Antisense oligonucleotides for the treatment of neurodegenerative diseases

Abstract

This disclosure relates to a guide oligonucleotide that is at least partially complementary to a portion of a target RELN nucleic acid sequence containing a target nucleotide, wherein the RELN nucleic acid sequence encodes a reelin protein, and the guide oligonucleotide is configured to form a double-stranded complex with the portion of the target RELN nucleic acid sequence under intracellular physiological conditions, and the double-stranded complex can mobilize nucleic acid editing entities to carry out editing of the target nucleotide to produce an edited RELN nucleic acid sequence containing the edited target nucleotide, as well as related compositions, vectors, and methods of use thereof.

Description

[Technical Field]

[0001] This disclosure relates to the field of medicine, particularly to neurodegenerative diseases such as Alzheimer's disease. This disclosure describes guide oligonucleotides that mediate nucleotide-specific editing in the RELN gene and / or encoding transcript, resulting in alterations of the encoding reelin protein that affect reelin protein activity. [Background technology]

[0002] Identifying effective treatments for neurodegenerative diseases such as Alzheimer's disease (AD) is an urgent and unmet medical need. AD is a progressive neurological disorder characterized by symptoms including dementia, cognitive decline, memory loss, and impairment of daily functioning. AD typically begins gradually and worsens progressively with age. Extensive research efforts have been made to identify treatments for AD, but have largely been unsuccessful in identifying effective treatments.

[0003] At the molecular level, both amyloid plaques, formed by the aggregation of amyloid precursor protein (APP), and so-called neurofibrillary tangles, composed of extensive phosphorylated tau protein deposits in the patient's brain, are characteristic features of Alzheimer's disease (AD). Tau protein is a crucial component of the cytoskeleton. Its normal function is to bind to tubulin and stabilize microtubule structures used by motor proteins in cells to organize cell transport. Highly phosphorylated tau is unable to perform this function very well, and tau dysregulation leads to neurodegenerative diseases such as parkinsonism and frontotemporal dementia, accompanied by disruption of neuronal migration and neuronal degeneration. Animal models created to express mutant tau variants develop neurodegeneration. Recent research (Lopera F et al. Nat Med. 2023, 29:1243-1252) has identified human subjects with amino acid mutations in the protein reelin encoded by the RELN gene that confer resilience to the development of symptoms from autosomal dominant Alzheimer's disease (ADAD). The effectiveness of this reelin variant in protecting against AD has been demonstrated in a mouse model.

[0004] Mechanistically, the protective effect of this reelin mutation can be reasonably explained by a model already hypothesized in 2002 (Ohkubo N et al. FASEB J. 2002, 17(2):295-297). According to this model, later refined by others, reelin is involved in tau phosphorylation through the apolipoprotein E receptor (APOEr) / disabled-1 (Dab1) / glycogen synthase kinase-3β (GSK3β) cascade. The binding of the reelin protein to the ApoE receptor and other cadherin-related neuronal receptors (CNRs) that cooperate with src family kinases as intracellular effector proteins phosphorylates Dab1, activating Dab1 and inhibiting two downstream kinases known to phosphorylate tau at identified sites in neurofibrillary tangles: GSK3β and CDK5. The outcome of this sequence, which involves the blockade of tau hyperphosphorylation and consequently the expected improvement or further normalization of tau function, supports the hypothesis that it is a gain-of-function aspect of the described reelin variant that results in the observed protection against ADAD.

[0005] It is inferred herein that other gain-of-function variants in reelin can be identified, and it is proposed that these gain-of-function variants be newly constructed using the methods described herein, with a view to producing clinically beneficial outcomes in patients with or prone to developing AD and other types of dementia, such as frontotemporal dementia, and / or neuropsychiatric disorders including schizophrenia, depression, autism, (temporal lobe) epilepsy, and / or other neurodegenerative disorders including parkinsonism, parkinson's disease, and spinocerebellar ataxia.

[0006] This disclosure aims to provide a guide oligonucleotide that can be used in the treatment of neurodegenerative diseases such as AD, wherein the guide oligonucleotide causes nucleic acid editing to occur by utilizing a nucleic acid editing mechanism to target and modify one or more target nucleotides in the RELN gene or encoding RELN transcript molecule, preferably premRNA and / or mRNA, thereby producing an edited RELN nucleic acid sequence. [Overview of the project]

[0007] This disclosure relates to a guide oligonucleotide that is at least partially complementary to a portion of a human RELN nucleic acid molecule containing a target nucleotide, wherein the RELN nucleic acid molecule encodes a reelin protein, and the guide oligonucleotide is configured to be able to form a double-stranded complex with the portion of the RELN nucleic acid under physiological conditions within a cell, preferably a brain cell, more preferably a neuron, and the double-stranded complex can carry out editing of the target nucleotide by recruiting a nucleic acid editing enzyme naturally present in the cell to produce an edited RELN nucleic acid containing the edited target nucleotide. Preferably, the editing of the target nucleotide results in increased activity of the encoding reelin protein. More preferably, the encoding reelin protein is given one or more of the following gain-of-function phenotypes: i) enhanced ability to trigger signaling, preferably in the APOEr / Dab1 / GSK3β pathway; ii) enhanced ability to increase Dab1 phosphorylation; iii) enhanced ability to reduce taur phosphorylation associated with neurofibrillary tangles; iv) enhanced ability to increase tubular structure formation and / or stability and / or neuron density; v) enhanced resistance to degradation by proteolysis; and / or vi) enhanced binding of the reelin protein to glycosaminoglycans, preferably heparin, and / or NRP1. In a further preferred embodiment, target nucleotide editing introduces an amino acid variant at one or more amino acid positions 3446-3460 of the encoding reelin protein, preferably, target nucleotide editing introduces a histidine-to-arginine change (H3447R) at amino acid position 3447 in the encoding reelin protein. Note that amino acid position 3447 is related to RELN isoform 203 (ensemble transcript ENST00000428762.6; RELN 203), and that RELN isoform 203 is 6 nucleotides longer than the transcript of isoform 201. In isoform 201, the histidine codon codes for amino acid 3445.For the sake of consistency, H3447R has been used throughout this disclosure, but the guide oligonucleotides disclosed herein can effect deamination of the target adenosine in both isoforms, providing amino acid changes, H3447R at 203 and H3445R at 201. Note that isoform 201 is the most abundant isoform of RELN present in the human iPSC forebrain neurons used in the attached examples, while isoform 203 is the second most abundant isoform present in these cells. Preferably, the target nucleotide is adenosine and the nucleic acid editing enzyme is an adenosine deaminase (ADAR) enzyme that acts on RNA. Preferably, the RELN nucleic acid molecule is mRNA or pre-mRNA.

[0008] In a preferred embodiment, the guide oligonucleotide

Table 1

[0009] This disclosure also relates to vectors, preferably viral vectors, more preferably adeno-associated virus (AAV) vectors, comprising nucleic acid molecules encoding the guide oligonucleotides disclosed herein. This disclosure also relates to the guide oligonucleotides disclosed herein for use in the treatment, alleviation, or slowing of the progression of neurodegenerative diseases, preferably AD, more preferably ADAD.

[0010] The disclosure also relates to a method for treating, alleviating, or slowing the progression of a neurodegenerative disease, preferably AD, more preferably ADAD, in a human subject in need, comprising administering a guide oligonucleotide disclosed herein to the subject, thereby editing a target RELN nucleic acid sequence to encode a reelin protein having the ability to delay the onset of one or more symptoms of the neurodegenerative disease.

[0011] One or more embodiments of the present disclosure are described below, by reference only to the accompanying drawings. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1A shows the nucleotide sequence in the 5' to 3' direction of a portion of the wild-type human RELN nucleic acid sequence (NCBI reference sequence: NM_005045.4), with the target adenosine (in bold font) indicated in the codon CAT (underlined) encoding histidine (H3447) at position 3447 in human reelin, along with the sequence number. Figure 1B shows the complementary sequence in the 3' to 5' direction of the nucleic acid sequence in Figure 1A, with the position of the orphan nucleotide (nucleotide opposite the target adenosine) indicated in bold font, along with the sequence number. Figure 1C shows the antisense sequence in the 5' to 3' direction of the nucleic acid sequence in Figure 1A, with the position of the orphan nucleotide (nucleotide opposite the target adenosine) indicated in bold font. It should be understood that if the target RELN nucleic acid is a premRNA or mRNA molecule, thymidine residues (T) should be read as uridine residues (U). Figure 1D is a diagram showing the transcript sequence from 5' to 3' of Figure 1A, representing the same portion of the RNA editing target sequence (SEQ ID NO: 106), with adenosine (in the middle of the underlined codon) shown in bold. It should also be understood that the orphan nucleotide in the guide oligonucleotide (disclosed herein) is preferably cytidine (C), a cytidine analog, uridine (U), or a uridine analog, rather than thymidine (T) as may be shown in Figures 1B and 1C. [Figure 2]Figure 2 shows the sequence of an example guide oligonucleotide disclosed herein. The chemical modifications in the guide oligonucleotide are as follows: Gm, Am, Um, and Cm are 2'-OMe modified guanosine, 2'-OMe modified adenosine, 2'-OMe modified uridine, and 2'-OMe modified 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 ("Te"; sometimes named 2'-MOE modified thymidine); Af, Uf Gf and Cf are 2'-F modified adenosine, 2'-F modified uridine, 2'-F modified guanosine, and 2'-F modified cytosine, respectively; Zd is a cytidine analog, also called a nucleoside, having a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base (a Benner base outlined herein) with a deoxy moiety (=DNA) at the 2'-ribose position; Id is deoxyinosine; * indicates a PS linkage; ! indicates a PNdmi linkage; ^ indicates an MP linkage; and θ indicates a PO linkage. [Figure 3] Figure 3 shows the sequence of one set of additional guide oligonucleotides, along with their respective RM names and sequence numbers. The chemical modifications are as provided in Figure 2. [Figure 4] Figure 4 shows the percentage of target adenosine editing in RELN target (pre)mRNA obtained after transfection with the guide oligonucleotides RM116817-RM116840 (shown below the graph) in human iPSC (WT04)-derived neural progenitor cells, 2 days post-transfection. A negative (untreated) control is also included (mock). [Figure 5]Figure 5 shows the percentage of target adenosine editing in RELN target (pre)mRNA obtained after gymnosis incorporation of the guide oligonucleotides shown below the graph, seven days after the start of gymnosis treatment with each guide oligonucleotide in human iPSC (WT04)-derived neural progenitor cells, as shown in Figure 1D. Negative (untreated; NT) controls are also included. [Figure 6] Figure 6 shows the percentage of target adenosine editing in RELN target (pre)mRNA obtained after gymnosis / saponin uptake of the guide oligonucleotide shown below the graph and co-treatment with triterpene glycoside AG1856 (saponin) in human iPSC (WT04)-derived neural progenitor cells, 7 days after the start of gymnosis / saponin treatment. A negative (untreated; NT) control was also included. [Figure 7-1] Figure 7 shows the sequences of further sets of guide oligonucleotides, along with their respective RM numbers and sequence numbers. RM118550–RM118867 are designed based on the sequence and modifications of oligonucleotide RM116835 (also known as sequence number 58, G3447-19), while RM118868–RM118880 are designed based on the sequence and modifications of oligonucleotide RM116838 (also known as sequence number 61, G3447-22). Chemical modifications are as provided in Figure 2, where # indicates PNms linkage. [Figure 7-2] Figure 7 shows the sequences of further sets of guide oligonucleotides, along with their respective RM numbers and sequence numbers. RM118550–RM118867 are designed based on the sequence and modifications of oligonucleotide RM116835 (also known as sequence number 58, G3447-19), while RM118868–RM118880 are designed based on the sequence and modifications of oligonucleotide RM116838 (also known as sequence number 61, G3447-22). Chemical modifications are as provided in Figure 2, where # indicates PNms linkage. [Figure 8-1]Figure 8 shows the percentage of target adenosine editing shown in Figure 1D in RELN target (pre)mRNA obtained after gymnosis uptake of the guide oligonucleotide shown in Figure 7 and simultaneous treatment with triterpene glycoside AG1856 (saponin) in human iPSC (WT04)-derived neural progenitor cells, 7 days after the start of gymnosis / saponin treatment. Figure 8A shows the results using a forward primer specific to the transcript sequence of isoform 201, and Figure 8B shows the results using a forward primer specific to the transcript of isoform 203. [Figure 8-2] Figure 8 shows the percentage of target adenosine editing shown in Figure 1D in RELN target (pre)mRNA obtained after gymnosis uptake of the guide oligonucleotide shown in Figure 7 and simultaneous treatment with triterpene glycoside AG1856 (saponin) in human iPSC (WT04)-derived neural progenitor cells, 7 days after the start of gymnosis / saponin treatment. Figure 8A shows the results using a forward primer specific to the transcript sequence of isoform 201, and Figure 8B shows the results using a forward primer specific to the transcript of isoform 203. [Modes for carrying out the invention]

[0013] This disclosure is believed to be the first disclosure herein of guide oligonucleotides capable of driving the editing of a target RELN nucleic acid sequence. Such guide oligonucleotides can be used as therapeutic agents to treat, alleviate, or slow the progression of neurodegenerative diseases such as AD. For example, it has been identified that a change of just one amino acid in the reelin protein may be sufficient to induce or enhance a protective pathway that slows the progression of AD. By targeting the RELN DNA sequence in a (pre)mRNA transcript molecule, this technology operates at the gene level. Thus, this disclosure opens up an entirely new field of use for specific gene editing technologies for the treatment of neurodegenerative diseases. Gene editing technologies are not particularly limited. Suitable technologies include DNA editing technologies such as Cas9-based technologies, as well as known gene therapy technologies that utilize guide oligonucleotides, including RNA editing technologies such as ADAR-mediated editing technologies. Both DNA editing technologies and RNA editing technologies have advantages and disadvantages. For example, DNA editing gene therapy can cause permanent changes in DNA molecules and therefore may only require a single treatment for a particular disorder. In certain circumstances, it may not be necessary or desirable to cause irreversible changes to DNA. In this case, RNA editing has the advantage of being transient; that is, only RNA is edited, and eventually the modified protein is produced. However, if the guide oligonucleotide is destroyed by metabolic processes and new mRNA is generated, the "old" version of the protein will be produced again. Depending on the type and severity of the injury, an appropriate mode of alteration of the nucleic acid encoding the reelin protein may be selected.

[0014] Embodiment According to a first aspect, the disclosure provides a guide oligonucleotide that is at least partially complementary to a portion of a human RELN nucleic acid molecule containing a target nucleotide, wherein the RELN nucleic acid molecule encodes a reelin protein, and the guide oligonucleotide is configured to form a double-stranded complex with the portion of the RELN nucleic acid under intracellular physiological conditions, and the double-stranded complex can carry out editing of the target nucleotide by recruiting a nucleic acid editing enzyme naturally present in the cell to produce an edited RELN nucleic acid containing the edited target nucleotide.

[0015] In some embodiments, editing of the target nucleotide preferably results in increased activity of an encoding reelin protein, selected from one or more of the following: - Enhancement of the ability to trigger signaling, preferably via the APOEr / Dab1 / GSK3β pathway; - Enhanced ability to increase Dab1 phosphorylation; - Enhanced ability to reduce taurinary tangle-related taurinary tangles; - Enhancement of the ability to form and / or stabilize tubular structures and / or increase neuronal density; - Enhanced resistance to degradation by proteolysis; and / or - Enhancement of the binding of reelin protein to glycosaminoglycans, preferably heparin, and / or NRP1.

[0016] In some embodiments, target nucleotide editing introduces an amino acid variant at one or more amino acid positions 3446-3460 of the encoding reelin protein, preferably, target nucleotide editing introduces a histidine-to-arginine change (H3447R) at amino acid position 3447 in the encoding reelin protein.

[0017] In some embodiments, the cells are brain cells, preferably neurons.

[0018] In some embodiments, the target nucleotide is adenosine, and the nucleic acid editing enzyme is ADAR enzyme.

[0019] In some embodiments, the RELN nucleic acid molecule is mRNA or premRNA.

[0020] In some embodiments, the orphan nucleotide is a nucleotide in a guide oligonucleotide opposite the target nucleotide, such that the nucleotide numbering is such that the orphan nucleotide is number 0, the nucleotides are positively (+) incremented toward the 5' end and negatively (-) incremented toward the 3' end, and at least one nucleic acid base, sugar, or nucleoside linkage is chemically modified. In some embodiments, the orphan nucleotide is a deoxycytidine, a cytidine analog, a deoxyuridine, or a uridine analog. The cytidine analog is preferably a deoxynucleotide containing a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base (also referred to herein and elsewhere as a “Benner base” or Z). The uridine analog is preferably a deoxynucleotide containing an isouracil nucleic acid base. In some embodiments, the guide oligonucleotide has a length of 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.

[0021] In some embodiments, the guide oligonucleotide is [Table 2] Includes.

[0022] In some embodiments, the guide oligonucleotide comprises one or more internucleoside linkage modifications, each independently selected from phosphorothioates (PS), phosphonoacetates, phosphorodithioates, methylsulfonates (MP), sulfonyl phosphoramidates, (1,3-dimethylimidazolidinedine-2-ylidene)phosphoamidates (PNdmi), or linkage modifications having a structure according to the following formula (I):

[0023] [ka] (wherein X=O or S; and R=aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C) 20 Alkyl, substituted C1-C 20 (Alkyl, C1-C6 alkenyl, C1-C6 substituted alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugate group). In preferred embodiments, X=O and R=methyl. In those embodiments, the linkage modification is generally called a mesylphosphoramidate (PNms).

[0024] In some embodiments, the nucleoside ligation numbering in the guide oligonucleotide is such that ligation number 0 is a ligation on the 5' side from the orphan nucleotide, and the ligation positions in the oligonucleotide are positively (+) incrementing toward the 5' end and negatively (-) incrementing toward the 3' end, with ligation position -2 being an MP or PNms ligation.

[0025] In some embodiments, the guide oligonucleotide comprises one or more monosubstituted or disubstituted nucleotides at the 2', 3', and / or 5' positions of ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, which may be interrupted by one or more heteroatoms, in a linear or branched lower (C1-C) chain. 10)alkyl, alkenyl, alkynyl, alkalyl, allyl, or aralkyl;-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;-dimethylaminooxyethoxy; and-dimethylaminoethoxyethoxy.

[0026] In some embodiments, a portion of the target RELN nucleic acid sequence is sequence numbers 1 and 2: [Table 3] The 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 derived from a nucleotide sequence selected from the above, and includes at least the underlined nucleotide region, where A in bold is the target nucleotide, the nucleic acid editing entity is ADAR, preferably human ADAR1 and / or human ADAR2, and more preferably the ADAR enzyme is spontaneously expressed in the cell (=endogenous).

[0027] In some embodiments, editing of the target nucleotide results in increased expression levels, increased activity, and / or increased stability of the reelin protein.

[0028] In some embodiments, the nucleic acid editing entity is a nucleic acid editing enzyme, preferably a deaminase enzyme, more preferably an adenosine deaminase such as human ADAR1 (hADAR1) and human ADAR2 (hADAR2), or a cytidine deaminase enzyme.

[0029] In some embodiments, nucleic acid editing entities are spontaneously expressed in cells (i.e., endogenous to the cell).

[0030] In some embodiments, the target RELN nucleic acid sequence is spontaneously expressed within the cell.

[0031] In some embodiments, the target RELN nucleic acid sequence is DNA.

[0032] In some embodiments, the nucleic acid editing entity is selected from the list including: Cas9 enzyme; base editor enzyme; dCas9-deaminase enzyme; dCas9-adenosine deaminase enzyme; dCas9-cytidine deaminase enzyme; prime editing enzyme; or Cas9 nickase enzyme.

[0033] In some embodiments, the linkage between the two terminal nucleotides at the 5' and / or 3' ends of the guide oligonucleotide is a PNdmi linkage or a PNms linkage, preferably both terminal links are PNms linkages.

[0034] In some embodiments, the first nucleotide (-1) from the 3' end of the orphan nucleotide is deoxyinosine.

[0035] In some embodiments, the guide oligonucleotide is: [Table 4] It includes 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 derived from a nucleotide sequence selected from, and includes at least the underlined nucleotide region.

[0036] In some embodiments, the guide oligonucleotide comprises at least the underlined region of SEQ ID NO: 6, where the orphan nucleotide (the C in bold in the underlined region) is a cytidine analog, preferably a deoxynucleotide having a Benner base (Zd instead of C; see International Patent Application Publication No. 2022 / 252376), and nucleotide position-1 is deoxyinosine (Id).

[0037] In some embodiments, the guide oligonucleotide includes at least the underlined region of SEQ ID NO: 6, where the orphan nucleotide (the C in bold in the underlined region) is deoxyuridine and nucleotide position-1 is deoxyinosine (Id).

[0038] In some embodiments, the guide oligonucleotide comprises at least the underlined region of SEQ ID NO: 6, where the orphan nucleotide (the C in bold in the underlined region) is a deoxynucleotide having a uridine analog, preferably isouracil, and nucleotide position-1 is deoxyinosine (Id).

[0039] In some embodiments, the guide oligonucleotide comprises at least the underlined region of SEQ ID NO: 6, where the orphan nucleotide (the C in the middle of the bold underlined region) is a deoxynucleotide having a Benner base (Zd instead of C), nucleotide position -1 is Id, nucleotide position +1 is deoxyadenosine (Ad), or adenosine (Ae) in which the 2' position of ribose is substituted with 2'-O-methoxyethyl (also called 2'-methoxyethoxy, 2'-O-MOE, or simply 2'-MOE), and preferably, ligation position -2 is an MP or PNms ligation, more preferably a PNms ligation.

[0040] In some embodiments, the guide oligonucleotide comprises at least the underlined region of SEQ ID NO: 6, the orphan nucleotide (the C in the middle of the bold underlined region) is a deoxynucleotide having a Benner base (Zd instead of C), nucleotide position -1 is Id, the length of the 5' portion immediately adjacent to the orphan nucleotide is 6, 7, or 8 nucleotides, and the length of the 3' portion immediately adjacent to the orphan nucleotide is at least 16 nucleotides, more preferably 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0041] In some embodiments, the guide oligonucleotide includes the following structure (5'→3'): N8N7N6N5N4N3N2N1 θ ZdId^M2M3M4M5M6M7M8M9M 10 M 11 M 12 M 13 M 14 M 15 M 16 M 17 M 18 M 19 M 20 M 21 M 22 M 23 M 24 (In the array, - Zd is an orphan nucleotide that is a deoxynucleotide having a Benner base at nucleotide position 0; - N1 is either Ae or Ad; - N2 is Af; - N3 and N5 are independently Am or Af; - N4 is Gf; - N6 is Uf; - N7 is either nonexistent (and if so, N8 also does not exist), Gm, or Gf; - N8 is either nonexistent or Um; - Id is deoxyinosine; - M2 is Um; - M3 is Cf; - M4, M 14 , and M 15 These are independently m5Ue or Um; - M5 and M7 are Gf; - M6 is Am or Af; - M8 and M 10 These are independently Cm or Cf; - M9 is Cf; - M 11 is Am; - M 12 is Um; - M 13is Gm; - M 16 is either Ge or Gm; - M 17 either does not exist (if so, M 18 ~M 24 also does not exist), or is m5Ue, or is Um; - M 18 either does not exist (if so, M 19 ~M 24 also does not exist), or is Cm, or is m5Ce; - M 19 either does not exist (if so, M 20 ~M 24 also does not exist), or is Gm, or is Ge; - M 20 either does not exist (if so, M 21 ~M 24 also does not exist), or is Um, or is m5Ue; - M 21 either does not exist (if so, M 22 ~M 24 also does not exist), or is Gm, or is Ge; - M 22 either does not exist (if so, M 23 and M 24 also does not exist), or is Am, or is Ae; - M 23 either does not exist (if so, M 24 also does not exist) or is Ae; - M 24 either does not exist or is Ae; - θ is at the linking position 0 and is a PO linking or a PNms linking; - ^ is at the linking position -2 and is an MP or a PNms linking; - All other linkings are either a PO linking, a PS linking, a PNdmi linking, or a PNms linking; and Gm, Am, Um, and Cm are 2'-O-methyl(2'-OMe) modified guanosine, 2'-OMe modified adenosine, 2'-OMe modified uridine, and 2'-OMe modified 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 ("Te"; sometimes named 2'-MOE modified thymidine); Af, Uf, Gf, and Cf are 2'-F modified adenosine, 2'-F modified uridine, 2'-F modified guanosine, and 2'-F modified cytosine, respectively.

[0042] In some embodiments, the guide oligonucleotide comprises or consists of any one sequence of SEQ ID NOs: 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. Preferably, the guide oligonucleotide comprises or consists of any one sequence of SEQ ID NOs: 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.

[0043] In some embodiments, the guide oligonucleotide is conjugated to a triterpene glycoside, preferably AG1856.

[0044] According to a second aspect, 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 to a first aspect of the Disclosure.

[0045] According to a third aspect, the Disclosure provides a pharmaceutical composition comprising a guide oligonucleotide according to a first aspect of the Disclosure or a vector according to a second aspect of the Disclosure, and a pharmaceutically acceptable carrier.

[0046] According to a fourth aspect, the present disclosure provides guide oligonucleotides according to a first aspect of the present disclosure, vectors according to a second aspect of the present disclosure, or pharmaceutical compositions according to a third aspect of the present disclosure for use in the treatment, alleviation, or slowing of the progression of neurodegenerative diseases, preferably AD, more preferably ADAD.

[0047] According to a fifth aspect, the Disclosure provides the use of a guide oligonucleotide according to a first aspect of the Disclosure, a vector according to a second aspect of the Disclosure, or a pharmaceutical composition according to a third aspect of the Disclosure for use in the manufacture of pharmaceuticals for treating, alleviating, or slowing the progression of neurodegenerative diseases, preferably AD, more preferably ADAD.

[0048] According to a sixth aspect, the Disclosure provides a method for treating, alleviating, or slowing the progression of a neurodegenerative disease, preferably AD, more preferably ADAD, in a human subject in need thereof, the method comprising the steps of administering to the subject a guide oligonucleotide according to a first aspect of the Disclosure, or a vector according to a second aspect of the Disclosure, or a pharmaceutical composition according to a third aspect of the Disclosure, thereby editing a target RELN nucleic acid sequence to encode a reelin protein having the ability to delay the onset of one or more symptoms of a neurodegenerative disease, preferably AD, more preferably ADAD.

[0049] According to a seventh aspect, the present disclosure provides an in vitro, ex vivo, or in vivo method for deamination of a target adenosine in a target RELN nucleic acid sequence in a cell, comprising the steps of (i) supplying a cell with a guide oligonucleotide according to a first aspect of the present disclosure, or a vector according to a second aspect of the present disclosure, or a pharmaceutical composition according to a third aspect of the present disclosure; (ii) enabling the uptake of the guide oligonucleotide or vector or composition by the cell; (iii) enabling the annealing of the guide oligonucleotide to a target RELN nucleic acid sequence; and (iv) enabling a nucleic acid editing entity to edit the target.

[0050] In some embodiments, the method includes the step of identifying the presence of an edited target nucleotide using functional readout information.

[0051] In some embodiments, the method includes a step of administering a triterpene glycoside before, after, or simultaneously with the step of administering a guide oligonucleotide. In some embodiments, the triterpene glycoside is AG1856. In preferred embodiments, the triterpene glycoside, such as AG1856, is (non)covalently bonded to the guide oligonucleotide to enable enhanced endosomal escape once the guide oligonucleotide has entered the target cell, which requires deamination of the target nucleotide to occur.

[0052] According to an eighth aspect, the present disclosure provides a method for editing a human RELN nucleic acid sequence in cells, preferably brain cells, wherein the human RELN nucleic acid sequence is a premRNA or mRNA, and the method comprises the step of contacting a target RELN nucleic acid sequence with a guide oligonucleotide capable of triggering ADAR-mediated adenosine-to-reelin deamination, thereby editing the target RELN nucleic acid sequence to encode a reelin protein having the ability to delay the onset of one or more symptoms of a neurodegenerative disease, preferably AD, more preferably ADAD.

[0053] According to the ninth aspect, the disclosure provides a guide oligonucleotide for editing a target adenosine in a human RELN premRNA or mRNA molecule by supplying a guide oligonucleotide which hybridizes with a human RELN premRNA or mRNA molecule, thereby enabling the ADAR enzyme to attract and deaminate the target adenosine, wherein the target region is SEQ ID NO: 106, and the target adenosine is the second nucleotide of the codon encoding histidine at position 3447 of the RELN-encoding human reelin protein. In some embodiments of the ninth aspect, the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 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. In some embodiments of the ninth aspect, the nucleic acid molecule comprises at least one non-naturally occurring chemical modification and / or one or more additional non-naturally occurring chemical modifications in the ribose, ligation, or base portion, provided that the orphan nucleotide, which is a nucleotide in the nucleic acid directly opposite the target adenosine in the target region, is not a cytidine containing a 2'-OMe ribose substitution.

[0054] definition Guide oligonucleotides as referred to herein are known or may be referred to as antisense oligonucleotides (AONs). They may also be called “editing oligonucleotides” or “EONs,” even though the editing event is carried out by a nucleic acid editing entity and the action of the oligonucleotide merely triggers the editing to occur. Whenever guide oligonucleotides, oligonucleotides, oligos, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA)-editing oligonucleotides, EONs, and RNA (antisense) oligonucleotides are referred to, both oligoribonucleotides and deoxyribonucleotides are meant unless otherwise indicated in the context. Potentially, oligonucleotides may be completely devoid of RNA and DNA nucleotides (where they appear naturally) and may be complete from modified nucleotides. Whenever “oligoribonucleotides” are referred to, this may include the bases A, G, C, U, or I. Whenever “deoxyribonucleotides” are referred to, this may include the bases A, G, C, T, or I. However, guide oligonucleotides disclosed herein may include mixtures of ribonucleotides and deoxyribonucleotides. When deoxyribonucleotides are used and therefore unmodified at the 2' position of the sugar, the nucleotide is often abbreviated as dA (or Ad), dC (or Cd), dG (or Gd), or T (where "d" indicates the deoxygenation of the nucleoside), while ribonucleosides, which are either normal RNA or modified at the 2' position, are often abbreviated without "d" and often with their respective modifications as described herein.

[0055] The term “nucleoside” refers to a nucleic acid base linked to a (deoxy)ribosyl sugar without a phosphate group. A “nucleotide” consists of a nucleoside and one or more phosphate groups. Therefore, the term “nucleotide” refers to each nucleic acid base-(deoxy)ribosyl-phospholinker, as well as any chemical modification of the ribose moiety or phospho group. Thus, the term includes nucleotides, for example, locked ribosyl moieties (including 2'-4' crosslinks containing a methylene group or any other group), unlocked nucleic acids (UNAs), threose nucleic acids (TNAs), phosphodiesters (POs), phosphonoacetates, phosphotryesters, PSs, phosphoro(di)thioates, MPs (or MePs), methylthiophosphonates, phosphoramidate linkages, PNdmis, and nucleotides containing linkages with the structure of formula (I) as described herein. In some cases, the terms nucleic acid base, nucleoside, and nucleotide are used interchangeably, except when it is clearly required by the context to be otherwise, for example, when a nucleoside is linked to an adjacent nucleoside and the linkage between these nucleosides is modified. As described herein, a nucleotide is a nucleoside plus one or more phosphate groups. The terms “ribonucleoside” and “deoxyribonucleoside,” or “ribose” and “deoxyribose,” are used as they are used in the art.

[0056] In some cases, the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, inosine and hypoxanthine are used interchangeably, on the one hand referring to the corresponding nucleic acid base, and on the other hand referring to the nucleoside or nucleotide. The nucleic acid base thymine (T) is also known as 5-methyluracil (m 5 Thymine and 5-methyluracil, also known as 5-methyluridine, are uracil (U) derivatives; they are interchangeable throughout this document. Similarly, the nucleotide thymidine, also known as 5-methyluridine, is a uridine derivative; thymidine and 5-methyluridine are interchangeable throughout this document.

[0057] Whenever referring to nucleotides in oligonucleotides such as cytosine, this includes 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine. Whenever referring to adenine, this includes N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine, and 7-methyladenine. Whenever referring to uracil, this includes dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudracil, 4-thiouracil, and 5-hydroxymethyluracil. Whenever referring to guanine, this includes 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. Whenever nucleosides or nucleotides are mentioned, other modifications include ribofuranose derivatives, e.g., 2'-deoxy, 2'-hydroxy, and 2'-O-substituted mutants, e.g., 2'-OMe, as well as 2'-4' crosslinking mutants. Whenever oligonucleotides are mentioned, one or more linkages may be naturally occurring phosphodiester linkages, while the remaining linkages between the two mononucleotides may be modified linkages. Examples of such modified linkages include phosphonoacetates, phosphotriesters, PS, phosphoro(di)thioates, MP, phosphoramidate linkages, phosphorylguanidine, thiophosphorylguanidine, sulfonophosphoramidates, PNdmi, and linkage structures by formula (I), which are further outlined in detail below.

[0058] The term "comprising" encompasses "including" and "consisting of." For example, a composition "comprising X" may be exclusive to X or may include something additional, such as X + Y. The term "approximately" in relation to a numerical value x is optional and means, for example, x ± 10%.

[0059] The term “substantially” does not exclude “completely”; for example, a composition “substantially containing Y” may contain Y completely. Where relevant, the term “substantially” may be omitted from the definitions in this disclosure or the claims.

[0060] The terms “contribute” or “mediate” can be used interchangeably with “enhance.” When used in relation to guide oligonucleotides that contribute to (or mediate) ADAR editing, this means that, after entering the cell, the guide oligonucleotide interacts with the target RNA sequence, thereby forming a double-stranded structure that is recognized by the ADAR enzyme, which can then deaminate the target adenosine into RNA. Thus, while the guide oligonucleotide itself does not possess enzymatic function (as the ADAR enzyme does), after binding to the target RNA molecule, it can trigger, induce, cause, organize, mediate, supply, give, generate, enhance, and / or result in RNA editing.

[0061] The term “mismatch” is used herein to refer to opposing nucleotides in a double-stranded RNA complex that do not form a perfect base pair according to the Watson-Crick base pairing rules. In a historical sense, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the guide oligonucleotide contains fewer than four mismatches with the target sequence, e.g., zero, one, or two mismatches. “Fluctuating” base pairs are GU, IU, IA, and IC base pairs. If U is placed opposite target A, there is no mismatch, and the guide oligonucleotide may be 100% complementary. If C is placed opposite target A, there is at least one mismatch between the guide oligonucleotide and the target sequence. A G:G pairing would be considered a mismatch, but this does not necessarily mean the interaction is unstable, meaning that a Hoogsteen base pairing may appear as a mismatch based on the origin of the nucleotides, but is still relatively stable, and therefore, based on the current disclosure, the term “mismatch” may be somewhat outdated. For example, isolated G:G pairings in double-stranded RNA are highly stable, yet they can still be defined as mismatches. Analysis of native targets of ADAR enzymes has shown that these generally contain mismatches between the two strands forming the RNA helix edited by ADAR1 or 2. These mismatches have been suggested to enhance the specificity of the editing reaction (Stefl et al. 2006. Structure 14(2):345-355, Tian et al. 2011. Nucleic Acids Res 39(13):5669-5681). Characterizing the optimal pattern of paired / mismatched nucleotides between guide oligonucleotides and target RNA also appears to be important for the development of efficient ADAR-based AON therapies.

[0062] As used herein, the term “complementary” refers to the fact that a guide oligonucleotide hybridizes with a second nucleic acid chain under physiological conditions. Examples include (i) when the guide oligonucleotide, as the first nucleic acid chain (=guide oligonucleotide), forms a heterodouble-stranded RNA editing oligonucleotide complex with a second complementary nucleic acid chain (in vitro), or (ii) when it forms a double-stranded complex with the target RNA molecule. This term does not necessarily mean that each nucleotide in the nucleic acid chain has a perfect pairing with its counterpart nucleotide in the opposing sequence. In other words, a guide oligonucleotide may be complementary to the target sequence, but there may be mismatches, fluctuations, and / or bulges between the guide oligonucleotide and the target sequence, while under physiological conditions, the guide oligonucleotide may still hybridize with the target sequence, as a result of the cellular RNA editing enzyme deaminating the target adenosine to inosine. Therefore, the term “substantially complementary” also means that, despite the presence of mismatches, fluctuations, and / or bulges, the guide oligonucleotide has nucleotides that match the target sequence well enough to hybridize with the target RNA molecule under physiological conditions. As shown herein, a guide oligonucleotide may be complementary to the target sequence, but may also contain one or more mismatches, fluctuations, and / or bulges, and under physiological conditions, the guide oligonucleotide can hybridize with its target.

[0063] The term "orphan nucleotide" refers to a nucleotide in a guide oligonucleotide that directly opposes a target adenosine, where the target adenosine is adenosine that is deaminated by a deaminationase. An orphan nucleotide may be a natural cytidine or deoxycytidine, or a uridine or deoxyuridine. It may also be a chemically modified nucleotide, as further detailed below, or a known or chemically modified analog of natural (deoxy)cytidine, for example, a nucleotide having the Benner base (6-amino-5-nitro-3-yl-2(1H)-pyridone), or a known or chemically modified analog of natural (deoxy)uridine, as further outlined below, for example, isouridine.

[0064] A "nucleotide analog" refers to an analog of a nucleic acid nucleotide. Nucleotide analogs are analogs of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine.

[0065] In relation to nucleic acid sequences, the term "downstream" means further along the sequence in the 3' direction, and the term "upstream" means the opposite. Therefore, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand, but downstream of the stop codon in the antisense strand. The same is true for the guide oligonucleotides disclosed herein. In antisense guide oligonucleotides, nucleotides upstream of the orphan nucleotide are located towards the 5' end, and nucleotides downstream of the orphan nucleotide are located towards the 3' end.

[0066] The nucleotide "numbering" in the guide oligonucleotides disclosed herein is such that the orphan nucleotide is numbered 0, and the nucleotide 5' from the orphan nucleotide is numbered +1. The counting further increases positively (+) toward the 5' end and decreases negatively (-) toward the 3' end, with the first nucleotide 3' from the orphan nucleotide being numbered -1. The internucleoside ligation numbering in the guide oligonucleotides is such that ligation number 0 is the ligation 5' from the orphan nucleotide, and the ligation position in the oligonucleotide further increases positively (+) toward the 5' end and decreases negatively (-) toward the 3' end.

[0067] The term "hybridization" typically refers to specific hybridization, excluding nonspecific hybridization. Specific hybridization can be performed using techniques well known in the art under selected experimental conditions to ensure that the most stable interaction between the probe and target occurs when the probe and target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0068] The term "splicing mutation" refers to a mutation in a gene encoding an mRNA precursor that causes dysfunction in the splicing mechanism, meaning that the splicing of introns from exons is disrupted due to abnormal splicing, subsequent translation is out of frame, and the encoded protein terminates prematurely. Such shortened proteins are often rapidly degraded and have no functional activity.

[0069] Whenever the “naked” form relating to the guide oligonucleotides disclosed herein is referred to, it means that the guide oligonucleotides are manufactured in a laboratory or manufacturing facility and therethrough, through which they are generally chemically modified to prevent rapid degradation after they enter the mammalian body or tissue or cell upon administration. Thus, the naked form of a guide oligonucleotide is different from the form in which the guide oligonucleotide is encoded (and delivered) by a viral genome or within a plasmid vector. When such a viral vector or plasmid vector is administered, 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 consists solely of naturally occurring nucleotides, preferably naturally occurring RNA nucleotides.

[0070] In particular, if the guide oligonucleotide includes the chemical modifications detailed herein, it can still be delivered by means of a delivery vehicle. Suitable delivery vehicles are nanoparticle delivery vehicles such as polymer nanoparticles, dendrimers, inorganic nanoparticles and nanocrystals, organic nanocrystals, and liposomes. Preferred nanoparticles are lipid nanoparticles (LNPs), which are nanosized lipid vehicles having the guide oligonucleotides of this disclosure and assisting in the delivery of target cells. When an LNP or any other similar type of carrier is applied, the guide oligonucleotide is still considered naked because it is not transcribed from the polynucleotide encoding it (for example, in the case of a plasmid or vector where the guide oligonucleotide is not considered "naked"). Thus, although a chemically modified guide oligonucleotide is encapsulated by a carrier, preferably an LNP, it is still considered naked because it is manufactured as such in a laboratory setting and then encapsulated in a carrier using methods known to those skilled in the art. This disclosure also relates to delivery vehicles, preferably LNPs, containing the "naked" and chemically modified guide oligonucleotides disclosed herein. Those skilled in the art will understand that when a delivery portion or attachment to a guide oligonucleotide is used (for example, a GalNAc portion that can target hepatocytes in the liver, and / or is attached / conjugated with a saponin as discussed herein), the guide oligonucleotide is still seen naked, just as when the saponin-guide is encapsulated within a delivery vehicle such as an LNP.In other words, a variety of non-restrictive administration methods are feasible: i) the guide oligonucleotide as is; ii) the guide oligonucleotide encapsulated in a delivery vehicle, preferably an LNP; iii) the guide oligonucleotide administered with or separately from a saponin such as AG1856 (but not bound to it); iv) the guide oligonucleotide conjugated with a saponin such as AG1856; v) the guide oligonucleotide conjugated with a saponin such as AG1856, wherein the saponin-guide conjugate is encapsulated in a delivery vehicle, preferably an LNP; or vi) the guide oligonucleotide through an encoding vector such as a plasmid or viral vector into which it is transcribed. The method of administration will be selected depending on the disease target and the cells that need to be targeted, but such a method is preferably administration in the form of the guide oligonucleotide, whether conjugated or not with a delivery portion (or endosomal escape agent), or whether encapsulated or not in a delivery vehicle such as an LNP.

[0071] The length of the guide oligonucleotides disclosed herein, when delivered in their naked form, 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. However, if the guide oligonucleotides disclosed herein are to be delivered through the expression of a viral vector, the guide oligonucleotides may also be longer, for example, 70, 80, 90, 100, 150, or 200 or more nucleotides in length.

[0072] The term "HEON" refers to a heteroduplex double-stranded complex molecule in which a guide oligonucleotide disclosed herein is hybridized with a partially or completely complementary, partially or completely overlapping sense oligonucleotide. Because the guide oligonucleotides disclosed herein often have specific chemical modifications different from those on the sense strand, the two strands form a heteroduplex RNA editing oligonucleotide complex. The sense strand may be chemically modified almost entirely, similar to or different from those carried out in the guide oligonucleotides disclosed herein, by supplying nucleotides having ribose sugar moieties with, for example, 2'-OMe substitutions, 2'-F substitutions, or 2'-MOE substitutions. It should be understood that the sense strand present in HEON is a distinct entity from the target RNA molecule in the cell. The sense strand in 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. HEON is often produced in vitro and used as a delivery tool to protect guide oligonucleotides from degradation when administered to cells. In other words, HEON is preferably formed before the guide oligonucleotide is administered to cells.

[0073] RELN nucleic acid sequence This disclosure relates to a guide oligonucleotide that mediates the editing of one or more target nucleotides present in a target RELN nucleic acid sequence. The RELN gene encodes the protein reelin. Preferably, the guide oligonucleotide mediates the editing of one or more target nucleotides present in a human RELN nucleic acid sequence. Particularly preferred is that the human RELN nucleic acid sequence is present in a human cell and that the editing occurs in that cell.

[0074] In some embodiments, the target nucleotide is any nucleotide whose editing results in a reelin protein having one or more of the following: a gain-of-function phenotype; enhanced ability to upregulate reelin-induced signaling pathways; enhanced ability to increase Dab1 phosphorylation, decrease taur phosphorylation, and / or increase neuronal density; and / or enhanced binding of the reelin protein to glycosaminoglycans, preferably heparin, and / or NRP1.

[0075] In some embodiments, the target nucleotide is any nucleotide whose edited target nucleotide produces a structural effect on any of the amino acids in the “α-GAG binding site,” “β-GAG binding site,” and / or neuropilin 1 (NRP1) binding site of the reelin protein. The α-GAG binding site spans six C-terminal amino acids (3455-3460) of the human reelin protein. The β-GAG binding site spans amino acids 3446-3451 of the human reelin protein. The target nucleotide to be edited may be in a codon for an amino acid outside these sites, provided that the mutation of that amino acid produces an effect within one of these sites. Preferably, the target nucleotide is in a codon encoding an amino acid within one of these sites. It is particularly preferable that the target nucleotide is in a codon encoding one of amino acids 3446-3460 of the human reelin protein, preferably one of amino acids 3446-3451 or 3455-3460 of the human reelin protein.

[0076] In particularly preferred embodiments, the target nucleotide is located within the codon for amino acid 3447 of the human reelin protein. Specifically, the target RELN nucleic acid sequence codes for an amino acid other than arginine at position 3447 of the human reelin protein, preferably histidine at position 3447 of the human reelin protein. In these embodiments, it is preferable that the edited RELN nucleic acid sequence codes for arginine at position 3447 of the human reelin protein, resulting in an altered protein variant referred to herein as H3447R.

[0077] In a particularly preferred embodiment, the target nucleotide is adenosine, and the nucleic acid editing entity is adenosine deaminase. In a further preferred embodiment, the target RELN nucleic acid sequence containing the target nucleotide is a RELN RNA transcript molecule (pre-mRNA and / or mRNA) containing the target adenosine, and the nucleic acid editing entity is an ADAR enzyme, more preferably human ADAR1 and / or human ADAR2. In these embodiments, it is preferable that the RELN RNA transcript molecule has a codon CAU encoding histidine at position 3447 of the human reelin protein, and the adenosine in the CAU codon is the target adenosine. In this embodiment, ADAR-mediated editing produces a CIU, and the translation mechanism interprets the CIU as a CGU encoding arginine. As such, in some embodiments, it is preferable that the cell is a human cell having reelin-encoding DNA having an amino acid other than arginine, preferably histidine, at position 3447 of the human reelin protein.

[0078] In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having 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 having the ability to upregulate or induce pathways that are protective against AD and / or one or more symptoms of AD. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having an enhanced ability to bind to glycosaminoglycans (GAGs), particularly in the C-terminal region of reelin. It is particularly preferred that the edited RELN nucleic acid sequence encodes a reelin protein having an enhanced ability to bind to heparin. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having an enhanced ability to bind to neurophilin 1. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having an enhanced ability to reduce tau pathology. In particular, in some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having an enhanced ability to reduce tau phosphorylation. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having an enhanced ability to increase Dab1 phosphorylation. In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having the ability to increase neuronal density.

[0079] In some embodiments, the edited RELN nucleic acid sequence encodes a reelin protein having a gain-of-function phenotype. Examples of gain-of-function variants include, in addition to the variants described in the examples, variants having: 1) enhanced ability to trigger signaling, preferably in the APOEr / Dab1 / GSK3β pathway; 2) enhanced ability to increase Dab1 phosphorylation; 3) enhanced ability to reduce taur phosphorylation found in neurofibrillary tangles; 4) enhanced ability to increase microtubule formation and / or microtubule stability and / or neuron density; 5) enhanced resistance to degradation by proteolysis; and / or enhanced binding to glycosaminoglycans, preferably heparin; and / or 6) enhanced binding to NRP1.

[0080] A preferred embodiment involves a reelin protein gaining function through reduced protease-mediated inactivation. For example, ADAMTS-3 has been identified as a protease that cleaves and inactivates reelin in the brain (e.g., the cerebral cortex and hippocampus). Knockdown of ADAMTS-3 in mice has been shown to reduce taur phosphorylation and increase dendritic branching and elongation (Ogino H et al., J Neurosci. 2017, 37(12):3181-3191). Therefore, altering the Pro-Ala sequence at the ADAMTS-3 cleavage site in reelin, for example, at the Nt site (Koie M et al., J Biol Chem. 2014, 289:12922-12930), is a viable strategy to upregulate reelin and generate reelin with a gain-of-function phenotype. This disclosure relates to various guide oligonucleotides intended for the deamination of 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. While we do not wish to be bound by theory, it is possible that synergistic or additive effects may be obtained by combining multiple target nucleotides, such as target adenosine, and the guide oligonucleotides disclosed herein to target multiple amino acids within a single reelin protein, thereby increasing the therapeutic effect.

[0081] Neurodegenerative diseases A target nucleotide is a nucleotide whose editing results in an edited RELN nucleic acid sequence that encodes an edited reelin protein having beneficial therapeutic effects compared to an unedited reelin protein. In particular, beneficial therapeutic effects may include the treatment, improvement, or reduction of one or more symptoms of neurodegenerative diseases such as AD, or of one or more symptoms of such diseases, such as mild cognitive impairment, cognitive decline, and / or dementia.

[0082] In some embodiments, the guide oligonucleotide is used in subjects diagnosed with AD. In other embodiments, the guide oligonucleotide is used prophylactically in subjects identified as being at risk of developing AD. In some embodiments, the guide oligonucleotide is used prophylactically in subjects with healthy cognitive function. In some embodiments, the guide oligonucleotide is used in subjects having the PSEN1-E280A mutation (PSEN1 is the gene encoding presenilin 1), which is a mutation associated with the development of mild cognitive impairment in AD. In some embodiments, the guide oligonucleotide is used in subjects having two copies of the APOE3 Christchurch (APOECh) (R136S) gene variant. In some embodiments, the guide oligonucleotide is used in subjects with ADAD.

[0083] In some embodiments, the cells are brain cells, preferably neurons. In some embodiments, the cells are located in the medial temporal lobe, preferably the heterocortex, more preferably the entorhinal cortex. In some embodiments, editing occurs in or around the endoplasmic reticulum of the cells.

[0084] DNA editing In some embodiments, this disclosure relates to “DNA editing.” DNA editing techniques compatible with those disclosed herein include DNA editing techniques based on the CRISPR-Cas9 nuclease enzyme. These techniques are well known in the art. These techniques involve the use of CRISPR-Cas9 to introduce double-strand breaks into DNA and can be programmed to occur at a specific site by using a guide RNA oligonucleotide having the necessary sequence to guide the CRISPR-Cas9 enzyme to the specific site. Such breaks can be used to remove, modify, and / or insert DNA sequences at a specific site in DNA. Another system derived from CRISPR-Cas9 is the use of a base editor (BE) system, which uses catalytically dead Cas9 (dCas9) fused with a functional enzyme such as DNA deaminase. dCas9 does not introduce double-strand breaks into DNA, but instead positions the DNA deaminase to produce deamination of a target DNA nucleotide, which is again guided to a specific site by a guide oligonucleotide. DNA deaminases can be cytidine deaminases (which induce C-to-T substitutions) or adenine deaminases (which induce A-to-G substitutions). dCas9 fused with cytidine deaminase enzymes is also known as a cytidine base editor (CBE), and dCas9 fused with adenine deaminase enzymes is also known as an adenine base editor (ABE). A further development is known as prime editing. Prime editing uses Cas9 nickases fused with reverse transcriptase. In this case as well, prime editing uses guide oligonucleotides to direct the enzyme to a specific site in the DNA. Prime editing also uses oligonucleotides containing prime editing guide RNA (pegRNA) that contains the primer binding site sequence and the sequence containing the desired edit. This may be part of the same molecule as the guide oligonucleotide. First, Cas9 nickases guided by the guide oligonucleotide cause a cleavage at a specific region on one strand of DNA.Next, reverse transcriptase uses pegRNA as a template for reverse transcription to transfer the edited sequence to DNA.

[0085] RNA editing In some embodiments, this disclosure relates to "RNA editing." RNA editing is a natural process by which eukaryotic cells alter the sequence of their RNA molecules, often in a site-specific and precise manner, thereby increasing the repertoire of RNA encoded by the genome by several orders of magnitude. RNA editing enzymes have been described for eukaryotes across the animal and plant kingdoms, and these processes play a crucial role in maintaining cellular homeostasis in metazoans, from the simplest organisms (such as nematodes (Caenorhabditis elegans)) to humans. Examples of RNA editing include the conversion of adenosine to inosine (A to I) and the conversion of cytidine to uridine, which occur through enzymes called adenosine deaminases acting on RNA (ADAR) and APOBEC / AID (cytidine deaminases acting on RNA), respectively.

[0086] ADAR is a multi-domain protein containing one catalytic domain and, depending on the enzyme, two to three double-stranded RNA recognition domains. Each recognition domain recognizes a specific double-stranded RNA (dsRNA) sequence and / or three-dimensional structure. While the catalytic domain also plays a role in recognizing and binding to portions of the dsRNA helix, its primary function is to convert A to I at a predetermined position nearby in the target RNA by deamination of nucleic acid bases. Inosine is read as guanosine by the cell's translation mechanism, meaning that if edited adenosine is in the coding region of mRNA or mRNA precursor, this can recode the protein sequence. Interestingly, the A-to-I conversion can also occur in the 5' non-coding sequence of the target mRNA, creating a new translation initiation site upstream of the initial start site, resulting in an N-terminus elongated protein, or in the 3' untranslated region (UTR) or other non-coding portion of the transcript, potentially affecting RNA processing and / or stability. Furthermore, the conversion from A to I occurs in the splicing elements of introns or exons in the mRNA precursor, thereby altering the splicing pattern. As a result, exons may be included or skipped. Enzymes that catalyze adenosine deamination are located within the ADAR enzyme family, which includes the human deaminationases hADAR1 and hADAR2, as well as hADAR3. However, deamination activity of hADAR3 has not been demonstrated.

[0087] The use of guide oligonucleotides (or antisense oligonucleotides; AON or EON) to edit target RNA and apply adenosine deaminase has been described (e.g., Woolf et al. Proc Natl Acad Sci USA. 1995, 92:8298-8302; Montiel-Gonzalez et al. Proc Natl Acad Sci USA. 2013, 110(45):18285-18290; Vogel et al. Angewandte Chemie 2014, Int Ed 53:267-271). A disadvantage of the method described by Montiel-Gonzalez et al. (2013) is the need for a fusion protein consisting of the box B recognition domain of bacteriophage lambda N-protein fused with the adenosine deaminose enzyme domain of the cleaved native ADAR protein. This requires either transducing the target cells using a fusion protein (which is a major obstacle) or transposing the target cells using a nucleic acid construct encoding a modified adenosine deaminose fusion protein for expression. The system described by Vogel et al. (2014) suffers from a similar drawback in that it is unclear how this system can be applied without first genetically modifying ADAR and then transposing or transforming cells containing the target RNA to provide the cells with this genetically modified protein. U.S. Patent No. 9,650,627 describes a similar system. The oligonucleotide by Woolf et al. (1995), which is 100% complementary to the target RNA sequence, suffered from a significant lack of specificity: almost all adenosine in the target RNA strand complementary to the guide oligonucleotide was edited out.

[0088] ADARs are known to act on any dsRNA. Through a process sometimes called "promiscuous editing," the enzyme edits multiple A atoms in the dsRNA. Therefore, there has been a need for methods and means to avoid such promiscuous editing and target only specific adenosines in the target RNA molecule to be therapeutically available. Vogel et al. (2014) showed that such off-target editing can be suppressed by using a 2'-OMe modified nucleoside in the guide oligonucleotide at a position opposite to the adenosine that should not be edited, and using an unmodified nucleoside directly opposite the specific targeted adenosine on the target RNA. However, it has not been shown that a specific editing effect at the target nucleotide occurs without the use of recombinant ADAR enzymes covalently bonded to the guide oligonucleotide. Currently, several publications have shown that the recruitment of endogenous ADARs (and therefore without the need for exogenous and / or recombinant sources) is possible while maintaining specificity to target a single adenosine in the target RNA molecule and deaminate it to inosine. International Publication No. 2016 / 097212 discloses an AON for targeted editing of RNA, characterized by the presence of a sequence complementary to the target RNA sequence (referred to as the “targeting region”) and a stem-loop / hairpin structure (referred to as the “recruiting region”) which is preferably complementary to the target RNA. Such oligonucleotides are called “self-loop-forming AONs.” The recruiting region acts to recruit a cellular (i.e., endogenously present) native ADAR enzyme to a dsRNA formed by hybridization of the target sequence and the targeting region. Thanks to the recruiting region, the presence of a conjugated entity or a modified recombinant ADAR enzyme is not required. International Patent Application Publication No. 2016 / 097212 describes the recruiting region as a Z-DNA structure known to be recognized by the native substrate of the ADAR enzyme (e.g., the GluB receptor) or the dsRNA-binding domain, or a stem-loop structure that mimics either the Z-DNA-binding domain or the native substrate of the ADAR enzyme (e.g., the GluB receptor).The stem-loop structure may be an intermolecular stem-loop structure formed by two separate nucleic acid strands, or an intramolecular stem-loop structure formed within a single nucleic acid strand. The described stem-loop structure of the recruiting moiety is an intramolecular stem-loop structure formed within the AON itself and is thought to attract (endogenous) ADAR. Systems containing similar stem-loop structures for RNA editing are subsequently described in International Patent Applications Publications 2017 / 050306, 2020 / 001793, 2017 / 010556, U.S. Patent No. 11,390,865, International Publication 2020 / 246560, and 2022 / 078995.

[0089] International Patent Application Publications 2017 / 220751 and 2018 / 041973 describe next-generation AONs that do not include such stem-loop structures, are (almost completely) complementary to the target region, and are still considered capable of attracting endogenous ADAR enzymes. In one embodiment, one or more mismatched nucleotides, fluctuations, or bulges are present between the oligonucleotide and the target sequence. A single mismatch may be at the nucleoside site opposite the target adenosine, while in other embodiments, the AON is described to have multiple bulges and / or fluctuations when attached to the target sequence region. It was considered possible to achieve in vitro, ex vivo, and in vivo RNA editing using AONs lacking stem-loop structures, and using endogenous ADAR enzymes if the sequence of the AON is carefully selected to attract / mobilize ADARs. An "orphan nucleoside," defined as a nucleoside in a guide oligonucleotide (or AON) positioned directly opposite the target adenosine in the target RNA molecule, was a nucleotide having an unmodified cytosine nucleic acid base without 2'-OMe modification. The orphan nucleoside could be a deoxyribonucleoside (DNA), the rest of the guide oligonucleotide could still have 2'-O-alkyl modifications (e.g., 2'-OMe) on the sugar entity, or the nucleotide directly surrounding the orphan nucleoside could contain further chemical modifications (e.g., DNA compared to RNA) that improved RNA editing efficiency and / or increased resistance to nucleases. Furthermore, such effects could be further enhanced by using a sense oligonucleotide (SON) that "protects" the AON from degradation during delivery to cells (as described in International Patent Application Publication 2018 / 134301 and U.S. Patent No. 11,274,300).

[0090] The use of chemical modifications and specific structures in oligonucleotides that can be used in the editing of specific adenosines in ADAR-mediated target RNAs has been the subject of numerous disclosures in the art, for example, in International Patent Application Publications 2019 / 111957, 2019 / 158475, 2020 / 165077, 2020 / 201406, 2020 / 211780, 2021 / 008447, and 2021 / 0205 These include specifications No. 50, No. 2021 / 060527, No. 2021 / 117729, No. 2021 / 136408, No. 2021 / 182474, No. 2021 / 216853, No. 2021 / 242778, No. 2021 / 242870, No. 2021 / 242889, No. 2022 / 007803, No. 2022 / 018207, No. 2022 / 026928, and No. 2022 / 124345, among others. The use of specific sugar moieties is disclosed, for example, in International Patent Application Publication Nos. 2020 / 154342, 2020 / 154343, 2020 / 154344, 2022 / 103839, and 2022 / 103852, while the use of sterically defined linker moieties (generally, for example, for exon skipping related to a wide variety of target sequences, in gapmers, in siRNA, or for oligonucleotides that can be specifically used for RNA editing oligonucleotides) is disclosed in International Patent Application Publication No. 2011 / 0057. Specification No. 61, Specification No. 2014 / 010250, Specification No. 2014 / 012081, Specification No. 2015 / 107425, No. 2017 / 015 Specification No. 575 (HTT), Specification No. 2017 / 062862, Specification No. 2017 / 160741, Specification No. 2017 / 192664, No. 201 Specification No. 7 / 192679 (DMD), Specification No. 2017 / 198775, Specification No. 2017 / 210647, Specification No. 2018 / 067973 Specification No. 2018 / 098264, Specification No. 2018 / 223056 (PNPLA3), Specification No. 2018 / 223073 (APOC3),Specification No. 2018 / 223081 (PNPLA3), Specification No. 2018 / 237194, Specification No. 2019 / 032607 (C9orf72), Specification No. 2019 / 055951, Specification No. 2019 / 075357 (SMA / ALS), Specification No. 2019 / 200185 Specification (DM1), Specification No. 2019 / 217784 (DM1), Specification No. 2019 / 219581, Specification No. 2020 / 118246 (DM1), Specification No. 2020 / 160336 (HTT), Specification No. 2020 / 191252, Specification No. 2020 / 196662, No. It is described in the following specifications: 2020 / 219981 (USH2A), 2020 / 219983 (RHO), 2020 / 227691 (C9orf72), 2021 / 071788 (C9orf72), 2021 / 071858, 2021 / 178237 (MAPT), 2021 / 234459, 2021 / 237223, 2022 / 099159, 2021 / 030778, 2022 / 174053, and 2023 / 278589.

[0091] Following these disclosures, a great number of publications relate to the targeting of specific RNA target molecules, or specific adenosine within such RNA target molecules, to repair mutations that result in immature stop codons, or other mutations that cause disease. Examples of such disclosures in which adenosine is targeted within a designated target RNA molecule include International Patent Application Publications 2020 / 157008 and 2021 / 136404 (USH2A), 2021 / 113270 (APP), 2021 / 113390 (CMT1A), 2021 / 209010 (IDUA, Hurler syndrome), 2021 / 231673 and 2021 / 242903 (LRRK2), 2021 / 231675 (ASS1), and 2021 / 23167 These are Specification No. 9 (GJB2), Specification No. 2019 / 071274 and Specification No. 2021 / 231680 (MECP2), Specification No. 2021 / 231685 and Specification No. 2021 / 231692 (OTOF, autosomal recessive non-syndromic hearing loss), Specification No. 2021 / 231691 (XLRS), Specification No. 2021 / 231698 (argininosuccinate deficiency), Specification No. 2021 / 130313 and Specification No. 2021 / 231830 (ABCA4), and Specification No. 2021 / 243023 (serpine A1).

[0092] It is particularly preferable that ADAR1 and / or ADAR2 are endogenously present in the cell. This is because such guide oligonucleotides, after binding to the target RNA molecule, can mediate the RNA editing of target adenosine present in the target RNA molecule, and a deaminationase is recruited to the double-stranded oligonucleotide / target RNA molecule complex, subsequently deaminating the target adenosine to inosine.

[0093] There is a constant need to improve the pharmacokinetic properties of guide oligonucleotides without negatively affecting the efficiency of target adenosine editing at target RNA and / or negatively affecting the stability of the guide oligonucleotide itself, which is constantly susceptible to degradation by nucleases present in native cells. Many chemical modifications are available for the production of oligonucleotides (and many chemical modifications have been applied in the art). However, many of these properties do not always align with the desire to achieve efficient RNA editing. In the search for better pharmacokinetic properties, it was previously found that 2'-MOE modification of ribose at some, but not all, nucleotides surprisingly appears to align with efficient ADAR association and editing (International Patent Application Publication 2019 / 158475). In a similar form, it was previously found that PS linkage at some, but not all, internucleoside linkages surprisingly appears to align with efficient ADAR association and editing (International Patent Application Publication 2019 / 219581). Furthermore, it has been previously found that phosphonoacetate linkage modifications and / or UNA-ribose modifications at some, but not all, positions in the guide oligonucleotide appear to be compatible with the efficient association and subsequent deamination of enzymes having nucleotide deamination activity (International Patent Application Publication 2020 / 165077). While the properties of phosphonoacetate and UNA modifications were known in this way, their compatibility with association and deamination reactions with enzymes having nucleotide deamination activity was unknown.

[0094] In some embodiments, the Disclosure provides guide oligonucleotides that can induce (mediate, induce, or trigger) RNA editing of a target adenosine in a target transcript molecule, such as premRNA and / or mRNA. The target transcript molecule may be encoded by a mutant gene, the mutation of which is the cause of the disease, and the editing reverses the mutation to produce a wild-type protein or a protein with wild-type function (for example, if the mutant amino acid is changed to an amino acid that does not cause the disease or gives an improved phenotype). As disclosed in more detail herein, the target transcript molecule may also be encoded by a wild-type gene, as in a preferred embodiment of the Disclosure, and the target RELN nucleic acid molecule is a transcript from a wild-type human RELN gene, as shown herein. In particular, the RNA editing encodes a modified reelin protein that improves the disease condition of the subject being treated. In particular, the target RELN nucleic acid sequence is a sequence that is naturally present in the subject. In other words, the target RELN nucleic acid sequence is the sequence before treatment with the guide oligonucleotide according to the Disclosure.

[0095] Non-limiting examples of transcript molecules targeted using RNA editing for various treatments include SERPINA1 (for the treatment of alpha-1 antitrypsin (A1AT) deficiency; see, for example, International Patent Application Publications 2016 / 097212, 2017 / 220751, 2018 / 041973, and 2021 / 243023), IDUA (for the treatment of Hurler syndrome; see, for example, International Patent Application Publications 2017 / 220751, 2018 / 041973, and 2021 / 209010). See, for example, International Patent Publication Nos. 2016 / 097212, 2017 / 220751, 2018 / 041973, 2021 / 231673, and 2021 / 242903), ABCA4 (for the treatment of Stargardt disease; for example, see International Patent Publication Nos. 2021 / 130313 and 2021 / 231830), USH2A (for the treatment of Usher syndrome; for example, International Patent Publication No. 2020 / 157008) See publications 2020 / 219981 and 2021 / 136404), APP (see, for example, International Patent Application Publication 2021 / 113270), CMT1A (see, for example, International Patent Application Publication 2021 / 113390), ASS1 (see, for example, International Publication 2021 / 231675), GJB2 (see, for example, International Patent Application Publication 2021 / 231679), MECP2 (for the treatment of Rett syndrome; see, for example, International Patent Application Publication 2019 / 071274 and 2021 / 2 These include 31680, OTOF (for the treatment of autosomal recessive non-symptomatic hearing loss; see, for example, International Patent Application Publications 2021 / 231685 and 2021 / 231692), XLRS (see, for example, International Patent Application Publication 2021 / 231691), PCSK9 (for the treatment of hypercholesterolemia; see, for example, International Publication 2023 / 152371), and HFE (for the treatment of hemochromatosis / iron overload; see, for example, International Patent Application Publication 2024 / 110565).

[0096] chemical modification Various chemicals and modifications that can be readily used in accordance with this disclosure are known in the field of oligonucleotides. The chemical modifications listed herein may be used in relation to guide oligonucleotides intended for DNA editing or RNA editing, as necessary and / or unless otherwise noted. All chemical modifications listed herein that may be used in guide oligonucleotides such as those disclosed herein may also be used in sense strands that are complementary to the guide oligonucleotide, except that the opposing sense strand does not have an orphan nucleotide, when the guide oligonucleotide and its complementary strand form a HEON complex, as described in and disclosed above in International Patent Application Publication No. 2024 / 084048. Thus, modifications related to orphan nucleotides relate not only to guide oligonucleotides such as those disclosed herein, but all other modifications relate to the guide oligonucleotides disclosed herein and any (protective) sense oligonucleotides that may be used together with the guide oligonucleotide in pharmaceuticals. This includes the use of hydrophobic moieties (e.g., tocopherol and cholesterol) that can bind to either the guide oligonucleotide or its opposing strand or both.

[0097] Those skilled in the art will know that oligonucleotides, such as guide oligonucleotides outlined herein, generally consist of repeating monomers. Such monomers are most frequently nucleotides or chemically modified nucleotides. The most common naturally occurring nucleotides in RNA are adenosine monophosphate (A), cytidine monophosphate (C), guanosine monophosphate (G), and uridine monophosphate (U). These consist of a pentose, a ribose, a 5'-linked phosphate group linked via a phosphate ester, and a 1'-linked base. The sugar links the base and phosphate and is therefore often called the “scaffold” of the nucleotide. Thus, modifications in the pentose are often called “scaffold modifications.” The original pentose may, in its whole form, be replaced by another part that links as well as the base and phosphate. Thus, it will be understood that while the pentose is often the scaffold, the scaffold is not necessarily a pentose. Examples of scaffold modifications that can be applied to guide oligonucleotide monomers, such as those disclosed herein, are disclosed in International Patent Application Publications 2020 / 154342, 2020 / 154343, and 2020 / 154344.

[0098] Nucleosides in guide oligonucleotides as disclosed herein may be natural nucleosides (deoxyribonucleotides or ribonucleosides) or non-natural nucleosides. For RNA editing, where double-stranded RNA is generally a substrate for enzymes with deamination activity (e.g., ADAR), ribonucleosides are considered "natural," while deoxyribonucleosides may, for convenience of discussion, be considered non-natural or modified, simply because DNA does not exist in the substrate configuration of an RNA-RNA double-stranded (natural) molecule. Those skilled in the art will understand that even if a nucleotide has a natural ribose moiety, it can still be non-naturally modified in its base and / or ligation.

[0099] Common limiting factors in oligonucleotide-based therapies are the ability of oligonucleotides to be taken up by cells (when delivered on their own or "naked" without the application of a delivery vehicle such as a viral vector or plasmid), their distribution in vivo, and their resistance to nuclease-mediated degradation. Those skilled in the art know that various chemical modifications can help overcome such limitations, and these have been described in detail in the art. Examples of such chemical modifications now commonly used are the 2'-OMe, 2'-F, and 2'-MOE modifications of sugars and the use of PS linkages between nucleosides, as described herein.

[0100] Ribose Modification Except for the ribose sugar portion of orphan nucleotides, which have certain limitations in terms of compatibility with RNA editing, the ribose 2' group of all nucleotides in guide oligonucleotides as disclosed herein can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4' linked (e.g., locked nucleic acid (LNA)), or other ribosyl 1'-substitutions, 2'-substitutions, 3'-substitutions, 4'-substitutions, or 5'-substitutions. Orphan nucleotides in guide oligonucleotides that do not contain ribose sugar, bases, or other chemical modifications to linkages preferably do not have 2'-OMe or 2'-MOE substitutions and may have 2'-F, 2',2'-difluoro (diF), or 2'-ala-F (FANA) substitutions, or may be DNA, if their nucleic acid base is naturally occurring cytosine. International Patent Application Publication No. 2024 / 013360 describes the modification of the 2' position of the ribose sugar moiety of orphan nucleotides by 2',2'-disubstituted substitutions such as diF, and the modification is also applicable to those disclosed herein. The 2'-4' linkage can be selected from many linkers known in the art, such as a methylene linker, an amide linker, or a restricted ethyl linker (cEt).

[0101] Guide oligonucleotides as disclosed herein may comprise one or more nucleotides having a 2'-MOE ribose modification. Alternatively, guide oligonucleotides as disclosed herein may comprise one or more nucleotides that do not have a 2'-MOE ribose modification, or where the 2'-MOE ribose modification is located at a position that does not interfere with an enzyme having adenosine deaminase activity to deaminate target adenosine. Guide oligonucleotides as disclosed herein may comprise a 2'-OMe ribose modification at a position that does not contain a 2'-MOE ribose modification. Guide oligonucleotides as disclosed herein may comprise a deoxyribonucleotide at a position that does not contain a 2'-MOE ribose modification, a 2'-OMe ribose modification, or any other 2'-ribose substitution. Guide oligonucleotides as disclosed herein may comprise one or more nucleotides including 2' substitutions such as 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, TNA, 2'-fluoro(2'-F), 2',2'-difluoro(diF) modification, 2'-fluoro-2'-C-methyl modification, or 2'-4' linkages (i.e., crosslinking nucleic acids such as LNA, or, for example, the example referred to in International Patent Application Publication No. 2018 / 007475). Other nucleic acid monomers that may be used in guide oligonucleotides as disclosed herein include, for example, arabino nucleic acids and 2'-deoxy-2'-fluoroarabino nucleic acids (FANA) for improved affinity. The 2'-4' linkage can be selected from linkers known in the art, for example, methylene linkers or restricted ethyl linkers. A wide variety of 2' modifications that may be present in guide oligonucleotides, such as those disclosed herein, are known in the art, including, but are not limited to, those outlined in detail in International Patent Application Publications 2016 / 097212, 2017 / 220751, 2018 / 041973, 2018 / 134301, 2019 / 219581, 2019 / 158475, and 2022 / 099159.In all cases, the modification should be adapted to RNA editing such that the guide oligonucleotide can perform its role as an oligonucleotide, capable of forming a double-stranded complex with the target RNA, thereby recruiting a deaminationase, which in turn can deaminate the target adenosine. If the monomer in a guide oligonucleotide as disclosed herein contains a UNA ribose modification, the monomer may have a 2' position containing the same modifications discussed above, such as 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, 2'-F, 2',2'-diF, 2'-fluoro-2'-C-methyl, arabino nucleic acid, FANA, or a 2'-4' linkage (i.e., a cross-linked nucleic acid such as LNA). In some embodiments, a guide oligonucleotide as disclosed herein contains at least one nucleotide containing a threose nucleic acid (TNA) ribose modification. In some embodiments, a guide oligonucleotide as disclosed herein contains at least one nucleotide having a sugar moiety containing a 2'-F modification. The preferred position for nucleotides having a 2'-F modification is position-3 in the guide oligonucleotide, and this modification may coexist with the same 2' modification in orphan nucleotides as discussed above.

[0102] Base modification The bases, sometimes called nucleic acid bases, are generally adenine, cytosine, guanine, thymine, or uracil, or their derivatives. A nucleic acid base is defined as a moiety that can bond with another nucleic acid base through hydrogen bonds, polar bonds (e.g., through the CF moiety), or aromatic-electrical interactions. Cytosine, thymine, and uracil are pyrimidine bases and are generally linked to a scaffold through their 1-nitrogen. Adenine and guanine are purine bases and are generally linked to a scaffold through their 9-nitrogen. As used herein, the terms “adenine,” “guanine,” “cytosine,” “thymine,” “uracil,” and “hypoxanthine” refer to the nucleic acid base itself. The terms “adenosine,” “guanosine,” “cytidine,” “thymidine,” “uridine,” and “inosine” refer to the nucleic acid base linked to a (deoxy)ribosyl sugar. The nucleic acid bases in guide oligonucleotides as disclosed herein may be adenosine, cytosine, guanine, thymine, or uracil, or any other moiety, which can interact with other nucleic acid bases through hydrogen bonds, polar bonds (e.g., CF), or aromatic-electrical interactions.Nucleic acid bases at any position in guide oligonucleotides as disclosed herein include adenine, cytosine, guanine, or modified forms of uracil, e.g., hypoxanthine (nucleic acid base in inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseuduracil, orotic acid, agmatidine, lysidine, 2-thiouracil, 2-thiothymine, 5-substituted pyrimidines (e.g., 5-halouracil, 5-halomethyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-formyluracil, 5-aminomethylcytosine, 5-formylcytosine), 5-hydroxymethylcytosine, 7-deazaguanine, 7-deazaadenine, 7-deaza-2, These may be 6-diaminopurines, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurines, 8-oxo-adenine, 3-deazapurines (e.g., 3-deaza-adenosine), pseudoisocytosine, N4-ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2-aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamps and their derivatives, super A, super T, super G, amino-modified nucleic acid bases or their derivatives, and degenerate or universal bases such as 2,6-difluorotoluene, or may not exist, such as debase sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, azaribose). Modified bases include synthetic and natural bases known or to be known in the art, such as inosine, xanthine, hypoxanthine, and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, and thioalkyl derivatives of pyrimidine and purine bases. Purine nucleotide bases and / or pyrimidine nucleotide bases may be modified to alter their properties, for example, by heterocyclic amination or deamination.The exact chemistry and form may vary between oligonucleotide constructs and applications, and may be formulated according to the wishes and preferences of those skilled in the art.

[0103] Scaffold modifications refer to the presence of modified versions of naturally occurring ribosyl moieties in RNA (i.e., pentose moieties), such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2'-modified sugars, 4'-modified sugars, 5'-modified sugars, and 4'-substituted sugars. Examples of appropriate modifications include 2'-O-modified RNA monomers, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, e.g., 2'-OMe, 2'-O-(2-cyanoethyl), 2'-MOE, 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(2-aminopropyl), 2'-O-(2-(dimethylamino)propyl), 2'-O-(2 -amino)ethyl, 2'-O-(2-(dimethylamino)ethyl), 2'-deoxy(DNA), 2'-O-(haloalkyl)methyl, e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM), 2'-O-alkoxycarbonyl, e.g., 2'-O-[2-(methoxycarbonyl)ethyl](MOCE), 2'-O-[2-N-methylcarbonyl] [Bamoyl)ethyl](MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl](DCME), 2'-halo, e.g., 2'-F, FANA, 2'-O-[2-(methylamino)-2-oxoethyl](NMA), bicyclic or cross-linked nucleic acid (BNA) scaffold modifications, e.g., structurally restricted nucleotide (CRN) monomers, LNA monomers, xylo-LNA monomers, α-LNA monomers, α -l-LNA monomer, β-d-LNA monomer, 2'-amino-LNA monomer, 2'-(alkylamino)-LNA monomer, 2'-(acylamino)-LNA monomer, 2'-N-substituted 2'-amino-LNA monomer, 2'-thio-LNA monomer, (2'-O,4'-C)-restricted ethyl (cEt)BNA monomer, (2'-O,4'-C)-restricted methoxyethyl (cMOE)BNA monomer, 2',4'-BNA NC(NH) monomer, 2’,4’-BNA NC (NMe) monomer, 2’,4’-BNA NC (NBn) monomer, ethylene-bridged nucleic acid (ENA) monomer, carbocyclic-LNA (cLNA) monomer, 3,4-dihydro-2H-pyran nucleic acid (DpNA) monomer, 2’-C-bridged bicyclic nucleotide (CBBN) monomer, oxo-CBBN monomer, heterocyclic-bridged BNA monomer (e.g., triazolyl or tetrazolyl linkage), amide-bridged BNA monomer (e.g., AmNA), urea-bridged BNA monomer, sulfonamide-bridged BNA monomer, bicyclic carbocyclic nucleotide monomer, TriNA monomer, α-l-TriNA monomer, bicyclo DNA (bcDNA) monomer, F-bcDNA monomer, tricyclo DNA (tcDNA) monomer, F-tcDNA monomer, alpha-anomer bicyclo DNA (abcDNA) monomer, oxetane nucleotide monomer, locked PMO monomer derived from 2’-amino LNA, guanidine-bridged nucleic acid (GuNA) monomer, spirocyclopropyl-bridged nucleic acid (scpBNA) monomer, and derivatives thereof, cyclohexenyl nucleic acid (CeNA) monomer, altritol nucleic acid (ANA) monomer, hexitol nucleic acid (HNA) monomer, fluorinated HNA (F-HNA) monomer, pyranosyl-RNA (p-RNA) monomer, 3’-deoxypyranosyl DNA (p-DNA), UNA, and inverted versions of any of the above monomers, but not limited thereto. All of these modifications are known to those skilled in the art.

[0104] Orphan nucleotide Mutagenicity studies of human ADAR2 have shown that a single mutation from glutamate to glutamine at residue 488 (E488Q) increases the deamination rate constant by 60-fold compared to the wild-type enzyme (Kuttan and Bass. Proc Natl Acad Sci USA. 2012, 109(48):3295-3304). During the deamination reaction, ADAR flips the base to be edited from its RNA double helix and places it within the enzyme's active site (Matthews et al. Nat Struct Mol Biol 2016. 23(5):426-433). When ADAR2 edits adenosine in a favorable situation (A:C mismatch), the nucleotide opposite the target adenosine is often called an "orphan nucleotide" (or sometimes "orphan cytidine"), as indicated above. Crystal structure analysis of ADAR2 E488Q bound to double-stranded RNA (dsRNA) reveals that the glutamine (Gln;Q) side chain at position 488 can contribute to hydrogen bonding with the N3 position of orphancytidine, leading to an increase in the catalytic rate of ADAR2 E488Q. In the wild-type enzyme, where glutamate (or glutamic acid;Glu;E) is present at position 488 instead of glutamine (Gln), the amide group of glutamine is absent, replaced by a carboxylic acid. To achieve the same contact between orphancytidine and the E488Q mutant, protonation would likely be required in the wild-type environment for this contact to occur. To utilize endogenously expressed ADAR2 to correct disease-related mutations, maximizing the editing efficiency of the wild-type ADAR2 enzyme present in cells is essential. International Patent Application Publication No. 2020 / 252376 discloses the use of a guide oligonucleotide having a modified RNA base at the position of an orphancytidine to mimic the hydrogen bonding pattern observed, in particular, by the E488Q ADAR2 variant.It was conceived that by replacing the nucleotide opposite the target adenosine in the guide oligonucleotide with a cytidine analog that acts as a hydrogen bond donor at N3, it might be possible to stabilize the same contact that is thought to increase the catalytic rate for the mutant enzyme. The following two cytidine analogs: pseudoisocytidine (also called "piC"; Lu et al. J Org Chem. 2009, 74(21):8021-8030; Burchenal et al. Cancer Res. 1976, 36:1520-1523) and Benner base Z (also called "dZ"; Yang et al. Nucl Acid Res. 2006, 34(21):6095-6101) were of particular interest and were initially selected because they provide a hydrogen bond contribution at N3 with minimal disruption to the shape of the nucleic acid base. Benner bases are also referred to by their chemical name 6-amino-5-nitro-3-yl-2(1H)-pyridone. The presence of cytidine analogs in guide oligonucleotides may be present in addition to modifications to the ribose 2' group. The ribose 2' group in orphan nucleotides can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4' linked (i.e., crosslinked nucleic acids such as LNA), or other 2' substitutions. The 2'-4' linkage can be selected from linkers known in the art, such as the methylene linker or the restricted ethyl linker.

[0105] The orphan nucleotide in guide oligonucleotides as disclosed herein is preferably cytidine or an analogue thereof (e.g., a nucleotide having a Benner base) or uridine or an analogue thereof (e.g., isouridine). The orphan nucleotide, whether it is cytidine or an analogue or uridine or an analogue, preferably contains deoxyribose (2'-H;=DNA), but may also contain diF modification at the 2' position of the sugar. In some embodiments, at least one of the adjacent (directly adjacent) nucleotides flanking the orphan nucleotide, and in other embodiments, both, do not contain 2'-OMe modification. Complete modification in which all nucleotides of the oligonucleotide retain the 2'-OMe modification (including the orphan nucleotide) along with the native base results in a non-functional oligonucleotide (known in the art) as long as RNA editing proceeds, presumably because it interferes with ADAR activity at the target site. Generally, adenosine in target RNA can be protected from editing by providing a 2'-OMe group to the opposing nucleotide (at least if no other chemical substitutions or modifications exist within the nucleotide), or by providing guanine or adenine as the opposing base (because these two nucleic acid bases can also reduce editing of the opposing adenosine).

[0106] Concatenation modification Nucleosides are generally linked to adjacent nucleosides through condensation of their 5'-phosphate moiety with the 3'-hydroxyl moiety of the adjacent nucleotide monomer. Similarly, their 3'-hydroxyl moiety is generally linked to the 5'-phosphate of a neighboring nucleotide monomer. This forms a PO bond. The PO and scaffold form an alternating copolymer. Bases are transferred onto this copolymer, i.e., to the scaffold portion. For this reason, the alternating copolymer formed by the linked scaffold of an oligonucleotide is often called the "backbone linkage." Because PO bonds link adjacent monomers together, they are often called "backbone linkages." It should be understood that if the phosphate group is modified to form an analogous portion such as PS instead, such a portion is still called a monomer backbone linkage. This is called a "backbone linkage modification." In general, the backbone of an oligonucleotide contains alternating scaffolds and backbone linkages.

[0107] As outlined in detail herein, a bare guide oligonucleotide, such as those disclosed herein, includes at least one, preferably more, ligation modifications. It is generally more preferable that a guide oligonucleotide, such as those disclosed herein, includes ligation modifications at most positions, and potentially at all positions, if the guide oligonucleotide can mediate editing. The ligation modifications may be, but are not limited to, modified versions of PO present in RNA, such as PS, chiral pure PS, (R)-PS, (S)-PS, MP, chiral pure MP, (R)-MP, (S)-MP, phosphorylguanidine (e.g., PNdmi), chiral pure phosphorylguanidine, (R)-phosphorylguanidine, (S)-phosphorylguanidine, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methylphosphorothioate, methylthiophosphonate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, boranoPS, methylboranophosphate, methylboranoPS, methylboranophosphonate, methylboranophosphothioate, phosphate, phosphate triesters, aminoalkyl phosphotryesters, and their derivatives. Other modifications include phosphoramidites, phosphoramidates, N3'→P5' phosphoramidates, phosphorodiamidates, phosphorothiodiamidates, sulfamates, diethylene sulfoxides, amides, sulfonates, siloxanes, sulfides, sulfones, formacetyl, alkenyls, methylenehydrazinos, sulfonamides, triazoles, oxalyls, carbamates, methyleneiminos (MMIs), and thioacetamide nucleic acids (TANAs), as well as their derivatives. Various salts, mixed salts, deprotonated forms, tautomerized forms, and free acid forms are also included, in addition to 3'→3' and 2'→5' linkages. Guide oligonucleotides such as those disclosed herein may also include one or more linkage modifications with the structure of formula (I) below:

[0108] [ka] [In the formula, X = O or S R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C 20 Alkyl, substituted C1-C 20 [Also an alkyl, C1-C6 alkenyl, C1-C6 substituted alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugate group]. In preferred embodiments, X=O and R=methyl, in which case the linkage modification is called MsPA or PNms. In other preferred embodiments, R is equal to one of the following structures (a), (b), (c), (d), (e), (f), (g), (h), or (i):

[0109] [ka]

[0110] Also disclosed herein are guide oligonucleotides that, after forming a double-stranded complex with a region of a target RNA nucleic acid molecule in a cell, can mediate adenosine deamination by mobilizing a deaminationase in the cell, wherein the region contains target adenosine, the deaminationase can deaminate the target adenosine to inosine, and the guide oligonucleotide comprises a portion having a structure according to formula (II) at one and / or both ends:

[0111] [ka] (In the formula, X = O or S; Y=O - or S - and R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C20 Alkyl, substituted C1-C 20 (Alkyl, C1-C6 alkenyl, C1-C6 substituted alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugate group). In preferred embodiments, X=O and R=methyl.

[0112] Guide oligonucleotides as disclosed herein may include the substitution of one of the non-crosslinked oxygens in the PO linkage. This modification slightly destabilizes base pairing but adds significant resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include PS, phosphonoacetates, phosphorodithioates, phosphate triesters, aminoalkyl phosphotryesters, H-phosphonates, methyl and other alkylphosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkyl phosphotryesters, selenophosphates, or boranophosphates. Particularly preferred are nucleoside linkages modified to contain PS. Particularly preferred are nucleoside linkages modified to contain PNms. Particularly preferred are nucleoside linkages modified to contain PNdmi. Typical nucleoside linkages between nucleotides are altered by monothiolation or dithiolation of the PO bond to produce PS esters or hofholodithiolate esters, respectively. Other modifications of nucleoside linkages are possible, such as amidation and peptide linkers. Those skilled in the art can determine which guide oligonucleotides for which target nucleic acid sequences contain certain linkage modifications at each linkage position of the guide oligonucleotides disclosed herein in order to produce the most effective and stable oligonucleotide compounds.

[0113] Many of the non-natural modifications of PS linkages are chiral. This means that there are Rp and Sp configurations known to those skilled in the art. In one embodiment, the chirality of the PS linkages is controlled, meaning that each linkage is either Rp or Sp configuration, whichever is preferred. The choice of Rp or Sp configuration at a specific linkage site may depend on the target sequence and the efficiency of binding and induction that causes editing of the target adenosine. However, if such is not particularly desired, the composition may include a guide oligonucleotide as an active compound having both Rp and Sp configurations at a particular specific linkage site. Mixtures of such guide oligonucleotides are also possible, where a particular site preferably has one of the configurations, while for other sites it is not important. In one embodiment, a guide oligonucleotide as disclosed herein comprises one or more (chiral pure or chiral mixed) PS linkages. In one embodiment, a guide oligonucleotide as disclosed herein comprises one or more (chiral pure or chiral mixed) phosphoramidate (PN) linkages. In one embodiment, a guide oligonucleotide as disclosed herein comprises one or more (chiral pure or chiral mixed) PNms linkages. In one embodiment, the PN linkage connects the two terminal nucleotides at each end of the guide oligonucleotide. A guide oligonucleotide as disclosed herein may also include linkage modifications at all positions where chirality is not controlled. A guide oligonucleotide as disclosed herein may also include one or more naturally occurring nucleoside linkages. The selection and number of modified linkages may depend on the specific target, sequence, length, and stability of the guide oligonucleotide (which can be assessed by methods known to those skilled in the art) observed in the particular cell type of interest.In one embodiment, the modified nucleoside linkages are at least one, at least two, at least three, or at least four nucleoside linkages between each of the two, three, four, or five nucleosides at the 5' and / or 3' ends of a guide oligonucleotide as disclosed herein. In one embodiment, the guide oligonucleotide as disclosed herein includes at least one MP nucleoside linkage having the structure of formula (III) below:

[0114] [ka]

[0115] As has been mentioned in the Art, the preferred site for MP linkage in a guide oligonucleotide is linkage position-2, which links the nucleoside at position-1 to the nucleoside at position-2. In preferred embodiments, this site in a guide oligonucleotide, as disclosed herein, includes linkage modification by the structure of formula (I) instead of MP linkage. International Patent Application Publication 2020 / 201406 discloses the use of MP linkage modification at a specific site surrounding an orphan nucleotide in a first nucleic acid chain. While the presence of MP linkage is compatible with RNA editing by human ADAR enzymes, introducing MP linkage during the manufacture of oligonucleotides is difficult in terms of additional manufacturing (purification) steps in the coupling and decoupling processes. In one embodiment, the guide oligonucleotide does not contain MP linkage.

[0116] In one embodiment, a guide oligonucleotide as disclosed herein preferably comprises at least one PNdmi linkage that links the two most terminal nucleosides at the 5' and / or 3' ends of the guide oligonucleotide. A PNdmi linkage preferably used in a guide oligonucleotide as disclosed herein has the structure of formula (IV):

[0117] [ka]

[0118] Other nucleoside linkages that can be used in guide oligonucleotides such as those disclosed herein are disclosed in International Patent Application Publication No. 2023 / 278589. In one embodiment, a guide oligonucleotide such as those disclosed herein comprises a nucleoside linkage of at least one phosphonoacetate and / or at least one phosphonoacetamide.

[0119] Conjugate Chemicals In one embodiment, the guide oligonucleotide is covalently or noncovalently bound to a triterpene glycoside, preferably AG1856, directly or through a linker. AG1856 is also referred to as a “saponin” (see International Patent Application No. PCT / EP2024 / 051278, unpublished; and International Patent Application Publication No. 2021 / 122998). Once taken up by target cells, the saponin may enable enhanced endosomal extrusion of the guide oligonucleotide, resulting in more efficient editing during treatment. The saponin, preferably AG1856, may be administered concurrently (or before / after administration of the guide oligonucleotide), but preferably conjugated with the guide oligonucleotide either directly or indirectly through one or more linkages. Based on these teachings, those skilled in the art can find the best form of such conjugation for achieving the objective of targeting RELN premRNA and / or mRNA, and for directing the guide oligonucleotide to target cells of choice.

[0120] In one embodiment, a guide oligonucleotide, as disclosed herein, or a sense chain (in HEON as disclosed herein) that can be annealed before entering a target cell, is bound to a hydrophobic moiety such as palmityl or its analogues, cholesterol or its analogues, or tocopherol or its analogues. This is preferably bound to the 5' end. If the hydrophobic moiety is bound to both the 5' and 3' ends, such hydrophobic moieties may be the same or different. The hydrophobic moiety bound to the oligonucleotide may be directly or indirectly mediated by another substance. If the hydrophobic moiety is directly bound, it is sufficient that the moiety is bound via covalent, ionic, hydrogen, or the like. If the hydrophobic moiety is indirectly bound, it may be bound via a linker. The linker may be cleavable or incleavable. A cleavable linker is one that can be cleaved under physiological conditions, for example, in a cell or animal body (e.g., the human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases, or by physiological conditions specific to a body part or cell, such as pH or a reducing environment (e.g., glutathione concentration). Examples of cleavable linkers include, but are not limited to, amides, esters, esters of one or both POs, phosphoesters, carbamates, and disulfide bonds, as well as natural DNA linkers. Cleavable linkers also include self-sacrificing linkers. Non-cleavable linkers are those that are not cleaved under physiological conditions, or are very slow to cleave compared to cleavable linkers, for example, in linkers consisting of PS links, modified or unmodified deoxyribonucleosides linked by PS links, spacers connected via PS links, and modified or unmodified ribonucleosides. When the linker is a nucleic acid such as DNA or an oligonucleotide, there is no limit to the length of the chain. However, this is usually a length of 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.There are no restrictions on the length or composition of the spacer connecting the ligand and oligonucleotide, and examples include ethylene glycol, triethylene glycol (TEG), HEG, alkyl chains, propyl, 6-aminohexyl, or dodecyl. One or more other types of molecules may be bound to the guide oligonucleotide through one or more linkers, and examples include peptides, sugars, vitamins, polymers, aptamers, antibodies (fragments), small molecules, etc.

[0121] General In addition to certain preferred chemical modifications at certain positions in compounds such as those disclosed herein, guide oligonucleotides such as those disclosed herein may include one or more (additional) modifications to the nucleic acid base, scaffold, and / or backbone linkage, which may or may not be present in the same monomer, for example, at the 3' and / or 5' positions. In one embodiment, a guide oligonucleotide such as those disclosed herein comprises at least one nucleoside linkage in the structure of formula (I), and / or the guide oligonucleotide further comprises at least one nucleotide having a sugar moiety comprising a 2'-OMe modification, and / or the guide oligonucleotide comprises at least one nucleotide having a sugar moiety comprising a 2'-MOE modification, and / or the guide oligonucleotide comprises at least one nucleotide having a sugar moiety comprising a 2'-F modification, and / or the guide oligonucleotide comprises an orphan nucleotide called a DNA nucleotide, even if the sugar moiety has 2'-H and therefore additional modifications may be present in the linkage with its base and / or its adjacent nucleoside. In one embodiment, the orphan nucleotide has 2'-F in the sugar moiety. In one embodiment, the orphan nucleotide has a diF substitution in the sugar moiety. In one embodiment, the orphan nucleotide has 2'-F and 2'-C-methyl in the sugar moiety. In one embodiment, the orphan nucleotide contains 2'-F in the sugar moiety in an arabinose configuration (FANA).

[0122] In one embodiment, the guide oligonucleotide is an antisense oligonucleotide capable of forming a double-stranded nucleic acid complex with a portion of a target RELN nucleic acid sequence containing target adenosine, wherein the double-stranded nucleic acid complex can recruit an adenosine deaminationase for deamination of target adenosine in the target RNA molecule, and the nucleotide in the guide oligonucleotide facing the target adenosine is an orphan nucleotide, and the orphan nucleotide has the structure of formula (V) below.

[0123] [ka] (wherein X is O, NH, OCH2, CH2, Se, or S; B is a nitrogen-containing base selected from the group consisting of cytosine, uracil, isouracil, N3-glycosylated uracil, pseudoisocytosine, 8-oxo-adenine, and 6-amino-5-nitro-3-yl-2(1H)-pyridone; R1 and R2 are both independently selected from H, OH, F, or CH3; R3 is a guide oligonucleotide portion at the 5' end of the orphan nucleotide, consisting of 6, 7, or 8 nucleotides; and R4 is a guide oligonucleotide portion at the 3' end of the orphan nucleotide, consisting of 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides). The nucleotides at the 3' and / or 5' end of the orphan nucleotide may be DNA, more preferably at 3'(position-1).

[0124] Other chemical modifications of guide oligonucleotides as disclosed herein include substitution of one or more hydrogen atoms with deuterium or tritium, examples of which can be found, for example, in International Patent Application Publication Nos. 2014 / 022566 or 2015 / 011694. In all cases, the modifications should be edited and adapted so that the guide oligonucleotide can serve as an oligonucleotide capable of recruiting an adenosine deaminase enzyme for the resulting double-stranded nucleic acid entity after binding to its target sequence. In all aspects of this disclosure, the enzyme having adenosine deaminase activity is preferably ADAR1, ADAR2, or ADAT.

[0125] Guide oligonucleotides as disclosed herein preferably do not contain 5'-terminal O6-benzylguanosine or 5'-terminal amino modification, and preferably are not covalently linked to a SNAP tag domain (manipulated O6-alkylguanosine-DNA-alkyltransferase). Guide oligonucleotides as disclosed herein preferably do not contain a boxB RNA hairpin sequence. In one embodiment, guide oligonucleotides as disclosed herein contain 0, 1, 2, or 3 fluctuation base pairs with the target sequence and / or 0, 1, 2, 3, 4, 5, 6, 7, or 8 mismatched bases with the target RNA sequence. If the orphan nucleotide is uridine, no mismatches exist, and in that case, they may be defined differently than when the orphan nucleotide is a uridine analog or derivative. One alternative to uridine is to position isouridine opposite the target adenosine, which may not pair in the same way that G pairs with U. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the guide oligonucleotide directly opposite the target adenosine.

[0126] As outlined above, guide oligonucleotides such as those disclosed herein utilize specific nucleotide modifications at predefined spots to ensure stability, as well as proper ADAR binding and activity. These modifications can vary and include modifications to the backbone of the guide oligonucleotide, the sugar portion of the nucleotide, as well as modifications to the nucleic acid base or PO linkage, as outlined in detail herein. They can also be distributed in various ways across the sequence of the guide oligonucleotide. Specific modifications may be necessary to support the interactions of various amino acid residues within the RNA-binding domain and the deaminoenzyme domain of the ADAR enzyme. For example, PS linkages or 2'-OMe or 2'-MOE modifications between nucleotides may be acceptable in some parts of the guide oligonucleotide, but in other parts, they should be avoided as they do not disrupt the crucial interactions between the phosphate and 2'-OH groups and the enzyme. Additionally, if the target sequence is not optimal for ADAR editing, specific nucleotide modifications may be necessary to enhance editing activity against the substrate RNA. Previous studies have established that certain sequence configurations are more receptive to editing. For example, the target sequence 5'-UAG-3' (having target A in the center) contains the most preferred nearest neighbor nucleotide of ADAR2, while the 5'-CAA-3' target sequence is undesirable (Schneider et al. Nucleic Acids Res. 2014, 42(10):e87). Structural analysis of the ADAR2 deaminoenzyme domain suggests the possibility of enhancing editing by carefully selecting the nucleotide opposite the target trinucleotide. For example, the pair of the 5'-CAA-3' target sequence with the 3'-GCU-5' sequence on the opposite strand (forming an AC mismatch in the center) is undesirable because the guanosine base sterically collides with the amino acid side chain of ADAR2. In such a situation, the guanosine opposite C is preferably replaced with inosine, more preferably Id, as further outlined in this disclosure (and therefore at position-1 in the guide oligonucleotide).

[0127] Guide oligonucleotides as disclosed herein, in contrast to those described with respect to siRNA, or gapmers and their relationship to RNase degradation and the use of such gapmers in double-stranded complexes (see, for example, European Patent Application Publication No. 3954395), do not contain stretches of DNA nucleotides that would make the target sequence (or sense nucleic acid strand) a target for RNase-mediated degradation. It is undesirable for the target transcript molecule to be degraded through binding of the guide oligonucleotide to the transcript molecule. In one embodiment, the guide oligonucleotide does not contain four or more consecutive DNA nucleotides anywhere in its sequence. In one embodiment, the guide oligonucleotide is composed of as many (chemically) modified nucleotides as possible to enhance resistance to RNase-mediated degradation while being as efficient as possible in producing the RNA editing effect. This also means that while orphan nucleotides and several other nucleotides in the guide oligonucleotide may be DNA, there are no stretches of four or more consecutive DNA nucleotides in the guide oligonucleotide. Thus, guide oligonucleotides as disclosed herein are not gapmers. Gapmers reduce the expression of a target transcript but do not result in RNA editing of specific adenosine within the target transcript. A gapmer is, in principle, a single-stranded nucleic acid consisting of a central region (a DNA gap region having at least four consecutive deoxyribonucleotides), and wing regions located immediately to the 5' end (5' wing region) and immediately to the 3' end (3' wing region). In contrast, guide oligonucleotides, such as those disclosed herein, can be any oligonucleotide that produces an RNA editing effect in which the target adenosine in the target RNA molecule is deaminated to inosine, and is therefore as resistant to RNase-mediated degradation as possible to produce this effect and allow the mRNA transcript to be translated into a protein.

[0128] Guide oligonucleotides, such as those disclosed herein, may also be administered in a manner that would help increase the entry of the guide oligonucleotide into target cells and / or, as soon as it is present in the cell, increase its endosomal extrusion. The portion that can be applied to such applications is a set of compounds (generally purified from nature) called, for example, “saponins” or “triterpene glycosides.” A preferred saponin that can be used in a manner such as that disclosed herein is AG1856, which is disclosed in International Patent Application Publication No. 2021 / 122998 and further described in International Patent Application No. PCT / EP2024 / 051278 (unpublished) for use in combination with oligonucleotides that produce RNA editing.

[0129] Compositions and methods Pharmaceutical compositions are also disclosed herein, comprising a guide oligonucleotide as disclosed herein, and further comprising a pharmaceutically acceptable carrier, solvent, diluent, and / or other additives (e.g., saponins or triterpene glycosides such as AG1856 (as discussed above), which may also be administered separately from the guide oligonucleotide in practice), the pharmaceutical composition may be dissolved in a pharmaceutically acceptable organic solvent, etc. The dosage form in which the guide oligonucleotide or pharmaceutical composition is administered may depend on the disorder to be treated and the tissue to be targeted, and may be selected according to general procedures in the art. The pharmaceutical composition may be administered by single-dose or multi-dose administration. This may be administered daily or at appropriate time intervals, which may be determined using general knowledge in the art and may be adjusted based on the disorder and the potency of the active ingredient.

[0130] In a preferred embodiment, the guide oligonucleotide, as disclosed herein, is a single-stranded oligonucleotide comprising an orphan nucleotide opposite a target nucleotide, the orphan nucleotide being chemically modified as described herein, and the remainder of the oligonucleotide being chemically modified to prevent it from nuclease degradation, as also disclosed herein. In another embodiment, any type of oligonucleotide or heterodouble-stranded oligonucleotide complex is disclosed, which may or may not be bound to a hairpin structure (internally or terminally) that can bind to a nucleic acid editing entity or its catalytic domain, or the oligonucleotide is in a cyclic form. In a preferred embodiment, the guide oligonucleotide, as disclosed herein, is preferably a “naked” oligonucleotide comprising at least one linkage in the structure of formula (I) as disclosed herein, which can hybridize with a target nucleic acid sequence or a portion thereof, which comprises a target adenosine, and which can mobilize an endogenous (naturally occurring) nucleic acid editing entity in a target cell for editing the target nucleotide, and which includes various chemical modifications to one or more ribose sugars and / or bases among the nucleotides in the sequence. In another embodiment, guide oligonucleotides such as those disclosed herein, delivered in a “naked” form, do not include a stem-loop structure for the recruitment of deaminationases, which allows for shorter guide oligonucleotides and improved cell delivery and transport.

[0131] RNA editing entities (such as human ADAR enzymes) are known in the art to edit dsRNA structures with varying degrees of specificity, depending on several factors. One important factor is the degree of complementarity between the two strands constituting the dsRNA sequence. Perfect complementarity between the two strands usually causes the catalytic domain of human ADAR to react with any adenosine it encounters, deaminating the adenosine in an indiscriminate manner. The specificity of hADAR1 and hADAR2 can be increased by introducing chemical modifications and / or ensuring some mismatches in the dsRNA, which may help to position the dsRNA-binding domain in ways that have not yet been clearly defined. Furthermore, the deamination reaction itself can be enhanced by providing oligonucleotides containing mismatches opposite the adenosine to be edited. Following the instructions in this application, those skilled in the art will be able to design complementary portions of oligonucleotides according to their requirements.

[0132] Those skilled in the art will understand that the degree to which editing entities, such as editing enzymes inside cells, are redirected to other target sites can be controlled by altering the affinity of the primary nucleic acid chain to the recognition domain of the editing entity. Precise modification can be determined through several trials and / or by computer-based methods based on the structural interaction between the guide oligonucleotide and the recognition domain of the editing enzyme. In addition, or alternatively, the degree to which cell-endogenous editing enzymes are recruited and redirected can be controlled by the dosing and regimen of the guide oligonucleotide. This is determined by experimenters (in vitro) or clinicians, typically in Phase I and / or Phase II clinical trials.

[0133] Site-directed editing of target adenosine in RNA sequences in eukaryotes, preferably metazoans, more preferably mammals, more preferably nerve cells, more preferably human nerve cells, most preferably human cells of the central nervous system, is disclosed herein. Target cells may be located in vitro, ex vivo, or in vivo. One advantage of guide oligonucleotides such as those disclosed herein is that they can be used in situ with cells in living organisms, but they can also be used with cells in culture. In some embodiments, cells are treated ex vivo and then introduced into living organisms (e.g., reintroduced into the organism from which they originally originated). Guide oligonucleotides such as those disclosed herein can also be used to edit target RNA sequences in graft-derived cells, or so-called organoids, such as cells in brain tissue organoids. Organoids are three-dimensional in vitro derived tissues, but can be thought of as being driven using specific conditions to produce individual isolated tissues. In therapeutic settings, these are useful because they can be induced in vitro from the patient's cells, and the organoids can then be reintroduced into the patient as autologous material that is less likely to be rejected than conventional grafts.

[0134] While we do not wish to be bound by theory, RNA editing via human ADAR2, for example, is thought to occur in the nucleus during transcription or splicing, on the primary transcript, or in the cytoplasm (for example, mature mRNA, miRNA, or ncRNA may be edited). Generally speaking, RNA editing can be used to create RNA sequences with a variety of properties. Such properties may be coding properties (creating proteins with various sequences or lengths, resulting in altered protein properties or functions) or binding properties (causing inhibition or overexpression of the RNA itself or its target or binding partner, or altering the entire expression pathway by recoding a miRNA or its homologous sequence on the target RNA). Protein function or localization can be freely altered by functional domains or recognition motifs, including, but not limited to, signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co-modification or post-translational modification, enzyme catalytic sites, binding sites for binding partners, and degradation or activation signals. These, as well as other forms of "manipulation" of RNA and proteins, are included in this disclosure as diagnostic, prophylactic, therapeutic, research tools, or otherwise in medicine or biotechnology, whether for the purpose of preventing, delaying, or treating disease, or for any other purpose.

[0135] The amount, dosage, and administration regimen of guide oligonucleotides may vary between cell types, the disease being treated, the target population, the mode of administration (e.g., systemic vs. topical), the severity of the disease, and the level of acceptable side effects. These can and should be evaluated through trial and error during in vitro studies, preclinical studies, and clinical trials. Trials are particularly evident when modified sequences result in readily detectable phenotypic changes or changes in the level or activity of specific biomarkers (e.g., plasma levels of bile acids). Higher doses of guide oligonucleotides may compete for binding with intracellular ADAR enzymes, thereby depleting the amount of enzymes freely involved in RNA editing; however, routine dosing trials will reveal any such effects for a given guide oligonucleotide and a given target.

[0136] One suitable investigational technique involves delivering a guide oligonucleotide to a cell line or test organism, and then collecting biopsy samples at various points in time thereafter. The target RNA sequence can be evaluated in the biopsy samples, and the percentage of cells with modification can be easily tracked. As mentioned above, plasma levels of bile acids in samples derived from the treated subject are suitable biomarkers for evaluating the function of a particular protein in the subject before and after treatment, or with or without treatment of the subject with the guide oligonucleotide as disclosed herein. After this trial is performed once, the knowledge can be retained, and future deliveries can be made without the need to collect biopsy samples. Thus, the methods disclosed herein may include a step of identifying the presence of a desired change in the target RNA sequence of cells, thereby confirming that the target RNA sequence is modified. This step typically involves sequencing the relevant portion of the target RNA, or its cDNA copy (or, if the target RNA is an mRNA precursor, the cDNA copy of its splicing product), as described above, and thus the sequence change can be easily confirmed. Alternatively, as described above, changes can be evaluated before, during, and / or after treatment or evaluation of any other potential markers for protein function, and measurements are preferably performed in vitro on samples obtained from the treated subject.

[0137] After RNA editing occurs in a cell, the modified RNA may be diluted over time due to factors such as cell division and the limited half-life of the edited RNA. Therefore, in practical therapeutic applications, methods such as those disclosed herein may involve repeated delivery of guide oligonucleotides until sufficient target RNA has been modified to provide a visible benefit to the patient and / or maintain that benefit over the long term.

[0138] Guide oligonucleotides such as those disclosed herein are particularly suitable for therapeutic use, and therefore, pharmaceutical compositions comprising guide oligonucleotides such as those disclosed herein and pharmaceutically acceptable carriers, solvents, or diluents are also disclosed. In some embodiments, the pharmaceutically acceptable carrier may be simply physiological saline, which may be usefully isotonic or hypotonic, particularly for pulmonary delivery. Guide oligonucleotides such as those disclosed herein are optionally additives, excipients, and other components and are appropriately administered in aqueous solutions, for example, in physiological saline or suspension, at concentrations ranging from 1 ng / ml to 1 g / ml, preferably from 10 ng / ml to 500 mg / ml, and more preferably from 100 ng / ml to 100 mg / ml, suitable for pharmaceutically acceptable use. The dosage may appropriately range from about 1 μg / kg to about 100 mg / kg, preferably about 10 μg / kg to about 10 mg / kg, and more preferably about 100 μg / kg to about 1 mg / kg. Administration may be by inhalation (e.g., through atomization), intranasal, oral, by injection or infusion, intravenously, subcutaneously, intradermally, intramuscularly, intratracheally, intraperitoneally, intrarectally, subarachnoidally, intravenously (e.g., intracerebral), parenterally, etc. Administration may be in solid form, powder form, pill, gel, liquid, sustained-release formulation, or any other form suitable for pharmaceutically acceptable use in humans.

[0139] In one embodiment, with respect to the deamination effect of conversion from A to I, the step of identifying whether editing has occurred includes: sequencing the target nucleic acid sequence; assessing the presence or absence of a non-functional or poorly functional protein; assessing whether the splicing of the premRNA has been altered by the deamination of target adenosine in the RNA; or using functional readout information, since the deaminated target nucleic acid should encode a protein with lower or no functionality, or conversely, increased, restored, or newly acquired functionality. Identification of deamination to inosine may be functional readout information using appropriate biomarkers. Functional assessment is generally by methods known to those skilled in the art. Appropriate methods for identifying the presence of inosine after deamination of target adenosine are dPCR or sequencing, using methods well known to those skilled in the art. However, vendors in the field of neurodegenerative diseases would preferably apply tests that monitor certain biomarkers related to neurological function.

[0140] In one embodiment, a method as disclosed herein includes the steps of: administering a guide oligonucleotide or a vector capable of expressing it, as disclosed herein, to a target to enable the formation of a double-stranded nucleic acid complex of the guide oligonucleotide with a target nucleic acid sequence in the target cell; enabling the association of a nucleotide editing entity, such as an endogenously present adenosine deaminationase, such as ADAR1 or ADAR2; and enabling the entity to edit a target nucleotide in the target nucleic acid sequence, thereby mitigating, treating, alleviating or slowing the progression of a disease.

[0141] The nucleotide editing entities present in cells are typically naturally occurring and proteinaceous, such as ADAR enzymes found in metazoans, including mammals. Particularly preferred are human ADAR, hADAR1, and hADAR2, including any of its isoforms. RNA editing enzymes known in the art, for which oligonucleotide constructs such as those disclosed herein can be conveniently designed, include adenosine deaminases (ADARs) that act on RNA, such as hADAR1 and hADAR2 in human or human cells, as well as cytidine deaminases. hADAR1 is known to exist in two isoforms, namely a longer 150 kDa interferon-inducible version and a shorter 110 kDa version, generated from a common mRNA precursor through alternative splicing. Consequently, the level of the 150 kDa isoform available in cells can be influenced by interferons, particularly interferon-gamma (IFN-γ). hADAR1 is also inducible by TNF-α. This provides an opportunity to develop combination therapies in which IFN-γ or TNF-α and guide oligonucleotides, such as those disclosed herein, are administered either as a combination or as separate products, simultaneously or sequentially in any order. Certain medical conditions may already coincide with elevated IFN-γ or TNF-α levels in specific tissues of the patient, creating further opportunities to make editing more tissue-specific. Those skilled in the art will understand that the extent to which intracellular editing entities are redirected to other target sites can be regulated by varying the affinity of the first nucleic acid chain to the recognition domain of the editing molecule.

[0142] In some embodiments, guide oligonucleotides, such as those disclosed herein, can utilize endogenous cellular pathways and naturally available ADAR enzymes to specifically edit a target adenosine in a target RNA sequence. Certain guide oligonucleotides, such as those disclosed herein, can recruit ADAR and form a complex with it, which then promotes the deamination of a (single) specific target adenosine nucleotide in the target RNA sequence to which the guide oligonucleotide is bound. In some embodiments, only one adenosine is deaminated. When guide oligonucleotides, such as those disclosed herein, form a complex with ADAR, preferably results in the deamination of a single target adenosine.

[0143] Guide oligonucleotides, such as those disclosed herein, are typically longer than 16 nucleotides, especially when they are in a naked form. In one embodiment, guide oligonucleotides, such as those disclosed herein, are longer than 20 nucleotides. Guide oligonucleotides, such as those disclosed herein, are preferably shorter than 100 nucleotides, more preferably shorter than 60 nucleotides, and even more preferably shorter than 50 nucleotides. In a preferred embodiment, guide oligonucleotides, such as those disclosed herein, comprise 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, and even more preferably 18 to 50 nucleotides. Therefore, in a particularly preferred embodiment, a guide oligonucleotide, such as those 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. In a particularly preferred embodiment, a guide oligonucleotide, such as those disclosed herein, has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides. Preferably, the guide oligonucleotide has an asymmetric design, as shown in the appended examples, when it targets the premRNA and / or mRNA of the human RELN gene. Preferably, the length of the 5' portion relative to the orphan nucleotide at position 0 is 6, 7, or 8 nucleotides. Preferably, the length of the 3' portion relative to the orphan nucleotide at position 0 is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0144] In some embodiments, the guide oligonucleotide is not used with an editing system that does not require a guide oligonucleotide. In some embodiments, the guide oligonucleotide is not used to induce the Reln-H3448R mutation in mice by homologous recombination. In some embodiments, the guide oligonucleotide is not used to induce the mouse Reln-H3448R mutation and / or the human RELN-H3447R mutation by homologous recombination. In some embodiments, the guide oligonucleotide is not used to create a Reln-H3448R-Tg knock-in mouse model having a Reln-COLBOS variant by homologous recombination.

[0145] In some embodiments, the nucleic acid editing enzyme does not edit using homologous recombination. In some embodiments, the nucleic acid editing enzyme does not produce mouse Reln-H3448R mutations and / or human RELN-H3447R mutations by homologous recombination. In some embodiments, the nucleic acid editing entity does not produce a Reln-H3448R-Tg knock-in mouse model having a Reln-COLBOS variant by homologous recombination. [Examples]

[0146] [Example 1] RNA editing of RELN transcripts using different guide oligonucleotides To target adenosine at the CAU codon (in human RELN transcript), various guide oligonucleotides were designed. This codon encodes histidine (H) (H3447) at amino acid position 3447 in the human reelin protein, and the deamination of adenosine to inosine (CIU) gives a codon that will translate to arginine (R) at this position. Thus, the amino acid change is commonly referred to herein as H3447R. The sequence of the target codon in human RELN DNA, as well as the surrounding sequence, is provided in Figure 1A. Clearly, the deamination of the target sequence using the guide oligonucleotides and endogenous ADAR enzymes of this disclosure occurs in the transcript transcribed from that DNA, and therefore the actual target sequence contains uridine (U) instead of thymidine residue (T). Thus, the sequence in Figure 1A represents its target transcript, with T replaced by U. The sequences, designs, and chemical modifications of the guide oligonucleotides are provided in Figures 2, 3, and 7. Chemical modifications are discussed in the brief description of the drawing.

[0147] First, human iPSCs (WT04) derived from neural progenitor cells were differentiated into mature cortical neurons using neural precursor medium, generally following protocols known to those skilled in the art. For the initial screening of guide oligonucleotides and to determine RNA editing efficiency, the following was performed: On day 0, mature neurons were placed in 12-well plates at 2.0 × 10⁶ per well. 5 Cells were plated at individual cell concentrations and grown until day 11. Treatment with guide oligonucleotides was performed using three different protocols, as follows: 1) On day 11, cells were transfected with 200 nM guide oligonucleotides using Lipofectamine® RNAiMAX reagent according to the manufacturer's instructions for use (N=2 or 3 biological replicas per guide oligonucleotide). Plates were incubated at 37°C and 5% CO2 for 48 hours until harvesting. On day 2 (48 hours after transfection), the supernatant was discarded and cells were prepared for RNA isolation as described below. 2) On day 11, cells were treated with 5 μM guide oligonucleotides dissolved in culture medium by gymnosis (gymnosis, without transfection) (N=2 biological replicas per guide oligonucleotide), and the plates were incubated at 37°C and 5% CO2 for 7 days until harvesting. On days 2 and 4 after the start of guide oligonucleotide treatment, cells were restored in 50% fresh medium without guide oligonucleotides. On day 7 after the start of treatment, the supernatant was discarded, and the cells were prepared for RNA isolation as described below. 3) On day 11, cells were treated by gymnosis with a 5 μM guide oligonucleotide dissolved in a medium also containing 1 μM triterpene glycoside AG1856 (saponin) (N=1 or 2 biological replicas per guide oligonucleotide). Subsequently, the plates were incubated at 37°C and 5% CO2 for 7 days until harvesting. On days 2 and 4 after the start of guide oligonucleotide + saponin treatment, cells were restored in 50% fresh medium without guide oligonucleotide or saponin. On day 7 after the start of treatment, the supernatant was discarded and the cells were prepared for RNA isolation as described below.

[0148] Cells were collected and used for RNA isolation using the ReliaPrep® RNA Cell Miniprep System (Promega-Z612) according to the manufacturer's instructions. Reverse transcription was performed using the Maxima reverse transcriptase (Thermo-EP0742) kit containing oligo dT primers, random hexamer primers, and a dNTP mixture (10 mM each). Quantitative PCR was then performed using a digital PCR system (QIAGEN, QIAcuity dPCR system) on 12 μl aliquots of a reaction mixture containing cDNA, appropriate primer pairs and probes, and a dPCR master mix (QIAGEN - QIAcuity Mastermix (4×)). Plate priming was performed using the QIAcuity system. The forward and reverse primers shown in Table 1 were used with the following PCR program: 2 minutes at 95°C; 40 cycles of 15 seconds at 95°C and 30 seconds at 63°C. The channels (HEX, FAM, and CY5; see Table 1 for probe sequences) were imaged for 500 milliseconds, 500 milliseconds, and 400 milliseconds, respectively, at gains of 6, 6, and 8.

[0149] [Table 5]

[0150] The editing percentage was calculated by pooling the biological replicas per transfection for the total number of A and G samples, and then scored according to the following formula: Score = SUM(G) / (SUM(A+G)*100

[0151] The results of the transfection experiments are shown in Figure 4. The results of experiments with gymnosis incorporation (without saponin), in which RNA editing can be easily detected, are shown in Figure 5. The results of experiments with gymnosis incorporation (with simultaneous saponin treatment), in which RNA editing can be easily detected, are shown in Figure 6. The transfection data showed that asymmetric guide oligonucleotides having a relatively short 5' portion of approximately 8 nucleotides and a relatively long 3' portion of approximately 16-22 nucleotides produced the highest editing efficiency, at approximately 10-12% (see RM116835, RM116836, and RM116837). These experiments show that RM116835, RM116836, RM116837, RM116838 (with a typical asymmetric design), in addition to RM116818 and RM116827 are preferred embodiments of those disclosed herein. It also indicates that an asymmetric design in which the 5' portion of the guide oligonucleotide calculated from the orphan position is relatively short and the 3' portion of the guide oligonucleotide calculated from the orphan position is relatively long is a preferred embodiment of the present disclosure.

[0152] Unexpectedly, RNA efficiency after gymnastic incorporation was lower than that observed with respect to transfection. Overall editing efficiency was less than 3%, but RM116827 and several asymmetric guide oligonucleotides (RM116836, RM116837, and RM116838) showed good performance. When using gymnastic incorporation in association with endosomal escape factor AG1856 (triterpene glycoside or saponin), the asymmetrically designed guide oligonucleotides RM116835, RM116836, RM116837, and RM116838, along with RM116821 and RM116827, showed particularly good performance, reaching approximately 9.5% editing.

[0153] [Example 2] RNA editing of RELN transcripts using an additional set of guide oligonucleotides Based on the results obtained in Example 1, the designs of RM116835 and RM116838 were adopted as a basis for designing additional guide oligonucleotides for testing. These 31 additional designs are shown in Figure 7, where RM118850-RM118867 are based on RM116835 (SEQ ID NO: 58), and RM118868-RM118880 are based on RM116838 (SEQ ID NO: 61). Along with the newly synthesized RM116835 and RM116838 oligonucleotides, these 31 new guide oligonucleotides were tested in gymnosis uptake experiments in association with saponins as described in the experimental setup 3) above, followed by RNA isolation, cDNA generation, and dPCR detection setup as described in Example 1. RNA editing of target adenosine was measured as described above. In particular, there are different RELN isoforms known as 201 and 203, in which isoform 210 lacks exon 64, which is present in isoform 203. Exon 64 is only 6 nucleotides long, and its sequence happens to match that of the forward primer "hRELN 201_e63-64 fw 1" (Table 1) used for cDNA generation in the experimental setup outlined above. To distinguish the effect of the guide oligonucleotide on isoform 201 and isoform 203 (and primarily to distinguish the cDNA generation of both isoforms), a further forward primer "hRELN_e63-64-65_fw 2" was constructed with the sequence 5'-ATG TGG AGG TCG TCC TAG TAA GC-3' (SEQ ID NO: 107). The effects on isoforms 201 and 203 were evaluated separately using these two different forward primers in two different cDNA generation scenarios, and then subsequently, these were evaluated separately for RNA editing.

[0154] The editing percentage results are provided in Figure 8, where Figure 8A shows the editing percentage using the forward primer for the 201 isoform, and Figure 8B shows the editing percentage using the forward primer for the 203 isoform. The editing percentage patterns between the two isoforms appear to be similar. Guide oligonucleotides originating from these two showed slightly better performance in this new experiment compared to the data shown in Figure 6, with RM116835 showing editing percentages of 13.9% (201) and 18.3% (203), and RM116838 showing editing percentages of 11.3% (201) and 13.2% (203). Interestingly, it was found that it is possible to design even more improved guide oligonucleotides that show a significant increase in editing efficiency, with the best performers being: RM118851 (Sequence ID 65) - 22.3% (201) and 23.2% (203) RM118852 (Sequence ID 66) - 20.4% (201) and 24.2% (203) RM118850 (Sequence ID 64) - 18.4% (201) and 20.3% (203) RM118874 (Sequence ID 88) - 20.0% (201) and 21.1% (203) RM118876 (Sequence ID 90) - 19.1% (201) and 25.4% (203) RM118875 (Sequence ID 89) - 17.2% (201) and 17.1% (203) RM118855 (Sequence ID 69) - 16.4% (201) and 18.1% (203) RM118853 (Sequence ID 67) - 16.3% (201) and 17.2% (203) RM118868 (Sequence ID 82) - 16.2% (201) and 16.6% (203) RM118869 (Sequence ID 83) - 14.2% (201) and 17.4% (203) RM118870 (Sequence ID 84) - 16.0% (201) and 17.6% (203)

[0155] In further experiments, these guide oligonucleotides, and newly designed guide oligonucleotides based on the designs of their best performers, will be tested in vivo. It is preferable to conjugate the guide oligonucleotide with a triterpene glycoside (preferably saponin AG1856, as used in the gymnosis / saponin experiments outlined above) to provide highly efficient endosomal escape once the guide oligonucleotide has entered the target cells requiring editing of the RELN transcript molecule. Conjugation of AG1856 to the guide oligonucleotide is generally carried out as described in international patent application PCT / EP2024 / 051278 (unpublished). In alternative experimental settings, the guide oligonucleotide is delivered in vivo using LNPs.

[0156] Next, the amount and / or rate of phosphorylated Dab1 protein is determined in relevant mammalian (primary) cell cultures using Western blotting and immunofluorescence staining.

Claims

1. A guide oligonucleotide that is at least partially complementary to a portion of a human RELN nucleic acid molecule containing a target nucleotide, wherein the RELN nucleic acid molecule encodes a reelin protein, and the guide oligonucleotide is configured to form a double-stranded complex with the portion of the RELN nucleic acid under physiological conditions within a cell, and the double-stranded complex can recruit a nucleic acid editing enzyme naturally present in the cell to perform editing of the target nucleotide to produce an edited RELN nucleic acid containing the edited target nucleotide.

2. The guide oligonucleotide according to claim 1, wherein editing of the target nucleotide results in an increase in the activity of the encoding reelin protein.

3. The guide oligonucleotide according to claim 2, wherein the encoding reelin protein is given a gain-of-function phenotype selected from one or more of the following: (i) Enhanced ability to trigger signaling, preferably the APOEr / Dab1 / GSK3β pathway; (ii) Enhanced ability to increase Dab1 phosphorylation; (iii) Enhanced ability to reduce taurinary tangle-related taurinary tangles; (iv) Enhanced ability to form and / or stabilize tubular structures and / or increase neuronal density; (v) Enhanced resistance to degradation by proteolysis; and / or (vi) Enhanced binding of reelin protein to glycosaminoglycans, preferably heparin, and / or NRP1.

4. The guide oligonucleotide according to any one of claims 1 to 3, wherein the editing of the target nucleotide introduces an amino acid variant at one or more amino acid positions 3446 to 3460 of the encoding reelin protein, preferably, the editing of the target nucleotide introduces a histidine-to-arginine change (H3447R) at amino acid position 3447 of the encoding reelin protein.

5. The guide oligonucleotide according to any one of claims 1 to 4, wherein the cells are brain cells, preferably neurons.

6. The guide oligonucleotide according to any one of claims 1 to 5, wherein the target nucleotide is adenosine and the nucleic acid editing enzyme is an RNA-acting adenosine deaminase (ADAR) enzyme.

7. The guide oligonucleotide according to any one of claims 1 to 6, wherein the RELN nucleic acid molecule is mRNA or premRNA.

8. The guide oligonucleotide according to any one of claims 1 to 7, wherein the orphan nucleotide is the nucleotide in the preceding guide oligonucleotide that is opposite to the preceding target nucleotide, the preceding nucleotide numbering is such that the orphan nucleotide is number 0, the nucleotide is positively (+) incremented toward the 5' end and negatively (-) incremented toward the 3' end, and at least one nucleic acid base, sugar, or nucleoside linkage is chemically modified.

9. The guide oligonucleotide according to claim 8, wherein the orphan nucleotide is deoxycytidine, a cytidine analog, deoxyuridine, or a uridine analog.

10. A guide oligonucleotide according to any one of claims 1 to 9, having a length of 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.

11. The guide oligonucleotide is 【Chemistry 1】 A guide oligonucleotide according to any one of claims 1 to 10, comprising a continuous stretch of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, wherein in the sequence, Z is a nucleotide comprising a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base and I is boar.

12. The guide oligonucleotides are, independently, phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylsulfonate (MP), sulfonyl phosphoramidate, (1,3-dimethylimidazolidinedine-2-ylidene)phosphoamidate (PNdmi), or formula (I): 【Chemistry 2】 (wherein X = O or S; and R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C １ ~C ６ alkoxy, substituted C １ ~C ６ alkoxy, C １ ~C ２０ alkyl, substituted C １ ~C ２０ alkyl, C １ ~C ６ alkenyl, C １ ~C ６ substituted alkenyl, C １ ~C ６ alkynyl, substituted C １ ~C ６ alkynyl, or conjugated group) A guide oligonucleotide according to any one of claims 1 to 11, comprising one or more internucleoside linkage modifications selected from linkage modifications having a structure by, preferably X=O and R=methyl, wherein the linkage modification is a mesylphosphoramidate (PNms).

13. The guide oligonucleotide according to any one of claims 1 to 12, wherein the nucleoside linkage numbering in the guide oligonucleotide is such that linkage number 0 is a linkage 5' from the orphan nucleotide, the linkage positions in the oligonucleotide are positively (+) incrementing toward the 5' end and negatively (-) incrementing toward the 3' end, and linkage position -2 is an MP or PNms linkage.

14. The guide oligonucleotide may have a linear or branched lower (C) molecule at the 2', 3', and / or 5' positions of ribose, independently of each other, interrupted by -OH; -F; or one or more heteroatoms. １ ~C １０ A guide oligonucleotide according to any one of claims 1 to 13, comprising one or more nucleotides, including monosubstituted or disubstituted ones, selected from the group consisting of alkyl, alkenyl, alkynyl, alkalil, allyl, or aralkyl; -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; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy.

15. Structure (5'→3'): N ８ N ７ N ６ N ５ N ４ N ３ N ２ N １ θ ZdId^M ２ M ３ M ４ M ５ M ６ M ７ M ８ M ９ M １０ M １１ M １２ M １３ M １４ M １５ M １６ M １７ M １８ M １９ M ２０ M ２１ M ２２ M ２３ M ２４ (In the array, - Zd is an orphan nucleotide that is a deoxynucleotide having a Benner base at nucleotide position 0; - N １ is Ae or Ad; - N ２ is Af; - N ３ and N ５ These are independently Am or Af; - N ４ is Gf; - N ６ is Uf; - N ７ It does not exist (if so, N ８ It is either (which also does not exist), Gm, or Gf; - N ８ It is either nonexistent or Um; - Id is deoxyinosine; - M ２ is Um; - M ３ is Cf; - M ４ M １４ , and M １５ These are independently m5Ue or Um; - M ５ and M ７ is Gf; - M ６ is Am or Af; - M ８ and M １０ These are independently Cm or Cf; - M ９ is Cf; - M １１ It is Am; - M １２ is Um; - M １３ is Gm; - M １６ is Ge or Gm; - M １７ It does not exist (if so, M １８ ~M ２４ It is either (which also does not exist), m5Ue, or Um; - M １８ It does not exist (if so, M １９ ~M ２４ It is either (which also does not exist), Cm, or m5Ce; - M １９ It does not exist (if so, M ２０ ~M ２４ It is either (which also does not exist), Gm, or Ge; - M ２０ It does not exist (if so, M ２１ ~M ２４ It is either (which also does not exist), Um, or m5Ue; - M ２１ It does not exist (if so, M ２２ ~M ２４ It is either (which also does not exist), Gm, or Ge; - M ２２ does not exist (if so, M ２３ and M ２４ also do not exist), either Am, or Ae; - M ２３ It does not exist (if so, M ２４ (and also does not exist) or Ae; - M ２４ It is either nonexistent or Ae; - θ It is in coupling position 0 and is a PO coupling or PNms coupling; - ^ is in coupling position -2 and is an MP or PNms coupling; - All other consolidations are either PO consolidations, PS consolidations, PNdmi consolidations, or PNms consolidations, and Gm, Am, Um, and Cm are 2'-O-methyl (2'-OMe) modified guanosine, 2'-OMe modified adenosine, 2'-OMe modified uridine, and 2'-OMe modified 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 ("Te"; sometimes named 2'-MOE modified thymidine); Af, Uf, Gf, and Cf are 2'-F modified adenosine, 2'-F modified uridine, 2'-F modified guanosine, and 2'-F modified cytosine, respectively. A guide oligonucleotide according to any one of claims 1 to 14, comprising:

16. A guide oligonucleotide according to any one of claims 1 to 15, comprising or consisting of one of the sequences 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.

17. A guide oligonucleotide according to any one of claims 1 to 16, which is bound to a triterpene glycoside, preferably AG1856, and preferably conjugated.

18. A vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding a guide oligonucleotide according to any one of claims 1 to 17.

19. A pharmaceutical composition comprising a guide oligonucleotide according to any one of claims 1 to 17 or a vector according to claim 18, and a pharmaceutically acceptable carrier.

20. A guide oligonucleotide according to any one of claims 1 to 17 for use in treating, alleviating, or slowing the progression of a neurodegenerative disease, preferably Alzheimer's disease, more preferably autosomal dominant Alzheimer's disease.

21. Use of a guide oligonucleotide according to any one of claims 1 to 17 for use in the manufacture of a pharmaceutical product for treating, alleviating, or slowing the progression of a neurodegenerative disease, preferably Alzheimer's disease, more preferably autosomal dominant Alzheimer's disease.

22. A method for treating, alleviating, or slowing the progression of a neurodegenerative disease, preferably Alzheimer's disease, more preferably autosomal dominant Alzheimer's disease, in a human subject in need thereof, comprising the step of administering to the subject a guide oligonucleotide according to any one of claims 1 to 17 or a vector according to claim 18, thereby editing a target RELN nucleic acid sequence to encode a reelin protein having the ability to delay the onset of one or more symptoms of the neurodegenerative disease.

23. An in vitro, ex vivo, or in vivo method for deamination of a target adenosine in a target RELN nucleic acid sequence in brain cells, preferably neurons, comprising the steps of (i) supplying the cells with a guide oligonucleotide according to any one of claims 1 to 17 or a vector according to claim 18; (ii) a step that enables the uptake of the guide oligonucleotide or vector by the cell; (iii) a step that enables the annealing of the guide oligonucleotide to the target RELN nucleic acid sequence; and (iv) A step that enables a nucleic acid editing entity to edit the target. Methods that include...

24. The method according to claim 23, further comprising the step of administering a triterpene glycoside, preferably AG1856, before, after, or simultaneously with the step of administering the guide oligonucleotide.

25. A method for editing a human RELN nucleic acid sequence in cells, preferably brain cells, wherein the human RELN nucleic acid sequence is premRNA or mRNA, and the method comprises the step of contacting a target RELN nucleic acid sequence with a guide oligonucleotide capable of triggering ADAR-mediated adenosine-to-reelin deamination, thereby editing the target RELN nucleic acid sequence to encode a reelin protein having the ability to delay the onset of one or more symptoms of a neurodegenerative disease, preferably Alzheimer's disease, more preferably autosomal dominant Alzheimer's disease.

26. The method according to claim 25, wherein the guide oligonucleotide is the guide oligonucleotide according to any one of claims 1 to 17.

27. A nucleic acid molecule for editing a target adenosine in a human RELN premRNA or mRNA molecule, wherein the target region is sequence number 106, and the target adenosine is the second nucleotide of the codon encoding histidine at position 3447 of the RELN-encoding human reelin protein.

28. The nucleic acid molecule according to claim 27, wherein the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 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.

29. The nucleic acid molecule according to claim 27 or 28, further comprising at least one non-naturally occurring chemical modification and / or comprising one or more additional non-naturally occurring chemical modifications in the ribose, ligation, or base portion, wherein the orphan nucleotide, which is a nucleotide in the nucleic acid directly opposite the target adenosine in the target region, is not a cytidine containing a 2'-OMe ribose substitution.

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