASO23.63 to a sequence that has no overlap with the nucleotide corresponding to the pathogenic variant p.Arg1080Cys (P.R1080C).
  • the ASO23.63.10 was produced with OMe backbone chemistry, and tested together with other ASO for its effect on inducing skipping of SORL1 exon 23 in HEK293 cells using the previously described herein above, with the difference that ASO was tested at a lower dose (12nM) to enhance the possibility to detect minor changes in ASO efficiencies.
  • results The inventors designed an ASO sequence being 2 nucleotides shorter than ASO23.63.10, thereby having nucleotides overlap with the codon for Arg-1084 but otherwise no overlap with any other known pathogenic variants within exon 23.
  • Table 3 below displays the SORL1 exon 23 sequence including the target sequence of ASO23.63 and the localization of the nucleotide that leads to substitution p.R1080C and is a known pathogenic variants causing Alzheimer’s disease within exon 23.
  • the target sequence of ASO23.63.10 deleted of two nucleotides and that have no overlap with the site of variation is also indicated.
  • Aim The aim of the present example was to demonstrate that treatment of cells with ASO23.63 has no unexpected impact on the overall SORL1 protein expression level.
  • HEK293 cells were transfected with 200nM of ASO23.17 (TD.17, SEQ ID NO: 15), ASO23.54 (TD.54, SEQ ID NO: 14), ASO23.63 (TD.63, SEQ ID NO: 2), EW.08 (SEQ ID NO: 7), EW.19 (SEQ ID NO: 18), EW.21 (SEQ ID NO: 19), or the negative control ASO targeting APP using Lipofectamine.
  • Cells were harvested 48 hrs post transfection, and cell lysates were then used for SDS-PAGE analysis followed by transfer to a nitrocellulose membrane using i Blot2.0 instrument.
  • the membrane was blocked in standard blocking buffer, and incubated overnight in a 1 :1 ,000 dilution of the LR11 mouse monoclonal antibody or with an anti-Actin antibody. Detection was carried out using the chemiluminescence femto kit and an i Bright instrument.
  • the inventors could clearly detect the endogenous SORL1 from HEK293 cells, and did not observe any significant decrease in SORL1 receptor expression by treatment with the tested ASOs ( Figure 6).
  • Example 007 Dose-dependent skipping of SORL1 exon 23: EC50 determination for three ASOs using concentration series in HEK293 cells by RT-PCR agarose gels
  • the aim of the present example was to determine the EC50 values to better compare exon 23 skipping efficiency for ASO23.63 (SEQ ID NO: 2), ASO23.63.10 (SEQ ID NO: 1), and EW.09 (SEQ ID NO: 3) also including lower concentrations.
  • HEK293 cells were transfected with ASO23.63, ASO23.63.10 or ASO EW.09 (with
  • OMe backbone chemistry at different concentrations, and 48 hrs post-transfection the cells were harvested, RNA isolated using PureLinkTM RNA from Invitrogen, cDNA synthesis made, and RT-PCR performed with primers as indicated in Example 001.
  • RT-PCR products were analyzed by 1% agarose gels and imaged for inspection of the skipping efficiency.
  • the inventors used RT-PCR and analyzed product by RT-PCR from HEK293 cells transfected with increasing concentrations of ASO23.63, ASO23.63.10 or ASO EW.09 from 0 nM to 400 nM to investigate dose-dependent skipping of SORL1 exon 23.
  • Aim The aim of the present example was to establish a quantitative qPCR analysis of SORL1 transcripts deleted of exon 23 using transfected SH-SY5Y cells for probe validation.
  • Taq-man probes designed to span the novel boundary between exons 22 and 24 (for exon 23 deleted transcripts) and forward/reverse primers within these exons were designed using the IDT PrimerQuestTM tool and ordered from IDT.
  • Standard taq-man assays for full-length probe spanning boundary between exons 3 and 4
  • the non-skipped transcript probe spanning boundary between exons 22 and 23
  • Standard taq-man assays were purchased from IDT (Assay ID: Hs. PT.58.40327368 and Hs.PT.58.23098607).
  • SH-SY5Y cells were either untransfected or transfected with expression plasmids for SORL1 including or excluding exon 23, and 48 hrs post-transfection used for RNA isolation (PureLinkTM RNA from Invitrogen), and cDNA synthesis following manufacturers procedure.
  • Quantitative qPCR was performed using the QuantStudioTM 7 Flex Real-Time PCR System instrument and taq-man assay reagents including nucleotides from TaqMan TM Universal PCR Master Mix from ThermoFisher Scientific (reagent cat.no 4304437).
  • Amplicon Length 102
  • the primers for amplification of a 102 bp fragment are listed as Forward (sense) and Reverse (antisense) as well as the Taq-man probe sense sequence in Table 4.
  • the binding sites of the primers and the probe are indicated in Table 5.
  • SH-SY5Y cells were transfected with pcDNA (blank) or plasmids encoding the full- length SORL1 (SORL1-FL) or exon 23 deleted SORL1 (SORL1-Aex23), and RNA/cDNA was prepared as described herein above. Then the inventors did qPCR with various conditions, optimized until the inventors only obtained a CT-value for the cells that were transfected with the SORL1-AEx23 plasmid.
  • Table 6 shows CT-values of qPCR assay run for SH-SY5Y with endogenous SORLA (WT SHSY5Y), or transfected with a cDNA for the SORL1 including SHSY5Y FL (transfected with plasmid overexpressing Full Length SORL1) or excluding SHSY5Y A23 (transfected with plasmid overexpressing SORL1 AEx23) exon 23. Only samples from SY5Y cells transfected with SORL1-AEx23 plasmids gave a CT-value above background which was established using water as control.
  • the inventors established a taq-man assay that was able to specifically detect transcripts of human SORL1 where exon 23 was deleted, while no signal was observed for transcripts that include exon 23 (i.e. non-skipped, full-length SORL1).
  • Example 009 IC50 determination for three ASOs using concentration series in
  • the aim of this example was to determine the EC50 values to better compare exon 23 skipping efficiency for ASO23.63 (SEQ ID NO: 2), ASO23.63.10 (SEQ ID NO: 1), and EW.09 (SEQ ID NO: 3) also including lower concentrations.
  • RT-PCR and products were analyzed by qPCR with taq-man probes including a housekeeping gene (HPRT), the validated probe spanning the boundary between exons 22 and 24, the standard assays for full-length (probe spanning boundary between exons 3 and 4), and the non-skipped transcript that contain exon 23 (probe spanning boundary between exons 22 and 23).
  • Standard taq-man assays were purchased from IDT (Hs. PT.58.40327368 and Hs.PT.58.23098607)
  • the inventors used qPCR for quantification of the skipped (AEx23) as well as the nonskipped transcript, and made this relative to the total amount of SORL1 transcripts as identified by the exon3-4 boundary probe as well as relative to HPRT levels to ensure equal amount of cDNA/RNA in the qPCR.
  • the inventors also quantified the level of all SORL1 transcripts that contain the boundary between exons 3 and 4, thus being the combined level of transcripts from the cells independent on the skipping event.
  • ASO23.63 was the most efficient ASO with an estimated IC50 of 1.26nM.
  • Example 010 ASO shortening according to overlap in identified sequences
  • ASO ASO was produced with OMe backbone chemistry, and used for transfection of HEK293 cells following protocols described above. RT-PCR and agarose gel analysis of products followed protocols already described.
  • the inventors designed and tested a total of 10 ASO sequences with shorter sequences than ASO23.63.
  • Example 011 Establishing RT-PCR protocols for detection of exon-exclusion of flanking exons in the human SORL1
  • the aim of the present example was to establish RT-PCR assays for analysis of exonexclusion for exons flanking exon 23 in the SORL1 and which show the most sequence similarity with exon 23.
  • the inventors designed primers that were suitable for amplification of fragments specific for the regions around exons 24, 25, 26, 30 or 31. And prepared cDNA for expression plasmids for said deletions constructs, which were then used for transfection of HEK293 cells following protocols described above. RT-PCR and agarose gel analysis of products were optimized for each set of the primers specific for the five splice events. Results:
  • the inventors determined which exons that have the highest sequence similarity to exon 23 from the human SORL1 gene, finding that exon 24, exon 25, exon 26, exon 30, and exon 31 were the five exons with the highest similarity (see Table 7).
  • the inventors prepared expression constructs allowing the preparation of positive control samples for cells with forced expression of SORL1 transcripts that lack each of the five different exons.
  • the inventors then used samples from cells either expressing full-length SORL1 (included all exons), or individually being deleted for one of the five different exons, and applied standard RT-PCR optimization protocols testing a series of temperatures for getting specific signals.
  • the inventors evaluated the obtained PCR-products by agarose gel analysis ( Figure 10).
  • Example 012 Testing for off-target effects of most promising ASOs for effects on splicing of flanking exons
  • the aim of the present example was to test if the ASO molecules had any impact on the inclusion of flanking exons encoding other CR-domains with high sequence similarity to SORL1 exon 23.
  • the inventors transfected HEK293 cells with each of the following four ASOs (EW19, EW21m ASO23.54 or ASO23.63) at 400 nM as previously described. RT-PCR and agarose gel analysis of products followed protocols already described.
  • each of the ASO including ASO23.63, showed high specificity towards skipping of exon 23 with no observed off-target of flanking exons.
  • the aim of this example was to compare the efficiency of ASOs described herein with previously described Exon 23 targeting ASOs.
  • RNA 48 h after transfection The inventors transfected HEK293 cells with 12 nM of ASO TD.63, EW.09, CA.63.10, (scrambled CA.63.10) scrCA.63.10, 23.1 (previously described ASO), 23.2 (previously described ASO), 23.3 (previously described ASO), or 23.4 (previously described ASO).
  • SEQ ID NO: 1 CA.10 ASO/ 23.63.10 ASO
  • SEQ ID NO: 6 CA.1 ASO
  • SEQ ID NO: 7 (EW.8 ASO) CAGCGATACTGGTTGCGAAGA
  • SEQ ID NO: 11 (CA.7 ASO) GCGATACTGGTT
  • SEQ ID NO: 12 (CA.8 ASO)
  • SEQ ID NO: 25 (CA.10 target / 23.63.10 target)
  • SEQ ID NO: 27 (EW.9 target) GCAACCAGTATCGCTGCA
  • SEQ ID NO: 28 (CA.4 target)
  • SEQ ID NO: 30 (CA.1 target) GCAACCAGTATCGCTG
  • SEQ ID NO: 31 (EW.8 target) TCTTCGCAACCAGTATCGCTG
  • SEQ ID NO: 34 (CA.6 target) AACCAGTATCGCTGCA
  • SEQ ID NO: 37 (C A.9 target) AACCAGTATCGCTG
  • SEQ ID NO: 40 (EW.5 target) ggtagAGAACACCTGTCTTCG
  • SEQ ID NO: 50 Fral length amplicon
  • SEQ ID NO: 63 (EW.15 ASO) CCACCAAATGCTGTTGATAC
  • SEQ ID NO: 64 (EW.16 ASO) ACACCACCAAATGCTGTT
  • SEQ ID NO: 65 (EW.17 ASO) CAAAGTCACACCACCAAATG
  • SEQ ID NO: 70 (EW.29 ASO) GTCCAATCCAGAAGACTCA
  • SEQ ID NO: 92 (SORL1 exon 23 sequence with flanking regions) tcccctgccgcactctgatgggtagagaacacctgtcttcgcaaccagtatcgctgcagcaacgggaactgtatcaacag catttggtggtgtgactttgacaacgactgtggagacatgagcgatgagagaaactgccgtgagtcttctggattggacgtt aaaa
  • SEQ ID NO: 110 SORL1 exon 23 sequence with flanking regions part 2
  • SEQ ID NO: 111 (SORL1 exon 23 sequence with flanking regions part 3) ttgacaacgactgtggagacatgagcgatgagagaaactgccgtgagtcttctggattggacgttaa
  • An antisense oligonucleotide comprising or consisting of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity.
  • An antisense oligonucleotide (ASO) binding or capable of binding to a target site comprising or consisting of a target polynucleotide sequence having at least 80% sequence identity to a target polynucleotide sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity
  • binding to a target site causes exon skipping of exon 23 encoding a complement-type repeat (OR) domain of SORLA
  • exon 23 comprises or consists of a polynucleotide sequence of SEQ ID NO: 49, or a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 49, such as at least 85%, 90%, 95%, 98% or 99% sequence identity.
  • exon 23 comprises or consists of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107 such as at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity.
  • exon 23 comprises a mutation selected from the list consisting of: p.C1078R (T>C), p.R1080C (C>T), P.R1084C (C>T), p.W1095C (G>0), p.W1096C (G>0), p.D1102N (G>A), P.C1103Y (G>A), p.D1105H (G>0), p.D1108N (G>A), and p.C1112Y (G>A).
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide further comprises one or more modifications at at least one nucleotide position, or at each nucleotide position.
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide comprises one or more modifications selected from the group consisting of: Peptide nucleic acid (PNA), Serinol nucleic acid (SNA), Phosphorodiamidate morpholino oligomer (PMO), Thiomorpholino oligonucleotide (TMO), and Morpholino nucleic acid (MNA).
  • PNA Peptide nucleic acid
  • SNA Serinol nucleic acid
  • PMO Phosphorodiamidate morpholino oligomer
  • TMO Thiomorpholino oligonucleotide
  • MNA Morpholino nucleic acid
  • antisense oligonucleotide comprises one or more modifications of the nucleic acid backbone selected from the group consisting of: Phosphorothioate (PS) modifications, Mesyl phosphoramidate (MsPA) modifications, p-toluenesulfonyl phosphoramidate (Ts) modifications, and 4- (trimythylammonio)butylsulfonyl phosphoramidate (N+) modifications.
  • PS Phosphorothioate
  • MsPA Mesyl phosphoramidate
  • Ts p-toluenesulfonyl phosphoramidate
  • N+ 4- (trimythylammonio)butylsulfonyl phosphoramidate
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide comprises one or more modifications of the ribose sugar selected from the group consisting of: 2’-O-methyl (2’-0me), 2’-O-methoxyethyl (2’MOE), 2’-fluoro (2’-F), Locked nucleic acid (LNA), 2’- 0,4’C-ethylene-bridged nucleic acid (ENA), 2’,4’-constrained 2’-0-ethyl (cEt), Amido-bridged nucleic acid (AmNA), Guanidine-bridged nucleic acid (GuNA), Cyclohexenyl nucleic acid (CeNA), Anhydrohexitol nucleic acid (HNA), Altritol nucleic acid (ANA), Tricyclo-DNA (tc-DNA), and 7’, 5’-alpha-bicyclo-DNA (7’5’-
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide comprises one or more 2’,4’-constrained 2'-0-Ethyl (cEt) modifications.
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide comprises one or more 2'-O-Methylation (2’-OMe) modifications.
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide comprises one or more phosphorothioate modifications.
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide comprises a 2'-O-methoxyethyl (2’MOE) sugar modification.
  • the antisense oligonucleotide is between 12 to 25 nucleotides in length, such as between 12 and 23 nucleotides, between 12 and 21 nucleotides, between 12 and 20 nucleotides, between 12 and 19 nucleotides, between 12 and 18 nucleotides, between 12 and 16 nucleotides, between 12 and 14 nucleotides, between 14 and 25 nucleotides, between 14 and 23 nucleotides, between 14 and 21 nucleotides, between 14 and 20 nucleotides, between 14 and 19 nucleotides, between 14 and 18 nucleotides, between 14 and 16 nucleotides, between 16 and 25 nucleotides, between 16 and 23 nucleotides, between 16 and 21 nucleotides, between 16 and 20 nucleotides, between 16 and 19 nucleotides, between 16 and 18 nucleotides, between 18 and
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide is conjugated to Triantennary N- acetylgalactosamine (GalNAc) moiety, TAT (SEQ ID NO: 108) and/or a peptide.
  • GalNAc Triantennary N- acetylgalactosamine
  • antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide is targeted to a 5’ splice site, a 3’ splice site and/or an exonic splice enhancer site (ESE).
  • ESE exonic splice enhancer site
  • composition comprising the antisense oligonucleotide according to any one of the preceding items, such as a pharmaceutical composition.
  • composition according to item 26 comprising one or more of said antisense oligonucleotides.
  • An antisense oligonucleotide (ASO) according to any one of items 1 to 25 and/or the composition according to any one of items 26 to 27, for use as a medicament.
  • An antisense oligonucleotide (ASO) according to any one of items 1 to 25 and/or the composition according to any one of items 26 to 27, for use in the prevention, treatment and/or alleviation of Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies, or a disease or disorder associated with Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies.
  • ASO antisense oligonucleotide
  • a method for treatment of Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies comprising administration of a therapeutically effective amount of an antisense oligonucleotide according to any one of items 1 to 25 to an subject in need thereof.
  • a method for mediating exon skipping in SORL1 transcripts in a cell, tissue or organ wherein the method comprises the step of contacting said cell, tissue or organ with the antisense oligonucleotide according to any one of items 1 to 25 and/or the composition according to any one of items 26 to 27, wherein the exon is exon 23 of SORL1.
  • ASOs such as 2 ASOs, such as 3 ASOs, such as 4 ASOs, such as 5 ASOs, such as 6 ASOs, such as 7 ASOs, such as 8 ASOs, such as 9 ASOs, such as 10 ASOs, such as 11 ASOs, such as 12 ASOs, such as 13 ASOs, such as 14 ASOs, such as 15 ASOs, such as 16 ASOs, such as 17 ASOs, such as 18 ASOs, such as 19 ASOs, such as 20 ASOs, such as 21 ASOs, such as 22 ASOs, such as 23 ASOs, or 24 ASOs.
  • exon skipping of one exon is mediated.
  • 20 nM or less such as 19 nM or less, such as 18 nM or less, such as 17 nM or less, such as 16 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less, such as 9 nM or less, such as
  • the amount of SORL1 transcripts in said cell, tissue or organ is at least 80% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition, such as at least 81%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition.
  • the amount of SORLA protein in said cell, tissue or organ is at least 80% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition, such as at least 81%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition.
  • a method of determining the efficiency of ASO mediated SORL1 exon skipping in a subject comprising the following steps: a) analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in a first sample comprising cerebrospinal fluid derived from a patient, obtained before treatment with an ASO, b) analyzing the levels of SORLA lacking the complement-type repeat encoded by exon 23 in a second sample comprising cerebrospinal fluid derived from the same patient as in a), obtained after treatment with an ASO, c) comparing the level of SORLA lacking the complement-type repeat encoded by exon 23 in the samples of a) and b), and d) determining exon skipping if the level of SORLA lacking the complement-type repeat encoded by exon 23 in b) is higher than in a).
  • a method for testing if a patient identified with a SORL1 mutation will benefit from treatment with an ASO mediating exon skipping comprising the steps of: a. determining if the mutation is in exon 23 of SORL1 , b. if yes, obtaining a cell comprising the SORL1 mutation identified in the patient, c. selecting an ASO that targets the exon 23, d. contacting a first aliquot of cells with medium comprising the selected ASO, and contacting a second aliquot of cells with medium not comprising an ASO, e. analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in the first and the second aliquot, f.
  • a method of determining the efficiency of ASO mediated SORL1 exon skipping comprising the following steps: a) analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in a first sample comprising cerebrospinal fluid, obtained before contacting with an ASO, b) analyzing the levels of SORLA lacking the complement-type repeat encoded by exon 23 in a second sample comprising cerebrospinal fluid as in a), obtained after contacting with an ASO, c) comparing the level of SORLA lacking the complement-type repeat encoded by exon 23 in the samples of a) and b), and d) determining exon skipping if the level of SORLA lacking the complement-type repeat encoded by exon 23 in b) is higher than in a).
  • a method for testing if a patient identified with a SORL1 mutation will benefit from treatment with an ASO mediating exon skipping comprising the steps of: a. determining if the mutation is in exon 23 of SORL1 , b. if yes, obtaining a cell comprising the SORL1 mutation identified in the patient, c. selecting an ASO that targets the exon 23, d. contacting a first aliquot of cells with medium comprising the selected ASO, and contacting a second aliquot of cells with medium not comprising an ASO, e. analyzing the levels of endoplasmic reticulum-resident SORLA in the first and the second aliquot, f.
  • the method according to item 51 wherein the mutation is a calcium-cage- mutation, an asx-turn mutation or an odd-numbered cysteines-mutation.
  • the mutation is a mutation in exon 23.
  • An antisense oligonucleotide comprising or consisting of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
  • ASO antisense oligonucleotide
  • An antisense oligonucleotide (ASO) binding or capable of binding to a target site comprising or consisting of a target polynucleotide sequence having at least 80% sequence identity to a target polynucleotide sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48.
  • ASO antisense oligonucleotide
  • binding to a target site causes exon skipping of exon 23 encoding a complement-type repeat (OR) domain of SORLA, wherein exon 23 comprises or consists of a polynucleotide sequence of SEQ ID NO: 49, or a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 49.
  • OR complement-type repeat
  • exon 23 comprises or consists of a polynucleotide sequence having at least 80% sequence identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107. 5.
  • antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide further comprises one or more modifications at at least one nucleotide position, or at each nucleotide position, wherein the modifications are modifications of the nucleic acid backbone, the nucleobase, the ribose sugar and/or 2'-ribose substitutions.
  • antisense oligonucleotide comprises one or more modifications of the nucleic acid backbone selected from the group consisting of: Phosphorothioate (PS) modifications, Mesyl phosphoramidate (MsPA) modifications, p-toluenesulfonyl phosphoramidate (Ts) modifications, and 4- (trimythylammonio)butylsulfonyl phosphoramidate (N+) modifications.
  • PS Phosphorothioate
  • MsPA Mesyl phosphoramidate
  • Ts p-toluenesulfonyl phosphoramidate
  • N+ 4- (trimythylammonio)butylsulfonyl phosphoramidate
  • antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide comprises one or more modifications of the ribose sugar selected from the group consisting of: 2’-O-methyl (2’-0me), 2’-O-methoxyethyl (2’MOE), 2’-fluoro (2’-F), Locked nucleic acid (LNA), 2’- 0,4’C-ethylene-bridged nucleic acid (ENA), 2’,4’-constrained 2’-0-ethyl (cEt), Amido-bridged nucleic acid (AmNA), Guanidine-bridged nucleic acid (GuNA), Cyclohexenyl nucleic acid (CeNA), Anhydrohexitol nucleic acid (HNA), Altritol nucleic acid (ANA), Tricyclo-DNA (tc-DNA), and 7’, 5’-alpha-bicyclo-DNA (7’5’- a-
  • antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide is conjugated to a moiety or to a nanoparticle formulation, such as wherein the moiety is a cell-targeting moiety and/or a cell-penetrating moiety and/or wherein the antisense oligonucleotide is conjugated to Triantennary N-acetylgalactosamine (GalNAc) moiety, TAT (SEQ ID NO: 108) and/or a peptide.
  • GalNAc Triantennary N-acetylgalactosamine
  • TAT SEQ ID NO: 108
  • a peptide an antisense oligonucleotide (ASO) according to any one of claims 1 to 9, for use as a medicament.
  • An antisense oligonucleotide (ASO) according to any one of claims 1 to 9, for use in the prevention, treatment and/or alleviation of Alzheimer’s Disease, Parkinson’s Disease, or Dementia with Lewy bodies.
  • An in vitro method for mediating exon skipping in SORL1 transcripts in a cell, tissue or organ wherein the method comprises the step of contacting said cell, tissue or organ with the antisense oligonucleotide according to any one of claims 1 to 9, wherein the exon is exon 23 of SORL1 , such as wherein one ASO is used or wherein more than one ASO is used.
  • the amount of SORL1 transcripts in said cell, tissue or organ is at least 80% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition, such as at least 100% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition and/or wherein the amount of SORLA protein in said cell, tissue or organ is at least 80% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition, such as at least 100% compared to said cell, tissue or organ without said contacting with the antisense oligonucleotide and/or the composition.
  • An in vitro method of determining the efficiency of ASO mediated SORL1 exon skipping comprising the following steps: a) analyzing the levels of SORLA lacking a complement-type repeat encoded by exon 23 in a first sample comprising cerebrospinal fluid, obtained before contacting with an ASO, b) analyzing the levels of SORLA lacking the complement-type repeat encoded by exon 23 in a second sample comprising cerebrospinal fluid as in a), obtained after contacting with an ASO, c) comparing the level of SORLA lacking the complement-type repeat encoded by exon 23 in the samples of a) and b), and d) determining exon skipping if the level of SORLA lacking the complement-type repeat encoded by exon 23 in b) is higher than in a).
  • a method of producing an ASO suitable for treatment of a patient with Alzheimer’s Disease, wherein the patient carries a mutation in an exon encoding a complement-type repeat (CR) domain of SORLA comprising the following steps: a. identifying an ASO according to any one of claims 1 to 9, b. determining if the target site of the ASO comprises the mutation, or if the target site of the ASO does not comprise the mutation, and c. determining that the ASO that binds to a target site not comprising the mutation is suitable for treatment of a patient with Alzheimer’s Disease.

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Abstract

La présente invention concerne des oligonucléotides antisens (ASO) se liant à un site cible sur l'exon 23 du pré-ARNm de SORL1. La présente invention concerne en outre une composition comprenant ledit ASO. La présente invention concerne en outre un ASO destiné à être utilisé en tant que médicament. La présente invention concerne en outre un ASO destiné à être utilisé dans la prévention, le traitement et/ou le soulagement de la maladie d'Alzheimer (AD), par exemple. La présente invention concerne en outre un procédé de médiation du saut d'exon de l'exon 23 dans des transcrits SORL1, un procédé de détermination de l'efficacité du saut d'exon 23 de SORL1 à médiation par ASO, un procédé destiné à vérifier si un patient identifié avec une mutation SORL1 bénéficiera d'un traitement avec un saut d'exon 23 à médiation par ASO, et un procédé de production d'un ASO approprié pour le traitement d'un patient atteint de la maladie d'Alzheimer.
PCT/EP2025/066815 2024-06-17 2025-06-17 Oligonucléotides antisens pour le traitement de la maladie d'alzheimer Pending WO2025262002A2 (fr)

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US6280934B1 (en) * 1998-05-13 2001-08-28 Millennium Pharmaceuticals, Inc. Assay for agents which alter G-protein coupled receptor activity
US20090123928A1 (en) * 2007-10-11 2009-05-14 The Johns Hopkins University Genomic Landscapes of Human Breast and Colorectal Cancers
EP2655621B1 (fr) * 2010-12-20 2018-05-23 The General Hospital Corporation Arn non codants associés à polycomb
US20170009304A1 (en) * 2015-07-07 2017-01-12 Splicingcodes.Com Method and kit for detecting fusion transcripts
WO2018224162A1 (fr) * 2017-06-09 2018-12-13 Biontech Rna Pharmaceuticals Gmbh Procédés de caractérisation de perte de présentation de l'antigène
EP4363578A1 (fr) * 2021-07-02 2024-05-08 Aarhus Universitet Méthodes de traitement de la maladie d'alzheimer

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"UniProt", Database accession no. Q92673

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