WO2024256620A1 - Oligonucléotides antisens pour le traitement des maladies neurodégénératives - Google Patents

Oligonucléotides antisens pour le traitement des maladies neurodégénératives Download PDF

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WO2024256620A1
WO2024256620A1 PCT/EP2024/066520 EP2024066520W WO2024256620A1 WO 2024256620 A1 WO2024256620 A1 WO 2024256620A1 EP 2024066520 W EP2024066520 W EP 2024066520W WO 2024256620 A1 WO2024256620 A1 WO 2024256620A1
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target
nucleotide
guide oligonucleotide
nucleic acid
guide
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Bart KLEIN
Gerardus Johannes Platenburg
Christopher Kuyler Doyle
Maarten HOLKERS
Laura CALO
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ProQR Therapeutics II BV
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ProQR Therapeutics II BV
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification

Definitions

  • This disclosure relates to the field of medicine, and in particular to the field of neurodegenerative diseases such as Alzheimer’s disease.
  • the disclosure describes guide oligonucleotides that mediate nucleotide-specific editing in the RELN gene and/or encoded transcript to bring about changes in the encoded reelin protein that influence reelin protein activity.
  • AD Alzheimer’s disease
  • amyloid plaques formed by aggregating Amyloid Precursor Protein (APP) and so-called neurofibrillary tangles made up of extensively phosphorylated Tau protein deposited in a patient’s brain are hallmarks of AD pathology.
  • Tau protein is an important component of the cytoskeleton. Its normal function is that it binds to tubulin, stabilizing microtubule structures used by motor proteins in the cell to organize cellular transport. Hyperphosphorylated Tau cannot perform this function very well and Tau dysregulation is associated with disturbed neuronal migration during development and neuronal degeneration, giving rise to neurodegenerative diseases such as Parkinsonism and Frontotemporal dementia and animal models generated to express mutated Tau variants give rise to neurodegeneration.
  • reelin is involved in the phosphorylation of Tau through the Apolipoprotein E receptor (APOEr)/disabled-1 (Dab1)/glycogen synthase kinase-3p (GSK3P) cascade.
  • APOEr Apolipoprotein E receptor
  • Dab1 disabled-1
  • GSK3P glycogen synthase kinase-3p
  • Dab1 By binding of reelin protein to the ApoE receptor and other cadherin-related neuronal receptors (CNRs) that co-operate with src family kinases as intracellular effector proteins, Dab1 is phosphorylated, activating Dab1 to inhibit two downstream kinases known to phosphorylate Tau in positions identified in neurofibrillary tangles: GSK3P and CDK5.
  • CNRs cadherin-related neuronal receptors
  • the present disclosure aims to provide guide oligonucleotides that can be used in the treatment of neurodegenerative diseases such as AD, wherein the guide oligonucleotides cause nucleic acid editing to generate an edited RELN nucleic acid sequence, by exploiting nucleic acid editing machinery to target and amend one or more target nucleotides in the RELN gene or the encoded RELN transcript molecules, preferably pre-mRNA and/or mRNA.
  • the present disclosure relates to a guide oligonucleotide that is at least partially complementary to a portion of a human RELN nucleic acid molecule comprising a target nucleotide, wherein the RELN nucleic acid molecule encodes a reelin protein, wherein the guide oligonucleotide is configured such that it is capable of forming a double stranded complex under physiological conditions within a cell, preferably a brain cell, more preferably a neuron, with the portion of the RELN nucleic acid, and the double stranded complex is capable of recruiting a nucleic acid editing enzyme that is naturally present in the cell, to perform editing of the target nucleotide to generate an edited RELN nucleic acid comprising an edited target nucleotide.
  • the encoded reelin protein is provided with a gain-of-function phenotype, selected from one or more of: i) an enhanced ability to trigger signalling, preferably of the APOEr/Dab1/GSK3p pathway; ii) an enhanced ability to increase Dab1 phosphorylation; iii) an enhanced ability to reduce Tau phosphorylation associated with neurofibrillary tangles; iv) an enhanced ability to increase tubular structure formation and/or stability and/or neuronal density; v) an enhanced resistance to degradation by proteolysis; and/or vi) enhanced binding of the reelin protein to glycosaminoglycans, preferably heparin, and/or to NRP1.
  • a gain-of-function phenotype selected from one or more of: i) an enhanced ability to trigger signalling, preferably of the APOEr/Dab1/GSK3p pathway; ii) an enhanced ability to increase Dab1 phosphorylation; iii) an enhanced ability to reduce Tau
  • editing of the target nucleotide introduces an amino acid variant at one or more of amino acid positions 3446 to 3460 of the encoded reelin protein, preferably wherein editing of the target nucleotide introduces a histidine to arginine change at amino acid position 3447 (H3447R) in the encoded reelin protein.
  • amino acid position 3447 relates to RELN isoform 203 (Ensemble transcript ENST00000428762.6; RELN 203), which is a transcript that is 6 nucleotides longer than the transcript of isoform 201.
  • isoform 201 the codon for histidine encodes amino acid 3445.
  • H3447R is used throughout the present disclosure, but the guide oligonucleotides disclosed herein can bring about the deamination of the target adenosine in both isoforms and provide the amino acid changes H3447R in 203 and H3445R in 201.
  • isoform 201 is the most abundant isoform of RELN present in the human iPSC forebrain neurons used in the accompanying examples
  • isoform 203 is the second most abundant isoform present in these cells.
  • the target nucleotide is adenosine
  • the nucleic acid editing enzyme is an Adenosine Deaminase Acting on RNA (ADAR) enzyme.
  • ADAR Adenosine Deaminase Acting on RNA
  • the RELN nucleic acid molecule is mRNA or pre-mRNA.
  • the guide oligonucleotide comprises a contiguous stretch of 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 nucleotides from SEQ ID NO:102 (5’-UG UA GAA ACI UCU GAG CCC AUG UUG UGG UGA AA-3’) or SEQ ID NO: 103 (5’-UG UA GAA AZI UCU GAG CCC AUG UUG UCGUGAAA-3’), and comprises at least the underlined section of nucleotides (represented by SEQ ID NO: 104 (5’-UA GAA ACI UCU GAG CCC AUG UUG-3’) and SEQ ID NQ:105 (5’-UA GAA AZI UCU GAG CCC AUG UUG-3’), respectively), wherein Z is a cytidine analog that is a nucleotide, preferably a deoxynucleotide, comprising a 6-amino-5-nitro-3-yl-2(1 H)-pyridone
  • the guide oligonucleotide as disclosed herein comprises the structure (from 5’ to 3’): N8N7N6N5N4N3N2Nl9Zd ld A M2M3M4M5M6M7M 8 M9Ml0Ml l Ml2Ml3Ml4Ml5Ml6Ml7Ml8Ml9M20M2l M22M23M24 wherein: Zd is the orphan nucleotide at nucleotide position 0, which is a deoxynucleotide carrying a Benner’s base; Ni is Ae or Ad; N2 is Af; N3 and Ns are each independently Am or Af; N4 is Gf; Ns is Uf; N7 is either absent (then Ns is also absent), Gm, or Gf; Ns is either absent or Um; Id is deoxyinosine; M2 is Um; M3 is Cf; M4, M14 and M15 are each independently m5
  • the present disclosure also relates to a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding a guide oligonucleotide as disclosed herein.
  • AAV adeno-associated virus
  • the present disclosure also relates to a guide oligonucleotide as disclosed herein for use in the treatment, amelioration, or slowing down the progression of a neurodegenerative disease, preferably AD, more preferably ADAD.
  • the present disclosure also relates to a method of treating, ameliorating, or slowing down the progression of a neurodegenerative disease, preferably AD, more preferably ADAD, in a human subject in need thereof, the method comprising administering to said subject a guide oligonucleotide as disclosed herein, thereby editing the target RELN nucleic acid sequence to encode a reelin protein with the ability to delay onset of one or more symptoms of the neurodegenerative disease.
  • a neurodegenerative disease preferably AD, more preferably ADAD
  • Fig. 1A shows a nucleotide sequence of a portion of a wild-type human RELN nucleic acid sequence (NCBI Reference Sequence: NM_005045.4), in the 5’ to 3’ direction, showing a target adenosine (in bold font) in the codon CAT (underlined) encoding histidine at position 3447 in human reelin (H3447) and the SEQ ID NO;
  • Fig. 1 B shows the complementary sequence of the nucleic acid sequence in Fig. 1A in the 3’ to 5’ direction, showing the position of the orphan nucleotide (the nucleotide opposite the target adenosine) in bold font, and the SEQ ID NO; Fig.
  • FIG. 1C shows the antisense sequence of the nucleic acid sequence in Fig. 1A in the 5’ to 3’ direction, showing the position of the orphan nucleotide (the nucleotide opposite the target adenosine) in bold font. It is to be understood that when the target RELN nucleic acid is a pre-mRNA or an mRNA molecule, the thymidine residues (T) should be read as uridine residues (U).
  • Fig. 1D shows the 5’ to 3’ transcript sequence of Fig. 1A and represents the same portion (SEQ ID NO: 106) of the target sequence for RNA editing and displays the adenosine in bold (middle of the underlined codon).
  • the orphan nucleotide in guide oligonucleotides is not a thymidine (T) as Fig. 1 B and Fig. 1C may suggest, but preferably a cytidine (C), a cytidine analog, a uridine (II) or a uridine analog.
  • Fig. 2 shows the sequences of example guide oligonucleotides disclosed herein.
  • the chemical modifications in the guide oligonucleotides are as follows: Gm, Am, Um, and Cm are 2’-0Me modified guanosine, adenosine, uridine, and cytidine, respectively; m5Ce is 2’-MOE modified 5-methylcytidine; Ge is 2’-MOE modified guanosine; Ae is 2’-MOE modified adenosine; m5Ue is 2’-MOE modified 5-methyluridine (also sometimes named “Te”; 2’-MOE modified thymidine); Af, Uf, Gf, and Cf are 2’-F modified adenosine, uridine, guanosine, and cytosine, respectively; Zd is the cytidine analog that is also referred to as a nucleoside carrying a 6-amino-5-nitro-3-yl-2
  • Fig. 3 shows the sequences of a set of additional guide oligonucleotides, with their respective RM names and SEQ ID NO’s.
  • the chemical modifications are as provided in Fig. 2.
  • Fig. 4 shows the editing percentage of the target adenosine shown in Fig. 1 D, in RELN target (pre-) mRNA obtained after transfection of the guide oligonucleotides RM116817 to RM 116840, as shown below the graph, in human iPSC (WT04) derived neural progenitor cells, at day 2 after transfection. A negative (non-treated) control was taken along (Mock).
  • Fig. 5 shows the editing percentage of the target adenosine shown in Fig. 1 D, in RELN target (pre-) mRNA obtained after gymnotic uptake of the guide oligonucleotides, as shown below the graph, in human iPSC (WT04) derived neural progenitor cells at day 7 after start of the gymnotic treatment with the respective guide oligonucleotides.
  • WT04 human iPSC
  • Fig. 6 shows the editing percentage of the target adenosine shown in Fig. 1 D, in RELN target (pre-) mRNA obtained after gymnotic uptake of the guide oligonucleotides, as shown below the graph, and co-treatment with the triterpene glycoside AG1856 (saponin), in human iPSC (WT04) derived neural progenitor cells, at day 7 after start of the gymnotic/saponin treatment. A negative (non-treated; NT) control was taken along.
  • Fig. 7 shows the sequences of a further set of guide oligonucleotides, with their respective RM numbers and SEQ ID NO’s.
  • RM 118550 to RM 118867 are designed based on the sequence and modifications of oligonucleotide RM116835 (SEQ ID NO:58, which is also referred to as G3447-19), whereas RM 118868 to RM 118880 are designed based on the sequence and modifications of oligonucleotide RM116838 (SEQ ID NO:61 , which is also referred to as G3447-22).
  • the chemical modifications are as provided in Fig. 2, wherein # refers to a PNms linkage.
  • Fig. 2 wherein # refers to a PNms linkage.
  • FIG. 8 shows the editing percentage of the target adenosine shown in Fig. 1 D, in RELN target (pre-) mRNA obtained after gymnotic uptake of the guide oligonucleotides of Fig. 7 and co-treatment with the triterpene glycoside AG1856 (saponin) in human iPSC (WT04) derived neural progenitor cells, at day 7 after start of the gymnotic/saponin treatment. A negative sap(onin) only control was taken along.
  • Fig. 8A shows the results using a forward primer specific for the transcript sequence of isoform 201 and
  • Fig. 8B shows the results using a forward primer specific for the transcript of isoform 203.
  • guide oligonucleotides that can drive editing of a target RELN nucleic acid sequence.
  • Such guide oligonucleotides can find use as therapeutic agents to treat, ameliorate or slow down the progression of a neurodegenerative disease such as AD. It has been identified, for instance, that a change of only a single amino acid in the reelin protein can be sufficient to initiate or enhance a protective pathway that slows down the progression of AD.
  • this technique operates at the genetic level.
  • the present disclosure therefore opens a whole new field of using specific genetic editing techniques for the treatment of neurodegenerative disease.
  • the genetic editing technique is not particularly limited.
  • Suitable techniques include known gene therapy techniques that utilize a guide oligonucleotide, which include DNA editing techniques such as Cas9-based techniques, as well as RNA editing techniques such as ADAR-mediated editing techniques.
  • DNA editing and RNA editing technologies have advantages and disadvantages.
  • DNA editing gene therapy can produce a permanent change in the DNA molecule and may therefore only require a single treatment for a particular disorder. In certain circumstances it may not be required or desired to have an irreversible change of the DNA.
  • RNA editing has the advantage of being transient: only the RNA is edited and over time amended proteins are being produced, but when the guide oligonucleotide has been broken down by metabolic processes and new mRNA is generated, the ‘old’ version of the protein is again being produced.
  • editing of the target nucleotide leads to elevated activity of the encoded reelin protein, preferably selected from one or more of: an enhanced ability to trigger signalling, preferably of the APOEr/Dab1/GSK3p pathway; an enhanced ability to increase Dab1 phosphorylation; an enhanced ability to reduce Tau phosphorylation associated with neurofibrillary tangles; an enhanced ability to increase tubular structure formation and/or stability and/or neuronal density; an enhanced resistance to degradation by proteolysis; and/or enhanced binding of the reelin protein to glycosaminoglycans, preferably heparin, and/or to NRP1.
  • editing of the target nucleotide introduces an amino acid variant at one or more of amino acid positions 3446 to 3460 of the encoded reelin protein, preferably wherein editing of the target nucleotide introduces a histidine to arginine change at amino acid position 3447 (H3447R) in the encoded reelin protein.
  • the cell is a brain cell, preferably a neuron.
  • the target nucleotide is adenosine
  • the nucleic acid editing enzyme is an ADAR enzyme
  • the RELN nucleic acid molecule is mRNA or pre-mRNA.
  • the orphan nucleotide is the nucleotide in the guide oligonucleotide that is opposite the target nucleotide, wherein the nucleotide numbering is such that the orphan nucleotide is number 0 and nucleotides are further positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end, and wherein at least one nucleobase, sugar, or internucleoside linkage, has been chemically modified.
  • the orphan nucleotide is a deoxycytidine, a cytidine analog, a deoxyuridine, or a uridine analog.
  • the cytidine analog is preferably a deoxynucleotide comprising a 6-amino-5-nitro-3-yl-2(1 H)-pyridone nucleobase (also referred to herein, and elsewhere as “Benner’s base”, or Z).
  • the uridine analog is preferably a deoxynucleotide comprising an iso-uracil nucleobase.
  • the guide oligonucleotide is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.
  • the guide oligonucleotide comprises a contiguous stretch of 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 nucleotides from SEQ ID NO: 102 (5’-UG UA GAA ACI UCU GAG CCC AUG UUG UCG UGA AA-3’) or SEQ ID NO: 103 (5’-UG UA GAA AZI UCU GAG CCC AUG UUG UCGUGAAA-3’), comprising at least the underlined section of nucleotides (SEQ ID NQ:104 (5’-UA GAA ACI UCU GAG CCC AUG UUG-3’ and SEQ ID NO: 105 (5’-UA GAA AZI UCU GAG CCC AUG UUG-3’), respectively), wherein Z is a nucleotide, preferably a deoxynucleotide, comprising a Benner’s base, and I is inosine, preferably deoxyinosine.
  • the internucleoside linkage numbering in the guide oligonucleotide is such that linkage number 0 is the linkage 5’ from the orphan nucleotide, and the linkage positions in the oligonucleotide are positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end, and wherein linkage position -2 is an MP or a PNms linkage.
  • the guide oligonucleotide comprises one or more nucleotides comprising a mono- or di-substitution at the 2', 3' and/or 5' position of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; - dimethylamino oxyethoxy; and -di
  • the portion of the target RELN nucleic acid sequence comprises a contiguous stretch of 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 nucleotides from a nucleotide sequence selected from SEQ ID NO:1 and 2:
  • editing of the target nucleotide leads to elevated levels of expression, elevated activity, and/or elevated stability, of the reelin protein.
  • the nucleic acid editing entity is a nucleic acid editing enzyme, preferably a deaminase enzyme, more preferably an adenosine deaminase enzyme, such as human ADAR1 (hADARI) and human ADAR2 (hADAR2), or a cytidine deaminase enzyme.
  • a deaminase enzyme preferably an adenosine deaminase enzyme, such as human ADAR1 (hADARI) and human ADAR2 (hADAR2), or a cytidine deaminase enzyme.
  • the nucleic acid editing entity is naturally expressed in the cell (/.e., endogenous to the cell).
  • the target RELN nucleic acid sequence is naturally expressed within the cell.
  • the target RELN nucleic acid sequence is DNA.
  • the nucleic acid editing entity is selected from the list comprising: a Cas9 enzyme; a base editor enzyme; a dCas9-deaminase enzyme; a dCas9- adenosine deaminase enzyme; a dCas9-cytidine deaminase enzyme; a prime editing enzyme; or a Cas9 Nickase enzyme.
  • the linkage between the most terminal two nucleotides on the 5’ and/or 3’ terminus of the guide oligonucleotide is a PNdmi linkage, or a PNms linkage, preferably wherein both most terminal linkages are PNms linkages.
  • the first nucleotide 3’ from the orphan nucleotide (-1) is a deoxyinosine.
  • the guide oligonucleotide comprises a contiguous stretch of 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 nucleotides from a nucleotide sequence selected from: 5 ' -.. ⁇ TCTTCTGTTGTAGAAACGTCTGAGCCCATGTTG...-3 ' (SEQ ID N0:3),
  • the guide oligonucleotide comprises at least the underlined section of SEQ ID NO:6, wherein the orphan nucleotide (in the underlined section the middle C in bold) is a deoxynucleotide carrying a cytidine analog, preferably a Benner’s base (Zd, instead of a C; see Inti. Patent Application Publication No. WO2022/252376), and wherein nucleotide position -1 is deoxyinosine (Id).
  • the guide oligonucleotide comprises at least the underlined section of SEQ ID NO:6, wherein the orphan nucleotide (in the underlined section the middle C in bold) is a deoxyuridine, and wherein nucleotide position -1 is deoxyinosine (Id).
  • the guide oligonucleotide comprises at least the underlined section of SEQ ID NO:6, wherein the orphan nucleotide (in the underlined section the middle C in bold) is a deoxynucleotide carrying a uridine analog, preferably an iso-uracil, and wherein nucleotide position -1 is deoxyinosine (Id).
  • the guide oligonucleotide comprises at least the underlined section of SEQ ID NO:6, wherein the orphan nucleotide (in the underlined section the middle C in bold) is a deoxynucleotide carrying a Benner’s base (Zd, instead of a C), wherein nucleotide position -1 is Id, and wherein nucleotide position +1 is deoxyadenosine (Ad) or an adenosine in which the 2’ position of the ribose is substituted with 2’-O-methoxyethyl (also referred to as 2’-methoxyethoxy, 2’-O-MOE, or simply 2’-MOE) (Ae), preferably wherein the linkage position -2 is an MP or a PNms linkage, more preferably a PNms linkage.
  • the guide oligonucleotide comprises at least the underlined section of SEQ ID NO:6, wherein the orphan nucleotide (in the underlined section the middle C in bold) is a deoxynucleotide carrying a Benner’s base (Zd, instead of a C), wherein nucleotide position -1 is Id, wherein the length of the 5’ part immediately adjacent to the orphan nucleotide is 6, 7, or 8 nucleotides, and wherein the length of the 3’ part immediately adjacent to the orphan nucleotide is at least 16 nucleotides, more preferably 16, 17, 18, 19, 20, 21 , 22, 23, or 24 nucleotides.
  • the guide oligonucleotide comprises the structure (from 5’ to 3’):
  • N8N7N6N5N4N3N2Nl9Zdld A M2M3M4M5M6M7M 8 M9Ml0MllMl2Ml3Ml4Ml5Ml6Ml7Ml8Ml9M20M2lM22M23M24 wherein:
  • Zd is the orphan nucleotide at nucleotide position 0, which is a deoxynucleotide carrying a Benner’s base;
  • Ni is Ae or Ad
  • N3 and Ns are each independently Am or Af;
  • N? is either absent (if so, then Ns is also absent), Gm, or Gf;
  • Ns is either absent or Um
  • Id is deoxyinosine
  • M3 is Cf
  • M 4 , M14 and M15 are each independently m5Ue or Um;
  • Ms and M7 are Gf;
  • Ms is Am or Af
  • Ms and M10 are each independently Cm or Cf;
  • M9 is Cf
  • M13 is Gm
  • M17 is either absent (if so, then Mis to M 24 are also absent), m5Ue, or Um;
  • M19 is either absent (if so, then M20 to M 24 are also absent), Gm, or Ge;
  • M20 is either absent (if so, then M21 to M 24 are also absent), Um, or m5Ue;
  • M21 is either absent (if so, then M22 to M 2 4are also absent), Gm, or Ge;
  • M22 is either absent (if so, then M23 and M 24 are also absent), Am, or Ae;
  • M23 is either absent (if so, then M 24 is also absent), or Ae;
  • M24 is either absent, or Ae
  • 0 is at linkage position 0, and is a PO linkage or a PNms linkage
  • A is at linkage position -2 and is an MP or a PNms linkage; all other linkages are either PO, PS, PNdmi, or PNms linkages; and wherein Gm, Am, Um, and Cm are 2’-O-methyl (2’-OMe) modified guanosine, adenosine, uridine, and cytidine, respectively; m5Ce is 2’-MOE modified 5-methylcytidine; Ge is 2’-MOE modified guanosine; Ae is 2’-MOE modified adenosine; m5Ue is 2’-MOE modified 5- methyluridine (also sometimes named “Te”; 2’-MOE modified thymidine); Af, Ilf, Gf, and Cf are 2’-F modified adenosine, uridine, guanosine, and cytosine, respectively.
  • the guide oligonucleotide comprises or consists of the sequence of any one of SEQ ID NO:41 , 44, 50, 58, 59, 60, 61 , 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, and 94.
  • the guide oligonucleotide comprises or consists of the sequence of any one of SEQ ID NO:65, 66, 90, 64, 88, 89, 69, 67, 82, 83, 84, 58, 61 , 59, 60, 41 , 44, 50, 68, 70, 71 , 72, 73, 78, 79, 80, 81 , 85, 86, and 87.
  • the guide oligonucleotide is bound, preferably conjugated, to a triterpene glycoside, preferably AG1856.
  • the disclosure provides a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding a guide oligonucleotide according the first aspect of the disclosure.
  • a viral vector preferably an adeno-associated virus (AAV) vector
  • AAV adeno-associated virus
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a guide oligonucleotide according to the first aspect of the disclosure, or a vector according to the second aspect of the disclosure, and a pharmaceutically acceptable carrier.
  • the disclosure provides a guide oligonucleotide according to the first aspect of the disclosure, or a vector according to a second aspect of the disclosure, or a pharmaceutical composition according to the third aspect of the disclosure, for use in the treatment, amelioration, or slowing down the progression of a neurodegenerative disease, preferably AD, more preferably ADAD.
  • a neurodegenerative disease preferably AD, more preferably ADAD.
  • the disclosure provides for the use of a guide oligonucleotide according to the first aspect of the disclosure, or a vector according to a second aspect of the disclosure, or a pharmaceutical composition according to the third aspect of the disclosure, for use in the manufacture of a medicament for the treatment, amelioration, or slowing down the progression of a neurodegenerative disease, preferably AD, more preferably ADAD.
  • a neurodegenerative disease preferably AD, more preferably ADAD.
  • the disclosure provides a method of treating, ameliorating, or slowing down the progression of a neurodegenerative disease, preferably AD, more preferably ADAD, in a human subject in need thereof, the method comprising administering to said subject a guide oligonucleotide according to the first aspect of the disclosure, or a vector according to a second aspect of the disclosure, or a pharmaceutical composition according to the third aspect of the disclosure, thereby editing the target RELN nucleic acid sequence to encode a reelin protein with the ability to delay onset of one or more symptoms of a neurodegenerative disease, preferably AD, more preferably ADAD.
  • a neurodegenerative disease preferably AD, more preferably ADAD
  • the disclosure provides an in vitro, ex vivo, or in vivo method for the deamination of a target adenosine in a target RELN nucleic acid sequence in a cell, the method comprising the steps of: (i) providing the cell with a guide oligonucleotide according to the first aspect of the disclosure, or a vector according to a second aspect of the disclosure, or a pharmaceutical composition according to the third aspect of the disclosure; (ii) allowing uptake by the cell of the guide oligonucleotide or vector or composition; (iii) allowing annealing of the guide oligonucleotide to the target RELN nucleic acid sequence; and (iv) allowing a nucleic acid editing entity to edit the target.
  • the method comprises step (v) of using a functional read-out to identify the presence of the edited target nucleotide.
  • the method comprises the step of administering a triterpene glycoside before, after or simultaneously with administering the guide oligonucleotide.
  • the triterpene glycoside is AG 1856.
  • the triterpene glycoside, such as AG1856 is (non)covalently bound to the guide oligonucleotide to allow improved endosomal escape once the guide oligonucleotide has entered a target cell in which deamination of the target nucleotide needs to take place.
  • the disclosure provides a method of editing a human RELN nucleic acid sequence in a cell, preferably a brain cell, wherein the human RELN nucleic acid sequence is pre-mRNA or mRNA, the method comprising contacting the target RELN nucleic acid sequence with a guide oligonucleotide capable of triggering an ADAR-mediated adenosine to inosine deamination, thereby editing the target RELN nucleic acid sequence to encode a reelin protein with the ability to delay onset of one or more symptoms of a neurodegenerative disease, preferably AD, more preferably ADAD.
  • a guide oligonucleotide capable of triggering an ADAR-mediated adenosine to inosine deamination
  • the disclosure provides a guide oligonucleotide for editing a target adenosine in a human RELN pre-mRNA or mRNA molecule by providing the guide oligonucleotide and allowing the guide oligonucleotide to hybridize to a human RELN pre- mRNA or mRNA molecule and thereby to attract an ADAR enzyme to deaminate the target adenosine , wherein the target region is SEQ ID NO: 106, and wherein the target adenosine is the second nucleotide of the codon encoding histidine at position 3447 of the REL/V-encoded human reelin protein.
  • the nucleic acid molecule is selected from the group consisting of SEQ ID NO:65, 66, 90, 64, 88, 89, 69, 67, 82, 83, 84, 58, 61 , 59, 60, 41 , 44, 50, 68, 70, 71 , 72, 73, 78, 79, 80, 81 , 85, 86, and 87.
  • the nucleic acid molecule comprises at least one non- naturally occurring chemical modification, and/or comprising one or more additional non- naturally occurring chemical modifications in a ribose, linkage or base moiety, with the proviso that the orphan nucleotide, which is the nucleotide in the nucleic acid that is directly opposite a target adenosine in the target region, is not a cytidine comprising a 2’-OMe ribose substitution.
  • the guide oligonucleotides referred to herein are sometimes known or referred to as antisense oligonucleotides (AONs). They are sometimes also referred to as ‘editing oligonucleotides’, or ‘EONs’, even though the editing event is performed by the nucleic acid editing entity and the action of the oligonucleotide only triggers the editing to take place.
  • AONs antisense oligonucleotides
  • EONs editing oligonucleotides
  • oligonucleotide oligonucleotide, oligo, ON, ASO, oligonucleotide composition, antisense oligonucleotide, AON, (RNA) editing oligonucleotide, EON, and RNA (antisense) oligonucleotide
  • oligonucleotide may completely lack RNA and DNA nucleotides (as they appear in nature) and may consist completely of modified nucleotides.
  • oligoribonucleotide Whenever reference is made to an ‘oligoribonucleotide’ it may comprise the bases A, G, C, II, or I. Whenever reference is made to a ‘deoxyoligoribonucleotide’ it may comprise the bases A, G, C, T, or I.
  • a guide oligonucleotide as disclosed herein may comprise a mix of ribonucleotides and deoxyribonucleotides.
  • the nucleotide When a deoxyribonucleotide is used, hence without a modification at the 2’ position of the sugar, the nucleotide is often abbreviated to dA (or Ad), dC (or Cd), dG (or Gd) or T in which the ‘d’ represents the deoxy nature of the nucleoside, while a ribonucleoside that is either normal RNA or modified at the 2’ position is often abbreviated without the ‘d’, and often abbreviated with their respective modifications and as explained herein.
  • nucleoside refers to the nucleobase linked to the (deoxy)ribosyl sugar, without phosphate groups.
  • a ‘nucleotide’ is composed of a nucleoside and one or more phosphate groups.
  • nucleotide thus refers to the respective nucleobase- (deoxy)ribosyl-phospholinker, as well as any chemical modifications of the ribose moiety or the phospho group.
  • nucleotide including a locked ribosyl moiety comprising a 2’-4’ bridge, comprising a methylene group or any other group
  • an unlocked nucleic acid (UNA) comprising a threose nucleic acid (TNA)
  • NAA threose nucleic acid
  • nucleobase nucleoside and nucleotide are used interchangeably, unless the context clearly requires differently, for instance when a nucleoside is linked to a neighbouring nucleoside and the linkage between these nucleosides is modified.
  • a nucleotide is a nucleoside plus one or more phosphate groups.
  • ribonucleoside and ‘deoxyribonucleoside’, or ‘ribose’ and ‘deoxyribose’ are as used in the art.
  • adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine/uridine, inosine, and hypoxanthine are used interchangeably to refer to the corresponding nucleobase on the one hand, and the nucleoside or nucleotide on the other.
  • the nucleobase thymine (T) is also known as 5- methyluracil (m 5 U) and is a uracil (II) derivative; thymine and 5-methyluracil can be interchanged throughout the document text.
  • the nucleotide thymidine is also known as 5-methyluridine and is a uridine derivative; thymidine and 5-methyluridine can be interchanged throughout the document text.
  • nucleotides in the oligonucleotide such as cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5- hydroxycytosine, and p-D-glucosyl-5-hydroxymethylcytosine are included.
  • cytosine such as cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5- hydroxycytosine, and p-D-glucosyl-5-hydroxymethylcytosine are included.
  • adenine N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine and 7-methyladenine are included.
  • uracil dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudouracil, 4-thiouracil and 5-hydroxymethyluracil are included.
  • guanine 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7- trimethylguanosine and N2,7-dimethylguanosine are included.
  • ribofuranose derivatives such as 2’-deoxy, 2’-hydroxy, and 2’- O-substituted variants, such as 2’-0Me, are included, as well as other modifications, including 2’-4’ bridged variants.
  • one or more linkages may be a naturally occurring phosphodieaster linkage, whereas the remaining linkages between two mononucleotides may be a modified linkage.
  • modified linkages are phosphonoacetate, phosphotriester, PS, phosphoro(di)thioate, MP, phosphoramidate linkages, phosphoryl guanidine, thiophosphoryl guanidine, sulfono phosphoramidate, PNdmi and the linkage structure according to formula (I), further outlined in detail below.
  • composition ‘comprising X’ may consist exclusively of X or may include something additional, e.g., X + Y.
  • the term ‘about’ in relation to a numerical value x is optional and means, e.g., x+10%.
  • the term ‘conducive to’ or ‘mediate’ can be used interchangeably with ‘capable of facilitating’.
  • the guide oligonucleotide itself does not have the enzymatic function (the ADAR enzyme has), but it can trigger, induce, cause, organize, mediate, provide, give, produce, facilitate, and/or result in RNA editing after binding to the target RNA molecule.
  • mismatched nucleotides are G-A, C-A, ll-C, A-A, G-G, C-C, Il-Il pairs.
  • guide oligonucleotides comprise fewer than four mismatches with the target sequence, for example 0, 1 or 2 mismatches.
  • ‘Wobble’ base pairs are G-ll, l-ll, l-A, and l-C base pairs.
  • a G:G pairing would be considered a mismatch, that does not necessarily mean that the interaction is unstable, which means that the term ‘mismatch’ may be somewhat outdated based on the current disclosure where a Hoogsteen base-pairing may be seen as a mismatch based on the origin of the nucleotide but still be relatively stable.
  • An isolated G:G pairing in duplex RNA can for instance be quite stable, but still be defined as a mismatch.
  • the term does not necessarily mean that each nucleotide in a nucleic acid strand has a perfect pairing with its opposite nucleotide in the opposite sequence.
  • a guide oligonucleotide may be complementary to a target sequence
  • there may be mismatches, wobbles and/or bulges between the guide oligonucleotide and the target sequence while under physiological conditions that guide oligonucleotide still hybridizes to the target sequence such that the cellular RNA editing enzymes can deaminate the target adenosine to an inosine.
  • the term ‘substantially complementary’ therefore also means that despite the presence of the mismatches, wobbles, and/or bulges, the guide oligonucleotide has enough matching nucleotides with the target sequence that under physiological conditions the guide oligonucleotide hybridizes to the target RNA molecule.
  • a guide oligonucleotide may be complementary, but may also comprise one or more mismatches, wobbles and/or bulges with the target sequence, if under physiological conditions the guide oligonucleotide is able to hybridize to its target.
  • orphan nucleotide relates to the nucleotide in the guide oligonucleotide that is directly opposite the target adenosine, which is the adenosine that is deaminated by the deaminating enzyme.
  • the orphan nucleotide may be a natural cytidine or deoxycytidine, or a uridine or deoxyuridine.
  • It may also be a chemically modified nucleotide, as further described in detail below, or a known or chemically modified analog of a natural (deoxy)cytidine, such as a nucleotide carrying a Benner’s base (6-amino-5-nitro-3-yl-2(1 H)-pyridone), or a known or chemically modified analog of a natural (deoxy)uridine, such as iso-uridine, as further outlined in detail below.
  • nucleotide analog refers to an analog of a nucleic acid nucleotide.
  • the nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine or deoxyuridine.
  • downstream in relation to a nucleic acid sequence means further along the sequence in the 3' direction; the term ‘upstream’ means the converse.
  • start codon is upstream of the stop codon in the sense strand but is downstream of the stop codon in the antisense strand.
  • guide oligonucleotides as disclosed herein. Nucleotides that are upstream of the orphan nucleotide in the antisense guide oligonucleotide are located towards the 5’ terminus, and nucleotides that are downstream of the orphan nucleotide are located towards the 3’ terminus.
  • nucleotide ‘numbering’ in a guide oligonucleotide as disclosed herein is such that the orphan nucleotide is number 0 and the nucleotide 5’ from the orphan nucleotide is number +1. Counting is further positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end, wherein the first nucleotide 3’ from the orphan nucleotide is number -1.
  • the internucleoside linkage numbering in the guide oligonucleotide is such that linkage number 0 is the linkage 5’ from the orphan nucleotide, and the linkage positions in the oligonucleotide are positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end.
  • splice mutation relates to a mutation in a gene that encodes fora pre-mRNA, wherein the splicing machinery is dysfunctional in the sense that splicing of introns from exons is disturbed and due to the aberrant splicing, the subsequent translation is out of frame resulting in premature termination of the encoded protein. Often such shortened proteins are degraded rapidly and do not have any functional activity.
  • the encoded guide oligonucleotide is expressed from the viral vector genome or from the plasmid in the cell to which the viral vector or plasmid vector is delivered. Consequently, the guide oligonucleotide is then not chemically modified, and comprises solely naturally occurring nucleotides, preferably naturally occurring RNA nucleotides.
  • the guide oligonucleotide is still considered naked because it is not transcribed from an encoding polynucleotide (such as in the case of a plasmid or a vector, in which the guide oligonucleotide is not regarded as ‘naked’). So, even though a chemically modified guide oligonucleotide is encapsulated by a carrier, preferably an LNP, it is still seen as naked, as it has been manufactured as such in a laboratory setting and encapsulated thereafter in the carrier using methods known to the person skilled in the art.
  • a carrier preferably an LNP
  • a naked guide oligonucleotide as is; ii) a naked guide oligonucleotide encapsulated in a delivery vehicle, preferably an LNP; iii) a naked guide oligonucleotide administered together or separately from (but not bound to) a saponin such as AG1856; iv) a naked guide oligonucleotide conjugated to a saponin such as AG 1856; v) a naked guide oligonucleotide conjugated to a saponin such as AG1856, and wherein the saponin-guide conjugate is encapsulated in a delivery vehicle, preferably an LNP; or vi) through an encoding vector, such as a plasmid or a viral vector from which the guide oligonucleotide is transcribed.
  • an encoding vector such as a plasmid or a viral vector from which the guide oligonucleotide is transcribed.
  • the sense strand may be chemically modified almost in its entirety, similar or different to what is performed in the guide oligonucleotide as disclosed herein, for example by providing nucleotides with a ribose sugar moiety carrying a 2’-OMe substitution, a 2’-F substitution, or a 2’-MOE substitution. It is to be understood that the sense strand present in the HEON is a different entity in comparison to the target RNA molecule in the cell.
  • the sense strand in an HEON is preferably 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.
  • the HEON is often generated in vitro and used as a delivery tool to protect the guide oligonucleotide from degradation when administered to the cell. In other words, the HEON is preferably formed before the guide oligonucleotide is administered to the cell.
  • the present disclosure relates to guide oligonucleotides that mediate editing of one or more target nucleotides present in a target RELN nucleic acid sequence.
  • the RELN gene encodes the protein reelin.
  • the guide oligonucleotide mediates editing of one or more target nucleotides present in a human RELN nucleic acid sequence. It is particularly preferred that the human RELN nucleic acid sequence is present in a human cell, wherein editing occurs in the cell.
  • the target nucleotide is any nucleotide whereby editing provides the reelin protein with one or more of: a gain-of-function phenotype; an enhanced ability to upregulate signalling pathways initiated by reelin; an enhanced ability to increase Dab1 phosphorylation, to reduce Tau phosphorylation, and/or to increase neuronal density; and/or enhanced binding of the reelin protein to glycosaminoglycans, preferably heparin, and/or to NRP1.
  • the target nucleotide is any nucleotide whereby the edited target nucleotide produces a structural effect on any of the amino acids in the ‘a-GAG binding site’, ‘P-GAG binding site’, and/or neuropilin 1 (NRP1) binding site, of the reelin protein.
  • the a-GAG binding site spans the six C-terminal amino acids of the human reelin protein (3455- 3460).
  • the p-GAG binding site spans amino acids 3446-3451 of the human reelin protein.
  • the edited target nucleotide can be within a codon for an amino acid that is outside of these sites, so long as mutation of the amino acid produces an effect within one of these sites.
  • the target nucleotide is adenosine
  • the nucleic acid editing entity is an adenosine deaminase.
  • the target RELN nucleic acid sequence comprising a target nucleotide is a RELN RNA transcript molecule (pre-mRNA and/or mRNA) comprising a target adenosine
  • the nucleic acid editing entity is an ADAR enzyme, more preferably human ADAR1 and/or human ADAR2.
  • the RELN RNA transcript molecule has a codon CAU encoding histidine at position 3447 of the human reelin protein, wherein the adenosine within the CAU codon is the target adenosine.
  • ADAR-mediated editing produces CIU, which the translation machinery interprets as CGU, encoding arginine.
  • the cell is a cell of a human having DNA encoding reelin with an amino acid other than arginine, preferably histidine, at position 3447 of the human reelin protein.
  • the edited RELN nucleic acid sequence encodes a reelin protein with an enhanced ability to protect against AD, and/or one or more symptoms of AD. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein with an ability to upregulate or initiate a pathway that is protective against AD, and/or one or more symptoms of AD. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein with an enhanced ability to bind glycosaminoglycans (GAGs), particularly at the C-terminal region of reelin. It is particularly preferred that the edited RELN nucleic acid sequence encodes a reelin protein with an enhanced ability to bind heparin.
  • GAGs glycosaminoglycans
  • the edited RELN nucleic acid sequence encodes a reelin protein with an enhanced ability to bind neuropilin 1. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein with an enhanced ability to lower Tau pathology. In particular, in some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein with enhanced ability to reduce Tau phosphorylation. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein with enhanced ability to increase Dab1 phosphorylation. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein with the ability to increase neuronal density.
  • the edited RELN nucleic acid sequence encodes a reelin protein with a gain-of-function phenotype.
  • gain-of-function are, in addition to the variant described in the examples, variants with: 1) an enhanced ability to trigger signalling, preferably of the APOEr/Dab1/GSK3p pathway; 2) an enhanced ability to increase Dab1 phosphorylation; 3) an enhanced ability to reduce Tau phosphorylation found in neurofibrillary tangles; 4) an enhanced ability to increase microtubular structure formation and/or stability of microtubule structures and/or neuronal density; 5) an enhanced resistance to degradation by proteolysis; and/or enhanced binding to glycosaminoglycans, preferably heparin; and/or 6) enhanced binding to NRP1.
  • a preferred embodiment is one wherein the reelin protein has a gain-of-function by way of reduced inactivation by proteases.
  • ADAMTS-3 was identified as the protease that cleaves and inactivates reelin in the brain ⁇ e.g., the cerebral cortex and hippocampus). Knock down of ADAMTS-3 in mice has been shown to reduce Tau phosphorylation and dendritic branching and elongation was increased (Ogino H et al., J Neurosci. 2017, 37(12):3181-3191). Therefore, altering the ADAMTS-3 cleavage site in reelin, such as the Pro-Ala sequence in the N-t site (Koie M et al., J Biol Chem.
  • the present disclosure relates to a variety of guide oligonucleotides that are aimed at deamination of a target adenosine in a target RELN nucleic acid sequence. However, it is not excluded that two or more adenosines may be targeted for deamination in a single treatment.
  • a synergistic or additive effect may be obtained by combining guide oligonucleotides as disclosed herein for targeting a multitude of target nucleotides, such as target adenosines, and thereby a multitude of amino acids within a single reelin protein, to increase the therapeutic effect.
  • the target nucleotide is a nucleotide whereby editing thereof produces an edited RELN nucleic acid sequence that encodes an edited reelin protein with beneficial therapeutic effects in comparison with the unedited reelin protein.
  • the beneficial therapeutic effects may include the treatment, alleviation, or reduction of neurodegenerative disease, such as AD, or one or more symptoms of such disease, including mild cognitive impairment, cognitive decline, and/or dementia.
  • the guide oligonucleotides are for use in a subject that has been diagnosed with AD. In other embodiments, the guide oligonucleotides are for use prophylactically in a subject that has been identified as at risk for developing AD. In some embodiments, the guide oligonucleotides are for use prophylactically in cognitively healthy subjects. In some embodiments, the guide oligonucleotides are for use in a subject having the PSEN 7-E280A mutation (wherein PSEN1 is the gene encoding presenilin 1), which is a mutation associated with development of mild cognitive impairment in AD.
  • the guide oligonucleotides are for use in a subject having two copies of the AP0E3 Wales (APOECh) (R136S) gene variant. In some embodiments, the guide oligonucleotides are for use in a subject having ADAD.
  • the cell is a brain cell, preferably a neuron. In some embodiments, the cell is in the medial temporal lobe, preferably the allocortex, more preferably the entorhinal cortex. In some embodiments, editing occurs in or around the endoplasmic reticulum of the cell.
  • DNA editing techniques that are compatible with what is disclosed herein include DNA editing techniques based on the CRISPR Cas9 nuclease enzyme. These techniques are well known in the art. These techniques include use of CRISPR-Cas9 to introduce a double-stranded break to DNA, which can be programmed to occur at a specified site by using a guide RNA oligonucleotide with the necessary sequence to guide the CRISPR-Cas9 enzyme to the specified site. Such breaks can be used to delete, modify, and/or insert DNA sequence at the specified site in DNA.
  • CRISPR-Cas9 Another system derived from CRISPR-Cas9 is the use of a Base Editor (BE) system, which uses a catalytically dead Cas9 (dCas9) that has been fused to a functional enzyme such as a DNA deaminase.
  • the dCas9 does not introduce double-stranded breaks to DNA, but instead locates the DNA deaminase such that it results in deamination of the target DNA nucleotide, again guided to the specific site by a guide oligonucleotide.
  • the DNA deaminase can be a cytidine deaminase (to induce C to T substitutions) or an adenine deaminase (to induce A to G substitutions).
  • dCas9 fused to a cytidine deaminase enzyme is also known as a Cytidine Base Editor (CBE)
  • dCas9 fused to an adenine deaminase enzyme is also known as an Adenine Base Editor (ABE).
  • a further development is known as Prime Editing.
  • Prime Editing uses a Cas9 nickase fused to a reverse transcriptase enzyme.
  • Prime Editing again uses a guide oligonucleotide to guide the enzyme to a specific site in the DNA.
  • Prime Editing also uses an oligonucleotide that comprises a prime editing guide RNA (pegRNA) which comprises a primer binding site sequence and a sequence containing the desired edit.
  • pegRNA prime editing guide RNA
  • WO2016/097212 discloses AONs for the targeted editing of RNA, wherein the AONs are characterized by a sequence that is complementary to a target RNA sequence (therein referred to as the ‘targeting portion’) and by the presence of a stem-loop I hairpin structure (therein referred to as the ‘recruitment portion’), which is preferably non- complementary to the target RNA.
  • the AONs are characterized by a sequence that is complementary to a target RNA sequence (therein referred to as the ‘targeting portion’) and by the presence of a stem-loop I hairpin structure (therein referred to as the ‘recruitment portion’), which is preferably non- complementary to the target RNA.
  • Such oligonucleotides are referred to as ‘self-looping AONs’.
  • the recruitment portion acts in recruiting a natural ADAR enzyme present in the cell (that is, endogenously present) to the dsRNA formed by hybridization of the target sequence with the targeting portion
  • the stem-loop structure of the recruitment portion as described is an intramolecular stem-loop structure, formed within the AON itself, and are thought to attract (endogenous) ADAR. Similar stem-loop structure-comprising systems for RNA editing have since then been described in Inti. Patent Application Publication Nos. WO2017/050306, W02020/001793, WO2017/010556, US11 ,390,865, WO2020/246560, and WO2022/078995.
  • Patent Application Publication Nos. WO2017/220751 and WO2018/041973 describe a next generation type of AONs that do not comprise such a stem-loop structure but that are (almost fully) complementary to the targeted area, and that appeared still capable of attracting endogenous ADAR enzymes.
  • one or more mismatching nucleotides, wobbles, or bulges exist between the oligonucleotide and the target sequence.
  • a sole mismatch may be at the site of the nucleoside opposite the target adenosine, but in other embodiments AONs were described with multiple bulges and/or wobbles when attached to the target sequence area.
  • the ‘orphan nucleoside’ which is defined as the nucleoside in the guide oligonucleotide (or AON) that is positioned directly opposite the target adenosine in the target RNA molecule, was a nucleotide with an unmodified cytosine nucleobase and that did not carry a 2’-OMe modification.
  • the orphan nucleoside can be a deoxyribonucleoside (DNA), wherein the remainder of the guide oligonucleotide could still carry 2’-O-alkyl modifications at the sugar entity (such as 2’-OMe), or the nucleotides directly surrounding the orphan nucleoside contained chemical modifications (such as DNA in comparison to RNA) that further improved the RNA editing efficiency and/or increased the resistance against nucleases.
  • Such effects could even be further improved by using sense oligonucleotides (SONs) that ‘protected’ the AONs against breakdown upon delivery to the cells (described in Inti. Patent Application Publication Nos. WO2018/134301 and US11 ,274, 300).
  • SONs sense oligonucleotides
  • WO2011/005761 WO2014/010250, W02014/012081 , WO2015/107425, WO2017/015575 (HTT), WO2017/062862, WO2017/160741 , WO2017/192664, WO2017/192679 (DMD), WO2017/198775, WO2017/210647, WO2018/067973, WO2018/098264, WO2018/223056 (PNPLA3), WO2018/223073 (APOC3), WO2018/223081 (PNPLA3), WO2018/237194, WO2019/032607 (C9orf72), WO2019/055951 , WO2019/075357 (SMA/ALS), W02019/200185 (DM1), WO2019/217784 (DM1), WO2019/219581 , W02020/118246 (DM1), W02020/160336 (HTT), WO2020/191252, W02020/196662, WO2020/219981 (USH
  • W02020/157008 and WO2021/136404 (USH2A); WO2021/113270 (APP); WO2021/113390 (CMT1A); W02021/209010 (IDUA, Hurler syndrome); WO2021/231673 and WO2021/242903 (LRRK2); WO2021/231675 (ASS1); WO2021/231679 (GJB2); WO2019/071274 and WO2021/231680 (MECP2); WO2021/231685 and WO2021/231692 (OTOF, autosomal recessive non- syndromic hearing loss); WO2021/231691 (XLRS); WO2021/231698 (argininosuccinate lyase deficiency); W02021/130313 and WO2021/231830 (ABCA4); and WO2021/243023 (SERPINA1).
  • the ADAR1 and/or ADAR2 are endogenously present in the cell.
  • Such guide oligonucleotide can mediate RNA editing of a target adenosine present in a target RNA molecule after it is bound to the target RNA molecule, since the deaminating enzymes are recruited to the double-stranded oligonucleotide/target RNA molecule complex and subsequently deaminate the target adenosine into an inosine.
  • the present disclosure provides guide oligonucleotides that can provide (mediate, cause, or trigger) RNA editing of a target adenosine in a target transcript molecule, such as pre-mRNA and/or mRNA.
  • the target transcript molecule may be encoded by a mutated gene, wherein the mutation is the cause of a disease and wherein the editing can reverse the mutation to give rise to a wildtype protein, or a protein with a wildtype function (for instance when the mutated amino acid is changed to an amino acid that does not cause the disease, or that provides an improved phenotype).
  • the target transcript molecule may also be encoded by a wildtype gene, such as in a preferred aspect of the present disclosure, wherein the target RELN nucleic acid molecule is a transcript from a wildtype human RELN gene as shown in the present disclosure.
  • the RNA editing encodes a modified reelin protein that improves the disease state of the treated subject.
  • the target RELN nucleic acid sequence is the sequence as naturally present in a subject.
  • the target RELN nucleic acid sequence is the sequence prior to treatment with guide oligonucleotides according to the disclosure.
  • Non-limiting examples of transcript molecules that are targeted using RNA editing for a variety of treatments are SERPINA 1 (for the treatment of alphal -antitrypsin (A1AT) deficiency; see e.g., Inti. Patent Application Publication Nos. WO2016/097212,
  • WO2018/041973, and W02021/209010 for the treatment of Parkinson’s disease; see e.g., Inti. Patent Application Publication Nos. WO2016/097212, WO2017/220751 , WO2018/041973, WO2021/231673 and WO2021/242903), ABCA4 (for the treatment of Stargardt disease; see e.g., Inti. Patent Application Publication Nos. W02021/130313 and WO2021/231830), USH2A (for the treatment of Usher syndrome; see e.g., Inti. Patent Application Publication Nos.
  • W02020/157008, WO2020/219981 and WO2021/136404 W02020/157008, WO2020/219981 and WO2021/136404
  • APP see e.g., Inti. Patent Application Publication No. WO2021/113270
  • CMT1A see e.g., Inti. Patent Application Publication No. WO2021/113390
  • ASS1 see e.g., Inti. Patent Application Publication No. WO2021/231675
  • GJB2 see e.g., Inti. Patent Application Publication No. WO2021/231679
  • MECP2 for the treatment of Rett syndrome; see e.g., Inti. Patent Application Publication Nos.
  • WO2019/071274 and WO2021/231680 for the treatment of autosomal recessive non-syndromic hearing loss; see e.g., Inti. Patent Application Publication Nos. WO2021/231685 and WO2021/231692
  • XLRS see e.g., Inti. Patent Application Publication No. WO2021/231691
  • PCSK9 for the treatment of hypercholesterolemia; see e.g., Inti. Patent Application Publication No. WO2023/152371
  • HFE for the treatment of hemochromatosis I iron overload; see e.g., Inti. Patent Application Publication No. WO2024/110565).
  • oligonucleotide Various chemistries and modifications are known in the field of oligonucleotides that can be readily used in accordance with the disclosure.
  • the chemical modifications listed herein may be used with guide oligonucleotides intended for DNA editing or RNA editing, as appropriate, and/or unless otherwise noted. All chemical modifications listed herein that may be used in the guide oligonucleotide as disclosed herein may also be used for a sense strand that is complementary to the guide oligonucleotide, when the guide oligonucleotide and the complementary strand form a HEON complex, such as described in Inti. Patent Application Publication No.
  • the modification related to the orphan nucleotide relate only to the guide oligonucleotide as disclosed herein, but all other modifications relate to the guide oligonucleotide as disclosed herein and any (protecting) sense oligonucleotide that may be used together with the guide oligonucleotide in a pharmaceutical product.
  • an oligonucleotide such as a guide oligonucleotide as outlined herein, generally consists of repeating monomers. Such a monomer is most often a nucleotide or a chemically modified nucleotide.
  • the most common naturally occurring nucleotides in RNA are adenosine monophosphate (A), cytidine monophosphate (C), guanosine monophosphate (G), and uridine monophosphate (II). These consist of a pentose sugar, a ribose, a 5’-linked phosphate group which is linked via a phosphate ester, and a T- linked base.
  • the linker may be a cleavable or an uncleavable linker.
  • a cleavable linker refers to a linker that can be cleaved under physiological conditions, for example, in a cell or an animal body (e.g., a human body).
  • An uncleavable linker refers to a linker that is not cleaved under physiological conditions, or very slowly compared to a cleavable linker, for example, in a PS linkage, modified or unmodified deoxyribonucleosides linked by a PS linkage, a spacer connected through a PS bond and a linker consisting of modified or unmodified ribonucleosides.
  • a linker is a nucleic acid such as DNA, or an oligonucleotide. However, it may be usually from 2 to 20 bases in length, from 3 to 10 bases in length, or from 4 to 6 bases in length.
  • a spacer that is connects the ligand and the oligonucleotide may include for example ethylene glycol, triethylene glycol (TEG), HEG, alkyl chains, propyl, 6-aminohexyl, or dodecyl.
  • TEG triethylene glycol
  • HEG alkyl chains
  • propyl 6-aminohexyl
  • dodecyl dodecyl
  • One or more other types of molecules may be bound to the guide oligonucleotide through one or more linkers, including peptides, sugars, vitamins, polymers, aptamers, (fragments of) antibodies, small molecules, and the like.
  • the orphan nucleotide carries a 2’-F in the sugar moiety. In one aspect, the orphan nucleotide carries a di F substitution in the sugar moiety. In one aspect, the orphan nucleotide carries a 2’-F and a 2’-C-methyl in the sugar moiety. In one aspect, the orphan nucleotide comprises a 2’-F in the arabinose configuration (FANA) in the sugar moiety.
  • FANA arabinose configuration
  • the modifications should be compatible with editing such that the guide oligonucleotide fulfils its role as an oligonucleotide that can, after binding to its target sequence, recruit an adenosine deaminase enzyme because of the double-stranded nucleic acid entity that arises.
  • the enzyme with adenosine deaminase activity is preferably ADAR1 , ADAR2, or ADAT.
  • uridine No mismatch exists when the orphan nucleotide is uridine, which may be defined differently when the orphan nucleotide is a uridine analog or derivative.
  • One alternative for uridine is positioning an iso-uridine opposite the target adenosine, which likely does not pair like G pairs with II.
  • the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the guide oligonucleotide that is directly opposite the target adenosine.
  • a guide oligonucleotide as disclosed herein makes use of specific nucleotide modifications at predefined spots to ensure stability as well as proper ADAR binding and activity. These changes may vary and may include modifications in the backbone of the guide oligonucleotide, in the sugar moiety of the nucleotides as well as in the nucleobases or the PO linkages, as outlined in detail herein. They may also be variably distributed throughout the sequence of the guide oligonucleotide. Specific modifications may be needed to support interactions of different amino acid residues within the RNA-binding domains of ADAR enzymes, as well as those in the deaminase domain.
  • a target sequence 5’- UAG-3’ contains the most preferred nearest-neighbor nucleotides for ADAR2, whereas a 5’-CAA-3’ target sequence is disfavored (Schneider et al. Nucleic Acids Res. 2014, 42(10):e87).
  • the structural analysis of ADAR2 deaminase domain hints at the possibility of enhancing editing by careful selection of the nucleotides that are opposite to the target trinucleotide.
  • the 5’-CAA-3’ target sequence paired to a 3’-GCU-5’ sequence on the opposing strand (with the A-C mismatch formed in the middle), is disfavored because the guanosine base sterically clashes with an amino acid side chain of ADAR2.
  • the guanosine opposite the C in such circumstances is preferably replaced by an inosine (hence, at the -1 position within the guide oligonucleotide), more preferably an Id, as further outlined in the present disclosure.
  • the guide oligonucleotide as disclosed herein in contrast to what has been described for siRNA, or gapmers and their relation towards RNase breakdown and the use of such gapmers in double-stranded complexes (see for instance EP 3954395 A1), does not comprise a stretch of DNA nucleotides which would make a target sequence (or a sense nucleic acid strand) a target for RNase-mediated breakdown. It is not desired that the target transcript molecule is degraded through the binding of the guide oligonucleotide to the transcript molecule.
  • the guide oligonucleotide does not comprise four or more consecutive DNA nucleotides anywhere within its sequence.
  • the guide oligonucleotide is composed of as much (chemically) modified nucleotides as possible to enhance the resistance towards RNase-mediated breakdown, while at the same time being as efficient as possible in producing an RNA editing effect.
  • the orphan nucleotide and several other nucleotides within the guide oligonucleotide may be DNA, but also that there is no stretch of four or more consecutive DNA nucleotides within the guide oligonucleotide.
  • the guide oligonucleotide as disclosed herein is not a gapmer. A gapmer reduces the expression of a target transcript but does not produce RNA editing of a specified adenosine within the target transcript.
  • a gapmer is in principle a single-stranded nucleic acid consisting of a central region (DNA gap region with at least four consecutive deoxyribonucleotides) and wing regions positioned directly at the 5’ end (5’ wing region) and the 3’ end (3’ wing region) thereof.
  • the guide oligonucleotide as disclosed herein may be any oligonucleotide that produces an RNA editing effect in which a target adenosine in a target RNA molecule is deaminated to an inosine, and accordingly is resistant to RNase- mediated breakdown as much as possible to yield this effect and to allow the mRNA transcript being translated into a protein.
  • the guide oligonucleotides as disclosed herein may also be administered in the context of aids that will increase the entry of the guide oligonucleotide into the target cell and/or its endosomal escape as soon as it is in the cell.
  • Moieties that can be applied for such applications are for example a set of chemical compounds (generally purified from nature) referred to as “saponins” or “triterpene glycosides”.
  • a preferred saponin that can be used in the methods as disclosed herein is AG1856, disclosed in Inti. Patent Application Publication No. WO2021/122998 and further described for use with RNA editing producing oligonucleotides in Inti. Patent Application No. PCT/EP2024/051278 (unpublished).
  • a pharmaceutical composition comprising the guide oligonucleotide as disclosed herein, and further comprising a pharmaceutically acceptable carrier, solvent, diluent, and/or other additive (such as a saponin or triterpene glycoside like AG1856 (as discussed above), which in fact may also be administered separately from the guide oligonucleotide) and may be dissolved in a pharmaceutically acceptable organic solvent, or the like.
  • Dosage forms in which the guide oligonucleotide or the pharmaceutical composition are administered may depend on the disorder to be treated and the tissue that needs to be targeted and can be selected according to common procedures in the art.
  • the pharmaceutical compositions may be administered by a single-dose administration or by multiple dose administration. It may be administered daily or at appropriate time intervals, which may be determined using common general knowledge in the field and may be adjusted based on the disorder and the efficacy of the active ingredient.
  • the guide oligonucleotide as disclosed herein is a single-stranded oligonucleotide comprising an orphan nucleotide opposite the target nucleotide, wherein the orphan nucleotide is chemically modified as disclosed herein, and wherein the remainder of the oligonucleotide is chemically modified to prevent it from nuclease breakdown also as disclosed herein, in another embodiment, disclosed is any kind of oligonucleotide or heteroduplex oligonucleotide complex, that may or may not be bound to hairpin structures (internally or at the terminal end(s)), that may be bound to nucleic acid editing entities or catalytic domains thereof, or wherein the oligonucleotide is in a circular format.
  • the guide oligonucleotide as disclosed herein is a ‘naked’ oligonucleotide, comprising a variety of chemical modifications in the ribose sugar and/or the base of one or more of the nucleotides within the sequence, that preferably comprises at least one linkage according to the structure of formula (I) as disclosed herein, that can hybridize to the target nucleic acid sequence or a part thereof that includes the target adenosine, and can recruit endogenous (naturally present) nucleic acid editing entity in the target cell for the editing of the target nucleotide.
  • the guide oligonucleotide as disclosed herein does not comprise a stem-loop structure for recruitment of the deaminating enzyme, which allows for a shorter guide oligonucleotide and improved cellular delivery and trafficking.
  • RNA editing entities such as human ADAR enzymes
  • RNA editing entities edit dsRNA structures with varying specificity, depending on several factors.
  • One important factor is the degree of complementarity of the two strands making up the dsRNA sequence. Perfect complementarity of the two strands usually causes the catalytic domain of human ADAR to deaminate adenosines in a non-discriminative manner, reacting with any adenosine it encounters.
  • the specificity of hADARI and 2 can be increased by introducing chemical modifications and/or ensuring several mismatches in the dsRNA, which presumably helps to position the dsRNA binding domains in a way that has not been clearly defined yet.
  • the deamination reaction itself can be enhanced by providing an oligonucleotide that comprises a mismatch opposite the adenosine to be edited.
  • an oligonucleotide that comprises a mismatch opposite the adenosine to be edited Following the instructions in the present application, those of skill in the art will be capable of designing the complementary portion of the oligonucleotide according to their needs.
  • the extent to which the editing entities, such as editing enzymes, inside the cell are redirected to other target sites may be regulated by varying the affinity of the first nucleic acid strand for the recognition domain of the editing entity.
  • the exact modification may be determined through some trial and error and/or through computational methods based on structural interactions between the guide oligonucleotide and the recognition domain of the editing enzyme.
  • the degree of recruiting and redirecting the editing enzyme resident in the cell may be regulated by the dosing and the dosing regimen of the guide oligonucleotide. This is something to be determined by the experimenter in vitro) or the clinician, usually in phase I and/or II clinical trials.
  • RNA sequences in eukaryotic, preferably metazoan, more preferably mammalian, more preferably neuronal cells, more preferably human neuronal cells, and most preferably human cells from the central nervous system.
  • the target cell can be located in vitro, ex vivo or in vivo.
  • One advantage of the guide oligonucleotide as disclosed herein is that it can be used with cells in situ in a living organism, but it can also be used with cells in culture. In some embodiments cells are treated ex vivo and are then introduced into a living organism ⁇ e.g., re-introduced into an organism from whom they were originally derived).
  • the guide oligonucleotide as disclosed herein can also be used to edit target RNA sequences in cells from a transplant or within a so-called organoid, e.g., a brain tissue organoid.
  • Organoids can be thought of as three-dimensional in v/fro-derived tissues but are driven using specific conditions to generate individual, isolated tissues. In a therapeutic setting they are useful because they can be derived in vitro from a patient’s cells, and the organoids can then be re-introduced to the patient as autologous material which is less likely to be rejected than a normal transplant.
  • RNA editing through human ADAR2 for example is thought to take place on primary transcripts in the nucleus, during transcription or splicing, or in the cytoplasm, where e.g., mature mRNA, miRNA or ncRNA can be edited.
  • RNA editing may be used to create RNA sequences with different properties. Such properties may be coding properties (creating proteins with different sequences or length, leading to altered protein properties or functions), or binding properties (causing inhibition or over-expression of the RNA itself or a target or binding partner; entire expression pathways may be altered by recoding miRNAs or their cognate sequences on target RNAs).
  • Protein function or localization may be changed at will, by functional domains or recognition motifs, including but not limited to signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co- or post-translational modification, catalytic sites of enzymes, binding sites for binding partners, signals for degradation or activation and so on.
  • RNA and protein “engineering” whether to prevent, delay or treat disease or for any other purpose, in medicine or biotechnology, as diagnostic, prophylactic, therapeutic, research tool or otherwise, are encompassed by the present disclosure.
  • the amount of guide oligonucleotide to be administered, the dosage and the dosing regimen can vary from cell type to cell type, the disease to be treated, the target population, the mode of administration (e.g., systemic versus local), the severity of disease and the acceptable level of side activity, but these can and should be assessed by trial and error during in vitro research, in pre-clinical and clinical trials.
  • the trials are particularly straightforward when the modified sequence leads to an easily detected phenotypic change, or a change in (the level of, or activity of) a specified biomarker (such as plasma levels of bile acids for example).
  • One suitable trial technique involves delivering the guide oligonucleotide to cell lines, or a test organism and then taking biopsy samples at various time points thereafter.
  • the sequence of the target RNA can be assessed in the biopsy sample and the proportion of cells having the modification can easily be followed.
  • plasma level concentrations of bile acids in a sample from a treated subject is a proper biomarker for assessing the function of certain proteins in the subject, before and after treatment, or with or without treating the subject with a guide oligonucleotide as disclosed herein.
  • a method as disclosed herein can thus include a step of identifying the presence of the desired change in the cell’s target RNA sequence, thereby verifying that the target RNA sequence has been modified.
  • This step will typically involve sequencing of the relevant part of the target RNA, or a cDNA copy thereof (or a cDNA copy of a splicing product thereof, in case the target RNA is a pre-mRNA), as discussed above, and the sequence change can thus be easily verified.
  • the change may be assessed on the function of the protein before, during, and/or after treatment or assessing any other potential marker, which measurements are preferably performed in vitro on samples obtained from the treated subject.
  • RNA editing After RNA editing has occurred in a cell, the modified RNA can become diluted over time, for example due to cell division, limited half-life of the edited RNAs, etc.
  • a method as disclosed herein may involve repeated delivery of a guide oligonucleotide until enough target RNAs have been modified to provide a tangible benefit to the patient and/or to maintain the benefits over time.
  • Guide oligonucleotides as disclosed herein are particularly suitable for therapeutic use, and so disclosed is also a pharmaceutical composition comprising a guide oligonucleotide as disclosed herein and a pharmaceutically acceptable carrier, solvent, or diluent.
  • the pharmaceutically acceptable carrier can simply be a saline solution. This can usefully be isotonic or hypotonic, particularly for pulmonary delivery.
  • the guide oligonucleotide as disclosed herein is suitably administrated in aqueous solution, e.g.
  • saline or in suspension, optionally comprising additives, excipients and other ingredients, compatible with pharmaceutical use, at concentrations ranging from 1 ng/ml to 1 g/ml, preferably from 10 ng/ml to 500 mg/ml, more preferably from 100 ng/ml to 100 mg/ml.
  • Dosage may suitably range from between about 1 pg/kg to about 100 mg/kg, preferably from about 10 pg/kg to about 10 mg/kg, more preferably from about 100 pg/kg to about 1 mg/kg.
  • Administration may be by inhalation (e.g., through nebulization), intranasally, orally, by injection or infusion, intravenously, subcutaneously, intradermally, intramuscularly, intra-tracheally, intraperitoneally, intrarectally, intrathecally, intracerebroventricularly (e.g. in the intra-cisterna magna), parenterally, and the like.
  • Administration may be in solid form, in the form of a powder, a pill, a gel, a solution, a slow-release formulation, or in any other form compatible with pharmaceutical use in humans.
  • the identification step of whether the editing has taken place comprises the following steps: sequencing the target nucleic acid sequence; assessing the presence or absence of a non-, or less-functional protein; assessing whether splicing of pre-mRNA was altered by the deamination of a target adenosine in RNA; or using a functional read-out, because the target nucleic acid after the deamination should encode a protein with a lower or absent functionality, or on the other hand, an increased, regained or newly gained functionality.
  • the identification of the deamination into inosine may be a functional read-out using a suitable biomarker.
  • a functional assessment will generally be according to methods known to the skilled person.
  • a suitable manner to identify the presence of an inosine after deamination of a target adenosine is dPCR or sequencing, using methods that are well-known to the person skilled in the art.
  • the person skilled in the art of neurodegenerative disease will preferably apply tests to monitor certain biomarkers related to neurological function(s).
  • a method as disclosed herein comprises the steps of administering to the subject a guide oligonucleotide or vector capable of expressing it, as disclosed herein, allowing the formation of a double stranded nucleic acid complex of the guide oligonucleotide with the target nucleic acid sequence in a cell in the subject; allowing the engagement of a nucleotide editing entity, such as an endogenously present adenosine deaminating enzyme, such as ADAR 1 or ADAR2; and allowing the entity to edit the target nucleotide in the target nucleic acid sequence, thereby alleviating, treating, ameliorating, or slowing down progression of the disease.
  • a nucleotide editing entity such as an endogenously present adenosine deaminating enzyme, such as ADAR 1 or ADAR2
  • Nucleotide editing entities present in a cell will usually be proteinaceous in nature, such as the ADAR enzymes found in metazoans, including mammals. Particularly preferred are the human ADARs, hADARI and hADAR2, including any isoforms thereof.
  • ADARs adenosine deaminases acting on RNA
  • hADARI exists in two isoforms; a long 150 kDa interferon inducible version and a shorter, 110 kDa version, that is produced through alternative splicing from a common pre-mRNA. Consequently, the level of the 150 kDa isoform available in the cell may be influenced by interferon, particularly interferon-gamma (IFN-y). hADARI is also inducible by TNF-a. This provides an opportunity to develop combination therapy, whereby IFN-y or TNF-a and guide oligonucleotides as disclosed herein are administered to a patient either as a combination product, or as separate products, either simultaneously or subsequently, in any order.
  • IFN-y or TNF-a and guide oligonucleotides as disclosed herein are administered to a patient either as a combination product, or as separate products, either simultaneously or subsequently, in any order.
  • Certain disease conditions may already coincide with increased IFN-y or TNF-a levels in certain tissues of a patient, creating further opportunities to make editing more specific for diseased tissues. It will be understood by a person having ordinary skill in the art that the extent to which the editing entities inside the cell are redirected to other target sites may be regulated by varying the affinity of the first nucleic acid strand for the recognition domain of the editing molecule.
  • a guide oligonucleotide as disclosed herein can utilise endogenous cellular pathways and naturally available ADAR enzymes to specifically edit a target adenosine in the target RNA sequence.
  • Certain guide oligonucleotides as disclosed herein are capable of recruiting ADAR and complexing with it, which then facilitates the deamination of a (single) specific target adenosine nucleotide in a target RNA sequence to which it is bound.
  • only one adenosine is deaminated.
  • a guide oligonucleotide as disclosed herein, when complexed to ADAR, preferably brings about the deamination of a single target adenosine.
  • a guide oligonucleotide as disclosed herein is normally longer than 16 nucleotides. In one aspect the guide oligonucleotide as disclosed herein is longer than 20 nucleotides.
  • the guide oligonucleotide as disclosed herein is preferably shorter than 100 nucleotides, still more preferably shorter than 60 nucleotides, still more preferably shorter than 50 nucleotides. In a preferred aspect, the guide oligonucleotide as disclosed herein comprises 18 to 70 nucleotides, more preferably comprises 18 to 60 nucleotides, and even more preferably comprises 18 to 50 nucleotides.
  • the guide oligonucleotide as disclosed herein comprises 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
  • the guide oligonucleotide as disclosed herein is 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 nucleotides in length.
  • the guide oligonucleotide when it targets pre-mRNA and/or mRNA of the human RELN gene, has an a-symmetrical design, as shown in the accompanying examples.
  • the length of the 5’ part - seen from the orphan position that is position 0 - is 6, 7, or 8 nucleotides.
  • the length of the 3’ part - seen from the orphan position that is position 0 - is 16, 17, 18, 19, 20, 21 , 22, 23, or 24 nucleotides.
  • the guide oligonucleotides are not for use with editing systems that do not require a guide oligonucleotide. In an embodiment, the guide oligonucleotides are not for use in generating a Re/n-H3448R mutation in mice by homologous recombination. In an embodiment, the guide oligonucleotides are not for use in generating a mouse Re/n-H3448R mutation and/or a human REL/V-H3447R mutation by homologous recombination.
  • the guide oligonucleotides are not for use in generating a Re/n-H3448R-Tg knock in mouse model carrying the Reln-COLBOS variant via homologous recombination.
  • the nucleic acid editing enzyme does not edit using homologous recombination.
  • the nucleic acid editing enzyme does not generate a mouse Re/n-H3448R mutation and/or a human REL/V-H3447R mutation by homologous recombination.
  • the nucleic acid editing entity does not generate a Reln- H3448R-Tg knock in mouse model carrying the Reln-COLBOS variant by homologous recombination.
  • Example 1 RNA editing of a RELN transcript using different guide oligonucleotides.
  • H3447R codon encodes histidine (H) at amino acid position 3447 in the human reelin (H3447) protein and wherein the deamination from adenosine to inosine (Clll), would provide a codon that would translate to arginine (R) at this position.
  • the amino acid change is herein generally referred to as H3447R.
  • the sequence of the target codon as well its surrounding sequence in the human RELN DNA is provided in Fig. 1A.
  • the target sequence for deamination using the guide oligonucleotides of the present disclosure and the endogenous ADAR enzyme takes place on the transcript that is transcribed from the DNA and therefore the real target sequence comprises uridines (Il’s) instead of thymidine residues (T’s).
  • the sequence of Fig. 1A represents that target transcript where T’s are replaced by Il’s.
  • the sequences, designs, and chemical modifications of the guide oligonucleotides are provided in Figs. 2, 3, and 7. The chemical modifications are discussed in the brief description of the drawings.
  • human iPSC (WT04) derived neural progenitor cells were differentiated into mature cortical neurons using neural progenitor medium and generally according to protocols known to the person skilled in the art.
  • WT04 human iPSC derived neural progenitor cells
  • the following was performed.
  • the mature neurons were plated in 12-well plates in a concentration of 2.0x10 5 cells per well and allowed to expand until day 11.
  • Treatment with guide oligonucleotides was carried out with three different protocols, as follows:

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Abstract

La présente invention concerne des oligonucléotides guides qui sont au moins partiellement complémentaires à une partie d'une séquence d'acide nucléique RELN cible contenant un nucléotide cible, la séquence d'acide nucléique RELN cible codant pour une protéine de la rééline, l'oligonucléotide guide étant conçu de manière à pouvoir constituer un complexe double brin dans des conditions physiologiques à l'intérieur d'une cellule avec la partie de la séquence d'acide nucléique RELN cible, et le complexe double brin étant capable de recruter une entité d'édition d'acide nucléique pour effectuer l'édition du nucléotide cible afin de générer une séquence d'acide nucléotidique RELN éditée comprenant un nucléotide cible édité ; ainsi que des compositions, des vecteurs et des procédés d'utilisation s'y rapportant.
PCT/EP2024/066520 2023-06-16 2024-06-14 Oligonucléotides antisens pour le traitement des maladies neurodégénératives Pending WO2024256620A1 (fr)

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