Antisense oligonucleotides for the treatment of cardiovascular disease

Abstract

The present invention relates to the field of diseases caused by high levels of LDL-C and / or fibrinogen, such as cardiovascular diseases. The present invention includes an oligonucleotide for RNA editing technology that deaminates a target adenosine nucleotide, such as adenosine at position 1055, in the transcript of the human B4GALT1 gene.

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, particularly cardiovascular disease (CVD), and involves using RNA editing technology to target transcripts encoding the enzyme β-1,4-galactosyltransferase 1 (B4GALT1) to create amino acid changes that reduce the function of B4GALT1. [Background technology]

[0002] CVD is one of the leading causes of death and disability worldwide. Therefore, providing treatments for CVD represents a significant unmet medical need. Various risk factors contribute to the development of CVD. These risk factors include elevated blood levels of low-density lipoprotein cholesterol (LDL-C) and / or elevated fibrinogen levels (Montasser et al. 2021. Science 374:1221-1227). Elevated LDL-C concentrations increase the likelihood of arterial plaque formation. Atherosclerosis and fibrinogen increase the risk of blood clotting and thrombosis. LDL-C is an established risk factor for coronary artery disease (CAD). Therefore, identifying mechanisms that lower blood levels of LDL-C and / or fibrinogen would provide promising targets for preventing, ameliorating, or treating CVD.

[0003] Recently identified targets in combating CVD include the enzyme B4GALT1, which is involved in the processing of biologically important biomolecules, including those involved in lipid metabolism and coagulation. B4GALT1 is ubiquitously expressed and plays a key role in the processing of N-linked oligosaccharide moieties in glycoproteins, transferring galactose from uridine diphosphate galactose (UDP-Gal) to specific glycoprotein substrates. B4GALT1 is therefore important for the biological activities associated with correctly assembled oligosaccharides.

[0004] A recent study identified an association between a B4GALT1 mutation present in the Old Order Amish population and reduced CVD in that population (Montasser et al. 2021, supra). Importantly, a gene-based analysis of B4GALT1 in nearly 600,000 subjects revealed an association with reduced CAD. This population was found to be enriched for a missense variant in the B4GALT1 protein (designated p.Asn352Ser (asparagine to serine; also referred to herein as p.N352S, or simply N352S)), and this variant was associated with reduced blood levels of LDL-C and fibrinogen. The effects of this change were systematically analyzed through various techniques. Knock-in mouse studies provided evidence that the N352S variant plays a causal role in reducing blood LDL-C and fibrinogen levels. N-linked glycan profiling of serum from human subjects with this mutation was associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. Enzyme assays revealed that this mutation resulted in a 50% decrease in galactosyltransferase activity compared to the unmutated protein. Structural studies showed that position 352 of B4GALT1 lies within the functional domain of B4GALT1, and the asparagine-to-serine mutation is understood to prevent a conformational change required for enzymatic activity, affecting the glycosylation efficiency of B4GALT1.

[0005] Thus, the B4GALT1 N352S mutation was observed to affect at least two distinct risk factors associated with CVD, namely, lowering blood LDL-C and fibrinogen levels, and may therefore be one of the first targets with pleiotropic potential to prevent CVD. Importantly, the B4GALT1 N352S missense variant was not associated with any severe phenotype.

[0006] Although targeted modulation of galactosylation has been identified as a promising therapeutic target for preventing, treating, or ameliorating CVD while avoiding significant side effects, no mechanism for modulating galactosylation has been proposed. The present disclosure aims to provide one or more alternative and / or improved techniques, compounds, and / or compositions for use in treating CVD. Summary of the Invention

[0007] The present disclosure provides an RNA editing oligonucleotide (EON) capable of forming a double-stranded complex with a region of a target RNA nucleic acid molecule in a human cell, wherein the double-stranded complex can recruit an endogenous ADAR enzyme naturally present in the cell, the region includes a target adenosine, the nucleotide in the EON opposite the target adenosine is a lone nucleotide, the ADAR enzyme deaminates the target adenosine to inosine, and the target RNA nucleic acid molecule is a transcription molecule of the human beta-1,4-galactosyltransferase 1 (B4GALT1) gene. In a preferred embodiment, the B4GALT1 transcription molecule is a pre-mRNA or mRNA molecule. In another preferred embodiment, the cell is a human liver cell, preferably a hepatocyte.

[0008] In one embodiment, the target adenosine in the B4GALT1 transcript molecule is located at a position that, if replaced by guanosine, would encode a B4GALT1 protein variant with a reduced enzyme turnover rate. In a preferred embodiment, the target adenosine is located at position c.1055A in the B4GALT1 transcript, and the deamination results in a change from asparagine (N; Asn; encoded by the AAU codon) to serine (S; Ser; encoded by the AGU codon) at position 352 in the human wild-type B4GALT1 amino acid sequence.

[0009] The present disclosure provides an EON as disclosed herein, wherein at least one nucleotide comprises one or more non-natural chemical modifications in the ribose moiety, linkage moiety, or base moiety, with the proviso that the isolated nucleotide is not a cytidine containing a 2'-OMe ribose substitution. In a preferred embodiment, the one or more modifications in the linkage are each independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonyl phosphoramidate, or PNdmi internucleoside linkage. In a preferred embodiment, the one or more modifications in the ribose moiety are mono- or di-substitutions at the 2', 3', and / or 5' positions of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C2) nucleotides. 10 ) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-aryl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy.

[0010] Provided herein are EONs, comprising or consisting of an EON selected from the group set forth in SEQ ID NOs: 3-42, 59-1069, and 1078-1190, preferably selected from the group consisting of SEQ ID NOs: 23, 19, 31, 27, 35, 39, 69, 70, 71, 72, 73, 93, 94, 95, 1079, 1084, 1093, 1095, 1100, 1102, 1115, 1121, 1123, 1124, and 1139-1190. Importantly, provided herein are EONs comprising a base nucleotide sequence according to these preferred EONs and further modified as described herein. This means that these preferred EONs can be further optimized using the teachings provided herein, and even more efficient RNA editing effects can be achieved. In a preferred embodiment, the isolated nucleotide in the EON disclosed herein is a deoxynucleotide having a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleobase (Benner's base Z) or an isouracil nucleobase (isoU).

[0011] Also provided herein is a vector, preferably a viral vector, more preferably an adeno-associated viral (AAV) vector, comprising a nucleic acid molecule encoding the EON disclosed herein.Also provided herein is a nanoparticle delivery vehicle formulation comprising the EON disclosed herein.In a preferred embodiment, the nanoparticle delivery vehicle is a lipid nanoparticle (LNP).Also provided herein is a pharmaceutical composition comprising the EON, vector, or nanoparticle delivery vehicle formulation disclosed herein and a pharmaceutically acceptable carrier.

[0012] In one aspect, the present disclosure provides an EON, vector, nanoparticle delivery vehicle formulation, or pharmaceutical composition disclosed herein for use in the treatment of CVD.

[0013] In one aspect, the disclosure provides for the use of an EON, vector, nanoparticle delivery vehicle formulation, or pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment of CVD.

[0014] In one aspect, the present disclosure provides an in vitro, ex vivo, or in vivo method for editing a B4GALT1 transcript molecule, the method comprising contacting the B4GALT1 transcript molecule or a portion thereof with an EON disclosed herein, thereby allowing the formation of a double-stranded complex between the EON and the B4GALT1 transcript molecule, thereby allowing recruitment of ADAR1 or ADAR2 deaminase that binds to the double-stranded complex, and thereby allowing adenosine in the B4GALT1 transcript molecule to be specifically edited to inosine by the deaminase.

[0015] In one aspect, the present disclosure provides a method of treating, delaying, or ameliorating CVD in a patient in need thereof, said method comprising contacting a B4GALT1 transcript molecule in a cell of the subject with an EON disclosed herein, thereby treating said patient.

[0016] In one aspect, the disclosure provides a method for deaminating a target adenosine in a B4GALT1 transcript molecule in a cell, the method comprising the steps of: (i) providing to said cells an EON, vector, or nanoparticle delivery vehicle formulation disclosed herein; (ii) uptake of the EON, the vector, or the nanoparticle delivery vehicle by the cells; (iii) annealing the EON to the B4GALT1 transcript molecule; (iv) allowing an endogenous ADAR enzyme naturally present in the cell to deaminate the target adenosine in the B4GALT1 transcript molecule to inosine; and (v) identifying the presence of the inosine in a target RNA molecule. In a preferred embodiment, the cell is a human cell, preferably a hepatocyte, more preferably a hepatocyte, wherein the target adenosine is present at position c.1055A in the B4GALT1 transcript, and wherein the deamination results in a change from asparagine (N; Asn) to serine (S; Ser) at position 352 in the human wild-type B4GALT1 amino acid sequence. In a preferred embodiment, step (v) of the method disclosed herein comprises: a) sequencing a B4GALT1 pre-mRNA or mRNA molecule, or a cDNA derivative thereof; b) assessing the presence of the 352Ser B4GALT1 protein variant; or c) Using a functional readout, preferably assessing the rate of depletion of UDP-Gal or assessing the glycosylation level of transferrin in serum.

[0017] [Brief description of the drawing] One or more embodiments will now be described, by way of example only, with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0018]

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[0019] It has been discovered that B4GALT1 transcripts can be targeted to regulate the activity of the B4GALT1 enzyme, thereby preventing, ameliorating, or treating CVD. This technology is commonly referred to as "RNA editing." Disclosed herein are oligonucleotides that can be used to specifically deaminate specific target adenosines in (human) B4GALT1 transcripts (pre-mRNA and / or mRNA) in vivo, preferably using endogenous deaminating enzymes to produce B4GALT1 enzyme variants with reduced galactosyltransferase activity. A particularly preferred target adenosine is found in the codon encoding asparagine at amino acid position 352 (Asn352), which upon deamination becomes a codon encoding serine (Ser352). However, the RNA editing technology disclosed herein can also be applied to other target adenosines within B4GALT1 that can be targeted to reduce the turnover rate of galactosyltransferase.

[0020] RNA editing is a natural process by which eukaryotic cells alter the sequences of their RNA molecules, often in a site-specific and precise manner, thereby expanding the genome-encoded RNA repertoire by several orders of magnitude. RNA editing enzymes have been reported in eukaryotic species throughout the animal and plant kingdoms, and these processes play a critical role in managing cellular homeostasis in metazoans, from the simplest organisms (e.g., Caenorhabditis elegans) to humans. Examples of RNA editing include the conversion of adenosine (A) to inosine (I) and cytidine (C) to uridine (U), which occur through enzymes called adenosine deamidase acting on RNA (ADAR) and APOBEC / AID (cytidine deamidase acting on RNA), respectively.

[0021] ADARs are multidomain proteins containing a catalytic domain and two to three double-stranded RNA recognition domains, depending on the enzyme in question. Each recognition domain recognizes a specific double-stranded RNA (dsRNA) sequence and / or conformation. While the catalytic domain also plays a role in recognizing and binding portions of the dsRNA helix, its primary function is to convert A to I by deaminating a nucleobase at a nearby (predefined) location in the target RNA. Inosine is read as guanosine by the cellular translation machinery, meaning that the edited adenosine can recode a protein sequence if present in the coding region of an mRNA or pre-mRNA. A to I conversion can also occur in the 5' non-coding sequence of the target mRNA, generating a new translation start site upstream of the original start site, which generates an N-terminally extended protein, or it can occur in the 3' UTR or other non-coding portion of the transcript, which can affect RNA processing and / or stability. Additionally, A-to-I conversion can occur at splice elements within introns or exons of pre-mRNA, thereby altering the splicing pattern, resulting in either inclusion or skipping of the exon. Enzymes that catalyze adenosine deamination belong to the ADAR enzyme family, which includes the human deaminases hADAR1, hADAR2, and hADAR3. However, hADAR3 has not demonstrated deaminating activity.

[0022] The use of oligonucleotides to apply adenosine deaminase to edit target RNA has been described (e.g., Woolf et al. 1995. Proc Natl Acad Sci USA 92:8298-8302; Montiel-Gonzalez et al. 2013. Proc Natl Acad Sci USA 110(45):18285-18290; Vogel et al. 2014. Angewandte Chemie Int Ed 53:267-271). A drawback of the method described in Montiel-Gonzalez et al. (2013, supra) is that it requires a fusion protein consisting of a genetic fusion of the boxB recognition domain of the bacteriophage lambda N protein with the adenosine deaminase domain of a truncated native ADAR protein. This method requires target cells to be transduced with the fusion protein (a major obstacle) or transfected with a nucleic acid construct encoding the modified adenosine deaminase fusion protein for expression. The system described by Vogel et al. (2014, supra) suffers from a similar drawback: it is unclear how to apply this system without first genetically modifying ADAR and then transfecting or transforming cells carrying the target RNA to provide the genetically modified protein. A similar system is also described in US 9,650,627. The oligonucleotides of Woolf et al. (1995, supra) were 100% complementary to the target RNA sequence, but suffered from a severe lack of specificity: nearly all adenosines in the target RNA strand complementary to the antisense oligonucleotide were edited.

[0023] ADARs are known to act on any dsRNA. Through a process sometimes referred to as "promiscuous editing," these enzymes edit multiple A's within dsRNA. Therefore, methods and tools were needed to circumvent such promiscuous editing and target specific adenosines within target RNA molecules for therapeutic applications. Vogel et al. (2014, supra) demonstrated that such off-target editing can be suppressed by using 2'-O-methyl (2'-OMe)-modified nucleosides in oligonucleotides opposite adenosines that should not be edited, and unmodified nucleosides in oligonucleotides directly opposite the specifically targeted adenosines on the target RNA. However, specific editing at the target nucleotide has not been shown to occur without the use of recombinant ADAR enzymes covalently linked to EONs. Several publications have shown that it is possible to recruit endogenous ADAR (thus not requiring exogenous and / or recombinant sources) while maintaining the specificity of targeting a single adenosine in a target RNA molecule and deaminating it to inosine. WO2016 / 097212 discloses an antisense oligonucleotide (AON) for targeted editing of RNA, which is characterized by a sequence complementary to the target RNA sequence (referred to as "targeting portion" in the publication) and the presence of a stem-loop / hairpin structure (referred to as "recruitment portion" in the publication), which is preferably non-complementary to the target RNA. Such oligonucleotides are referred to as "self-looping AONs". The recruitment portion acts to recruit the natural ADAR enzyme present in cells to the dsRNA formed by hybridization of the target sequence with the targeting portion. The presence of the recruitment moiety does not require the presence of the conjugate or modified recombinant ADAR enzyme.WO2016 / 097212 describes the recruitment moiety as a stem-loop structure that mimics a natural substrate (e.g., GluB receptor) or a Z-DNA structure (known to be recognized by the dsRNA-binding domain or Z-DNA-binding domain of ADAR enzymes). The stem-loop structure can 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 stem-loop structure of the recruitment moiety is an intramolecular stem-loop structure formed within the AON itself, which is thought to attract (endogenous) ADAR. Systems containing similar stem-loop structures for RNA editing are described in WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995.

[0024] WO2017 / 220751 and WO2018 / 041973 describe next-generation AONs, which do not contain such stem-loop structures but are (almost perfectly) complementary to target regions.In one embodiment, one or more mismatched nucleotides, wobbles, or bulges exist between the oligonucleotide and the target sequence.Sometimes, the mismatch only exists at the nucleoside site opposite the target adenosine, while in other embodiments, AONs (or RNA editing oligonucleotides, abbreviated as "EONs") have multiple bulges and / or wobbles when added to target sequence regions.It appears that if the sequence of EONs is carefully selected to attract / recruit ADAR, it may be possible to achieve RNA editing in vitro, ex vivo, and in vivo using EONs that lack stem-loop structures and endogenous ADAR enzymes. An "orphan nucleoside" is defined as a nucleoside within an EON directly opposite a target adenosine in a target RNA molecule, but which did not have a 2'-OMe modification. The orphan nucleoside may be a deoxyribonucleoside (DNA), with the remainder of the EON still having a 2'-O-alkyl modification (e.g., 2'-OMe) in the sugar moiety, or the nucleotides immediately surrounding the orphan nucleoside may contain chemical modifications (e.g., DNA compared to RNA), resulting in further improved RNA editing efficiency and / or increased resistance to nucleases. Such effects can be further improved by the use of sense oligonucleotides (SONs) to "protect" the EON from degradation (as described in WO 2018 / 134301).The use of chemical modifications and specific structures in oligonucleotides that can be used for ADAR-mediated editing of specific adenosines in target RNA has been the subject of numerous publications in the field, for example, WO2019 / 111957, WO2019 / 158475, WO2020 / 165077, WO2020 / 201406, WO2020 / 211780, WO2021 / 008447, WO2 021 / 020550, WO2021 / 060527, WO2021 / 117729, WO2021 / 136408, WO2021 / 182474, WO2021 / 216853, WO2021 / 242778, WO2021 / 242870, WO2021 / 242889, WO2022 / 007803, WO2022 / 018207, WO2022 / 026928, and WO2022 / 124345. The use of certain sugar moieties is disclosed, for example, in WO2020 / 154342, WO2020 / 154343, WO2020 / 154344, WO2022 / 103839, and WO2022 / 103852.On the other hand, the use of sterically defined linker moieties (generally for oligonucleotides that can be used, for example, for exon skipping, gapmers, siRNA, or specifically for RNA editing oligonucleotides related to a wide variety of target sequences) has been described in WO2011 / 005761, WO2014 / 010250, WO2014 / 012081, WO2015 / 1 07425, WO2017 / 015575(HTT), WO2017 / 062862, WO2017 / 160741, WO2017 / 192664, WO2017 / 192679(DMD), WO201 7 / 198775, WO2017 / 210647, WO2018 / 067973, WO2018 / 098264, WO2018 / 223056(PNPLA3), WO2018 / 223073(APOC 3), WO2018 / 223081(PNPLA3), WO2018 / 237194, WO2019 / 032607(C9orf72), WO2019 / 055951, WO2019 / 075357( SMA / ALS), WO2019 / 200185(DM1), WO2019 / 217784(DM1), WO2019 / 219581, WO2020 / 118246(DM1), WO2020 / 1912 52, WO2020 / 196662, WO2020 / 219981 (USH2A), WO2020 / 219983 (RHO), WO2020 / 227691 (C9orf72), WO2021 / 071788 (C9orf72), WO2021 / 071858, WO2021 / 178237 (MAPT), WO2021 / 234459, WO2021 / 237223, and WO2022 / 099159. In addition to these disclosures, there are numerous publications relating to targeting specific RNA target molecules, or specific adenosines within such RNA target molecules, to repair mutations that result in premature stop codons or other disease-causing mutations.Examples of such disclosures targeting adenosines within specific RNA target molecules include: WO2020 / 157008 and WO2021 / 136404 (USH2A); WO2021 / 113270 (APP); WO2021 / 113390 (CMT1A); WO2021 / 209010 (IDUA, Hurler syndrome); WO2021 / 231673 and WO2021 / 242903 (LRRK2); WO2021 / 231675 (ASS1); WO2021 / 23 1679 (GJB2); WO2019 / 071274 and WO2021 / 231680 (MECP2); WO2021 / 231685 and WO2021 / 231692 (OTOF, autosomal recessive non-syndromic hearing loss); WO2021 / 231691 (XLRS); WO2021 / 231698 (argininosuccinate lyase deficiency); WO2021 / 130313 and WO2021 / 231830 (ABCA4); and WO2021 / 243023 (SERPINA1).

[0025] [Embodiment] According to one aspect, the present disclosure provides an EON capable of forming a double-stranded complex with a region of an endogenous human B4GALT1 transcript molecule in a cell, wherein the region or portion thereof of the B4GALT1 transcript molecule includes a target adenosine, the nucleotide in the EON opposite the target adenosine is a lone nucleotide, and the double-stranded complex can recruit an endogenous ADAR enzyme present in the cell to deaminate the target adenosine to inosine, thereby editing the B4GALT1 transcript molecule.

[0026] According to one aspect, the present disclosure provides an EON capable of forming a double-stranded complex with a region of an endogenous human B4GALT1 transcript molecule in a cell, wherein the region or portion thereof of the B4GALT1 transcript molecule includes a target adenosine, wherein the nucleotide in the EON opposite the target adenosine is a lone nucleotide, and wherein the double-stranded complex can recruit an endogenous ADAR enzyme present in the cell to deaminate the target adenosine to inosine, thereby editing the B4GALT1 transcript molecule, and wherein the EON causes a splice regulation event, preferably exon 5, to be skipped from pre-mRNA, thereby removing the exon containing the target adenosine (edited or not) from the transcript and leaving an mRNA that is not translated into a functional B4GALT1 protein. The splice regulation event may be exon 5 skipping, aberrant exon skipping, exon 4 + exon 5 skipping, intron retention, and (partial) exon 6 skipping.

[0027] In one embodiment, the B4GALT1 transcript molecule is a pre-mRNA or mRNA molecule.

[0028] In one embodiment, the cells are human liver cells, preferably hepatocytes.

[0029] In one embodiment, the target adenosine is present in the B4GALT1 transcript at a position where the guanosine results in a B4GALT1 protein variant with a reduced enzyme turnover rate.

[0030] In one embodiment, the target adenosine is present at position c.1055A in the B4GALT1 transcript.

[0031] In one embodiment, said region or portion of said B4GALT1 transcript molecule has the sequence: 5'-...CCCA A UCCU...containing -3', where Ais the target adenosine.

[0032] In one embodiment, the EON comprises or consists of an EON individually selected from the group consisting of EONs detailed in SEQ ID NOs: 3-42, 59-1069, and 1078-1190, preferably selected from the group consisting of SEQ ID NOs: 23, 19, 31, 27, 35, 39, 69, 70, 71, 72, 73, 93, 94, 95, 1079, 1084, 1093, 1095, 1100, 1102, 1115, 1121, 1123, 1124, and 1139-1190.

[0033] In one embodiment, the EON comprises or consists of a basic nucleotide sequence set forth in any of SEQ ID NOs: 1139-1190, wherein: - the EON may comprise a GalNAc moiety (e.g., L001 as disclosed herein), and the GalNAc moiety may be linked to the EON via a linker (e.g., the L103 TEG linker as disclosed herein); - the EON may comprise a nucleotide with a modified sugar moiety as disclosed herein, preferably selected from the group consisting of LNA, 2'-MOE, 2'-OMe, 2'-F, and 2'-H (DNA); - said EON may comprise a nucleotide in which the nucleobase moiety is modified as disclosed herein; - said EON may comprise nucleosides linked to each other by bonds disclosed herein, and is preferably selected from the group consisting of PS, PNdmi, MP, and PNms; - the EON comprises an isolated nucleotide, the isolated nucleotide being preferably a deoxynucleotide comprising a cytosine base, a uracil base, an isouracil base, or a cytosine analog, more preferably a benar base; - said EON comprises a nucleotide at position -1, said nucleotide being preferably a deoxynucleotide; - said EON comprises a nucleotide at position +1, said nucleotide being preferably a deoxynucleotide or a 2'-MOE modified nucleotide; - said EON comprises a bond at bond position -2, said bond being preferably an MP bond or a PNms bond; - said EON comprises a bond at bond position -1, said bond being preferably a PS bond; said EON comprises, at positions +1, 0 and -1, the sequence 5'-AXU-3', wherein: a) A is a nucleoside having an adenine base, preferably A is deoxyadenosine (Ad) or 2'-MOE modified adenosine (Ae); b) X is an isolated nucleotide, said isolated nucleotide being a deoxynucleotide containing a modification disclosed herein, preferably containing a Benner base (Zd) or isouracil (8d); and c) U is a nucleoside having a uracil base, preferably U is deoxyuridine (Ud, or m5Ud); - the EON may contain a PNdmi bond linking the 5'-most nucleoside to its adjacent nucleoside; and / or - said EON may contain a PNdmi bond linking the 3'-most nucleoside to its adjacent nucleoside.

[0034] SEQ ID NOs: 1139-1151 are as follows (5'→3'), where X is a lone nucleotide, preferably a deoxynucleotide having a benign base (Zd), and the sequences at positions +1, 0, and -1 (sometimes referred to as the "Central Triplet") are underlined: 1139 CCUCUGAGG AXU GGGUUCAUUU 1140 CCUCUGAGG AXU GGGUUCAUUUUU 1141 AACCUCUGAGG AXU GGGUUCAUUU 1142 AACCUCUGAGG AXU GGGUUCAUUUU 1143 ACCUCUGAGG AXU GGGUUCAUUUUU 1144 AACCUCUGAGG AXU GGGUUCAUUUUU 1145 CACCUCUGAGG AXU GGGUUCAUUUUU 1146 GCACCUCUGAGG AXU GGGUUCAUUUUU 1147 CAAACCUCUGAGG AXU GGGUUCAUUUUU 1148 CGGUCAAACCUCUGAGG AXU GGGUUCAU 1149 GUCAAACCUCUGAGG AXU GGGUUCAUUUUU 1150 CGGUCAAACCUCUGAGG AXU GGGUUCAUUU 1151 CGGUCAAACCUCUGAGG AXU GGGUUCAUUUUU

[0035] SEQ ID NOs: 1152-1164 are as follows (5'→3'), where X is a lone nucleotide, preferably deoxycytidine, and the sequences at positions +1, 0, and -1 (sometimes referred to as the "central triplet") are underlined: 1152 CCUCUGAGG AXU GGGUUCAUUU 1153 CCUCUGAGG AXU GGGUUCAUUUUU 1154 AACCUCUGAGG AXU GGGUUCAUUU 1155 AACCUCUGAGG AXU GGGUUCAUUUU 1156 ACCUCUGAGG AXU GGGUUCAUUUUU 1157 AACCUCUGAGGAXU GGGUUCAUUUUU 1158 CACCUCUGAGG AXU GGGUUCAUUUUU 1159 GCACCUCUGAGG AXU GGGUUCAUUUUU 1160 CAAACCUCUGAGG AXU GGGUUCAUUUUU 1161 CGGUCAAACCUCUGAGG AXU GGGUUCAU 1162 GUCAAACCUCUGAGG AXU GGGUUCAUUUUU 1163 CGGUCAAACCUCUGAGG AXU GGGUUCAUUU 1164 CGGUCAAACCUCUGAGG AXU GGGUUCAUUUUU

[0036] SEQ ID NOs: 1165-1177 are as follows (5'→3'), where X is a lone nucleotide, preferably deoxyuridine, and the sequences at positions +1, 0, and -1 (sometimes referred to as the "central triplet") are underlined: 1165 CCUCUGAGG AXU GGGUUCAUUU 1166 CCUCUGAGG AXU GGGUUCAUUUUU 1167 AACCUCUGAGG AXU GGGUUCAUUU 1168 AACCUCUGAGG AXU GGGUUCAUUUU 1169 ACCUCUGAGG AXU GGGUUCAUUUUU 1170 AACCUCUGAGG AXU GGGUUCAUUUUU 1171 CACCUCUGAGG AXU GGGUUCAUUUUU 1172 GCACCUCUGAGG AXUGGGUUCAUUUUU 1173 CAAACCUCUGAGG AXU GGGUUCAUUUUU 1174 CGGUCAAACCUCUGAGG AXU GGGUUCAU 1175 GUCAAACCUCUGAGG AXU GGGUUCAUUUUU 1176 CGGUCAAACCUCUGAGG AXU GGGUUCAUUU 1177 CGGUCAAACCUCUGAGG AXU GGGUUCAUUUUU

[0037] SEQ ID NOs: 1178-1190 are as follows (5'→3'), where X is a lone nucleotide, preferably a deoxynucleotide having an isouracil base, and the sequences at positions +1, 0, and -1 (sometimes referred to as the "central triplet") are underlined: 1178 CCUCUGAGG AXU GGGUUCAUUU 1179 CCUCUGAGG AXU GGGUUCAUUUUU 1180 AACCUCUGAGG AXU GGGUUCAUUU 1181 AACCUCUGAGG AXU GGGUUCAUUUU 1182 ACCUCUGAGG AXU GGGUUCAUUUUU 1183 AACCUCUGAGG AXU GGGUUCAUUUUU 1184 CACCUCUGAGG AXU GGGUUCAUUUUU 1185 GCACCUCUGAGG AXU GGGUUCAUUUUU 1186 CAAACCUCUGAGG AXU GGGUUCAUUUUU 1187 CGGUCAAACCUCUGAGG AXUGGGUUCAU 1188 GUCAAACCUCUGAGG AXU GGGUUCAUUUUU 1189 CGGUCAAACCUCUGAGG AXU GGGUUCAUUU 1190 CGGUCAAACCUCUGAGG AXU GGGUUCAUUUUU

[0038] In one embodiment, at least one nucleotide of an EON disclosed herein comprises one or more non-natural chemical modifications in the ribose moiety, linkage moiety, or base moiety, or one or more additional non-natural chemical modifications, provided that the isolated nucleotide is not a cytidine containing a 2'-OMe ribose substitution.

[0039] In one embodiment, the one or more additional modifications in the linking moiety are each independently selected from a PS, phosphonoacetate, phosphorodithioate, MP, sulfonyl phosphoramidate, or PNdmi internucleotide linkage.

[0040] In one embodiment, the EON disclosed herein comprises one or more nucleotides containing mono- or di-substitutions at the 2′, 3′, and / or 5′ positions of ribose, each independently selected from the group consisting of: —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C 10 ) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-aryl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy.

[0041] According to one aspect, disclosed herein is a vector, preferably a viral vector, more preferably an AAV vector, comprising a nucleic acid molecule encoding an EON disclosed herein.

[0042] According to one aspect, disclosed herein is a nanoparticle delivery vehicle formulation comprising the EON disclosed herein. Preferably, the nanoparticle delivery vehicle formulation disclosed herein is an LNP formulation.

[0043] According to one aspect, disclosed herein is a method for editing a B4GALT1 polynucleotide, the method comprising contacting the B4GALT1 polynucleotide with an EON disclosed herein to induce CVD-associated adenosine to inosine conversion mediated by adenosine deaminase acting on RNA (ADAR), thereby editing the B4GALT1 polynucleotide. The polynucleotide is preferably a pre-mRNA or mRNA molecule.

[0044] According to one aspect, disclosed herein is a method of treating CVD in a patient in need thereof, the method comprising contacting a B4GALT1 polynucleotide in the subject's cells with an EON, vector, nanoparticle delivery vehicle formulation, or pharmaceutical composition disclosed herein to cause ADAR-mediated adenosine to inosine conversion of adenosine associated with CVD, thereby treating the patient.

[0045] According to one aspect, disclosed herein is a method of treating CVD, said method comprising administering to a patient in need thereof a therapeutically effective amount of an EON, vector, nanoparticle delivery vehicle formulation, or pharmaceutical composition disclosed herein, thereby treating said CVD.

[0046] According to one aspect, the present disclosure provides a method for deaminating a target adenosine in a human B4GALT1 pre-mRNA or mRNA molecule in a cell, the method comprising the steps of: (i) providing to said cell an EON disclosed herein; (ii) allowing the EON to be taken up by the cells; (iii) annealing the EON to the B4GALT1 pre-mRNA or mRNA molecule; (iv) allowing an endogenous ADAR enzyme to deaminate the target adenosine to inosine in the target RNA molecule; and (v) identifying the presence of said inosine in said target RNA molecule. In one embodiment, said target adenosine is present at position c.1055A in said B4GALT1 pre-mRNA or mRNA molecule. In one embodiment, step (v) comprises: a) determining the sequence of the B4GALT1 pre-mRNA or mRNA molecule; b) assessing the presence of the 352Ser B4GALT1 protein variant; or c) Using a functional readout, preferably assessing the rate of depletion of UDP-Gal or assessing the glycosylation level of transferrin in serum.

[0047] The present disclosure provides an EON capable of RNA editing target adenosines in human B4GALT1 transcripts (pre-mRNA and / or mRNA) to produce B4GALT1 enzyme variants with reduced turnover rates. In one embodiment, the EON results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of B4GALT1 transcript molecules encoding variant B4GALT1. The target adenosine may be at any position where editing reduces turnover rate. A reduced turnover rate relative to the wild-type enzyme may be beneficial. In one embodiment, the reduced turnover rate of the B4GALT1 variant is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduced compared to the turnover rate of the variant relative to the wild-type enzyme. In one embodiment, the reduced turnover rate can be measured by determining the rate at which the B4GALT1 enzyme can transfer galactose from UDP-Gal to GlcNAc as an acceptor, as described in Montasser et al. (2021, supra). Editing of the targeted adenosine can reduce the turnover rate of the B4GALT1 enzyme variant through various mechanisms, including, for example, mutation of the amino acid residue, generation of a premature stop codon, or alteration of a pre-mRNA splice site.

[0048] It should be further noted that the reduction in turnover rate throughout a system (e.g., in the intracellular environment) can depend on multiple factors, including the reduction in the turnover rate of any enzyme variant and the ratio of the enzyme variant to the wild-type enzyme produced in that system, and balancing these can produce the desired effect. For example, Montasser et al. (2021, supra) describe the use of a different editing technique, namely gene editing using CRISPR-Cas9 knockdown of the B4GALT1 gene (i.e., to encode a completely inactivated B4GALT1). In this model, gene editing was not complete, with 20.7% of the transcripts being active wild-type B4GALT1. This also resulted in a significant reduction (50%) in LDL-C. One advantage of the RNA editing approach disclosed herein is that RNA editing is transient, making it much easier to tailor the level of the edited transcript. This can be particularly useful when the reduction level needs to be continually reassessed and adjusted, for example, in response to disease progression / regression and / or changes in substrate levels within the system.

[0049] In one embodiment, the reduction in the turnover rate of the entire system is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% reduction in the turnover rate of the system containing EON compared to the system without EON. The ratio of the enzyme variant to the wild-type enzyme produced in the system can be controlled by selecting reaction conditions (e.g., selecting the concentration or temperature of the reactants), but in therapeutic situations, this often depends on the dose of EON provided. Those skilled in the art can achieve an optimal reduction in the turnover rate of the entire system by appropriately manipulating and optimizing these factors. For example, even if the B4GALT1 enzyme variant is non-functional, the dose of EON can be controlled so that the entire system still contains a certain level of functional wild-type B4GALT1. This is an important advantage of RNA editing over DNA editing, because editing DNA to encode a non-functional B4GALT1 would render all B4GALT1 enzyme molecules non-functional. In one embodiment, the wild-type enzyme has the sequence shown in Figure 1B.

[0050] In one embodiment, the EON causes the deamination of an adenosine present at position 1055 of human mRNA, thereby producing inosine. In other words, the AAU codon encoding asparagine at amino acid position 352 is converted to an AIU codon, which is read as AGU, encoding serine. In another embodiment, the EON disclosed herein causes the deamination of another adenosine present in the B4GALT1 transcript, which can be any adenosine that, when deaminated to inosine, results in a B4GALT1 enzyme with a reduced turnover rate. Other mutations that reduce normal B4GALT1 function, which can be mediated through RNA editing, can also be added to the B4GALT1 gene (and transcript). It should be understood that "causing," "triggering," or "producing" deamination does not imply that the EON itself edits adenosines (or has enzymatic activity). The double-stranded complex between the EON and the target RNA molecule is bound to the ADAR enzyme, which acts as a deaminating entity. ADAR is the enzyme that carries out the deamination, while the EON is responsible for targeting it to the desired site.

[0051] In one embodiment, the EON disclosed herein is a single-stranded oligonucleotide comprising an isolated nucleotide opposite a target adenosine, wherein the isolated nucleotide is chemically modified as disclosed herein, and the remainder of the oligonucleotide is chemically modified as disclosed herein to prevent degradation by nucleases. In one embodiment, the present disclosure relates to any kind of oligonucleotide or heteroduplex oligonucleotide complex, which may or may not be bound to a hairpin structure (internal or at terminal(s)), may bind to ADAR or its catalytic domain, or the oligonucleotide may be expressed through a vector such as AAV, or may be circular. Any kind of oligonucleotide-based RNA editing should be understood to be included as long as it is associated with the deamination of nucleotides in B4GALT1 transcripts, preferably the generation of the enzyme variant p.Asn352Ser, which causes a decrease in B4GALT1 enzyme function. The protein mutation designated p.Asn352Ser may also be referred to as N352S, while the adenosine to guanosine change at position 1055 in the B4GALT1 transcript may also be referred to as c.1055A>G. In one embodiment, the EONs disclosed herein are "naked" oligonucleotides that contain various chemical modifications to the ribose sugar, base, and / or internucleoside linkage of one or more nucleotides within the sequence and are capable of hybridizing to a B4GALT1 transcript or portion thereof containing a target adenosine and recruiting endogenous ADARs for deamination of the target adenosine.

[0052] [Definition] The term "nucleoside" refers to a nucleic acid base linked to a (deoxy)ribose without a phosphate group. A "nucleotide" is composed of a nucleoside and one or more phosphate groups. The term "nucleotide" therefore refers to the respective nucleic acid base-(deoxy)ribose-phospholinker and any chemical modification of the ribose moiety or phospho group. The term therefore includes: nucleotides containing locked ribose moieties (including 2'-4' bridges containing methylene groups or any other groups), unlocked nucleic acids (UNA), threose nucleic acids (TNA), nucleotides containing linkers containing phosphodiesters, phosphonoacetates, phosphotriesters, PS, phosphoro(di)thioates, MP, methylthiophosphonates, phosphoramidate bonds, etc. The terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, and inosine and hypoxanthine are sometimes used interchangeably to refer to the corresponding nucleobases on the one hand and the nucleosides or nucleotides on the other hand. Thymine (T) is a nucleotide that is substituted with 5-methyluracil (m 5 Thymine, 5-methyluracil, and uracil are also known as 5-methyl-uridine and are derivatives of uridine; thymidine, 5-methyl-uridine, and uridine are also used interchangeably throughout this disclosure. The terms nucleobase, nucleoside, and nucleotide are sometimes used interchangeably unless the context clearly dictates otherwise (e.g., when a nucleoside is linked to adjacent nucleosides and the bond between these nucleosides is modified). As used 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 in the art.

[0053] Whenever oligonucleotides, oligos, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA) editing oligonucleotides, EONs, and RNA (antisense) oligonucleotides are mentioned, unless the context dictates otherwise, to refer to both oligoribonucleotides and deoxyoligoribonucleotides. In some cases, oligonucleotides may be completely devoid of RNA or DNA nucleotides (as they naturally occur) and consist entirely of modified nucleotides. Whenever "oligoribonucleotides" are mentioned, they may contain the bases A, G, C, U, or I. Whenever "deoxyoligoribonucleotides" are mentioned, they may contain the bases A, G, C, T, or I. However, the oligonucleotides disclosed herein may contain a mixture of ribonucleosides and deoxyribonucleosides. When deoxyribonucleotides are used, i.e., when there is no modification at the 2' position of the sugar, the nucleotide is often abbreviated as Ad, Cd, Gd, Id, Ud, or T, where "d" denotes the deoxy nature of the nucleoside. On the other hand, normal RNA or ribonucleosides modified at the 2' position are often abbreviated without the "d" and often abbreviated with their respective modifications as described herein.

[0054] Whenever nucleotide is mentioned in oligonucleotide, for example, cytosine includes 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine.Whenever adenine is mentioned, it includes N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine, and 7-methyladenine.Whenever uracil is mentioned, it includes dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudouracil, 4-thiouracil, and 5-hydroxymethyluracil.Whenever guanine is mentioned, it includes 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. Whenever a nucleoside or nucleotide is referred to, ribofuranose derivatives such as 2'-deoxy, 2'-hydroxy, and 2'-O-substituted variants (e.g., 2'-OMe) are included, as well as other modifications, including 2'-4' bridged variants. Whenever an oligonucleotide is referred to, the linkage between two mononucleotides may be a phosphodiester bond, and modifications thereof include phosphonoacetate, phosphotriester, PS, phosphoro(di)thioate, MP, phosphoramidate linker, phosphorylguanidine, thiophosphorylguanidine, sulfonophosphoamidate, and the like.

[0055] The term "comprising" encompasses "including" and "consisting of," e.g., a composition "comprising X" may consist solely of X or may include something additional (e.g., X+Y). The term "about" in reference to a numerical value x is optional, and means, for example, x±10%.

[0056] The word "substantially" does not exclude "completely", for example a composition that is "substantially free of Y" may be completely free of Y. Where relevant, the word "substantially" may be omitted from the definition.

[0057] The term "complementary" as used herein refers to the fact that an EON hybridizes with a second nucleic acid strand under physiological conditions (e.g., when an oligonucleotide, as a first nucleic acid strand (=guide oligonucleotide), forms a heteroduplex RNA-editing oligonucleotide complex (i.e., HEON) with another complementary nucleic acid strand, or when it forms a double-stranded complex with a target RNA sequence). This term does not necessarily mean that each nucleotide in a nucleic acid strand perfectly pairs with the opposite nucleotide in the opposite sequence. In other words, an EON can be complementary to a target sequence, but there may be mismatches, wobble, and / or bulges between the oligonucleotide and the target sequence, while the EON still hybridizes with the target sequence under physiological conditions, allowing RNA-editing enzymes in cells to edit the target adenosine. Thus, the term "substantially complementary" also means that the EON has sufficient nucleotide matches between the EON and the target sequence such that the EON can hybridize to the target RNA under physiological conditions, even if mismatches, wobble, and / or bulges are present. As provided herein, an EON can be complementary but contain one or more mismatches, wobble, and / or bulge with the target sequence, provided that the EON can hybridize to its target under physiological conditions.

[0058] The term "downstream," with respect to a nucleic acid sequence, means further along the sequence in the 3' direction; the term "upstream" means the opposite. Thus, in any sequence that encodes a polypeptide, the start codon is upstream of the stop codon on the sense strand, but downstream of the stop codon on the antisense strand.

[0059] References to "hybridization" typically refer to specific hybridization and exclude non-specific hybridization, which may occur under selected experimental conditions using techniques well known in the art to ensure that the most stable interaction between probe and target is when the probe and target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0060] As used herein, the term "mismatch" refers 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 the traditional sense, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the EONs disclosed herein contain fewer than four mismatches with the target sequence, e.g., 0, 1, or 2 mismatches. "Wobble" base pairs are GU, IU, IA, and IC base pairs. Although G:G pairing is considered a mismatch, this does not necessarily mean that the interaction is unstable; the term "mismatch" may be somewhat outdated based on prior art and current disclosures that show that Hoogsteen base pairings, even when considered mismatches based on the origin of the nucleotides, are still relatively stable. An isolated G:G pairing within a duplex RNA, for example, may be very stable but is still defined as a mismatch.

[0061] The term "splice mutation" refers to a mutation in a gene encoding a pre-mRNA in which the splicing machinery is dysfunctional in the sense that splicing of an intron from an exon is disrupted and this aberrant splicing causes subsequent translation to frame out, resulting in premature termination of the encoded protein. Such truncated proteins are often rapidly degraded and lack any functional activity.

[0062] The EONs disclosed herein (and the complementary nucleic acid strands when two oligonucleotides form an HEON) can be chemically modified throughout, for example, by providing ribose sugar moieties with 2'-OMe, 2'-F, or 2'-O-methoxyethyl (2'-MOE) substitutions at the nucleotides. The isolated nucleotides in the EONs are preferably cytidine or its analogs (e.g., nucleotides with a benign base), or uridine or its analogs (e.g., isouridine), and / or, in one embodiment, contain a diF modification at the 2'-sugar position of the sugar, in another embodiment, deoxyribose (2'-H, DNA), and in yet another embodiment, at least one, and in another embodiment, both, of the two adjacent nucleotides flanking the isolated nucleotide do not contain a 2'-OMe modification. Full modification, in which all nucleotides of an oligonucleotide have natural bases but retain 2'-OMe modifications, results in an oligonucleotide that does not function in RNA editing (as is known in the art), presumably because it prevents ADAR activity at the target position. Generally, an adenosine in a target RNA can be protected from editing by providing a 2'-OMe group at the opposing nucleotide (at least in the absence of other chemical substitutions or modifications within the nucleotide) or by providing guanine or adenine as the opposing base, as these two nucleobases can also reduce editing of the opposing adenosine.

[0063] A variety of chemical techniques and modifications are known in the field of oligonucleotides that can be readily used in accordance with the present disclosure. The normal internucleoside linkage between nucleotides can be altered by mono- or dithiolation of the phosphodiester bond to produce PS esters or phosphorodithioate esters, respectively. Other modifications of the internucleoside linkage are possible, including amidation and peptide linkers.

[0064] In one embodiment, the EON disclosed in the present invention comprises 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.

[0065] It is known in the art that RNA editors (e.g., human ADAR enzymes) edit dsRNA structures with varying specificity, depending on several factors. One important factor is the degree of complementarity between the two strands that make up the dsRNA sequence. When the two strands are completely complementary, the catalytic domain of human ADAR usually reacts with all adenosines it encounters, causing indiscriminate adenosine deamination. The specificity of hADAR1 and 2 can be enhanced by introducing chemical modifications and / or ensuring multiple mismatches within the dsRNA, which is presumably useful for positioning the dsRNA binding domain in a yet-to-be-defined manner. Furthermore, the deamination reaction itself can be promoted by providing an oligonucleotide containing a mismatch opposite the adenosine to be edited. By following the instructions of the present disclosure, those skilled in the art can design the complementary portion of an oligonucleotide according to their needs.

[0066] The most interesting intracellular RNA editing proteins to be used with the EON of the present disclosure are human ADAR1 and / or ADAR2. Those skilled in the art will understand that the degree to which the editing product in the cell is redirected to other target sites can be adjusted by changing the affinity of the EON for the recognition domain of the editing enzyme. The modification itself can be determined through some trial and error and / or through a calculation method based on the structural interaction between the EON and the recognition domain of the editing enzyme. Additionally or alternatively, the degree of recruitment and redirection of the editing enzyme present in the cell can be adjusted by the dosage and administration regimen of the EON. This is determined by experimenters (in vitro) or clinicians, usually in phase I and / or phase II clinical trials.

[0067] The present disclosure provides for the modification of target RNA sequences in eukaryotic, preferably metazoan, more preferably mammalian, and most preferably human cells. The present disclosure is particularly suitable for modifying RNA sequences in cells and tissues in which B4GALT1 is expressed and in which the enzyme acts. B4GALT1 is an enzyme that plays a key role in the synthesis of complex carbohydrates called glycoconjugates. It has been shown that reducing B4GALT1 enzymatic activity can have a protective effect against CVD, but the exact mechanism by which this occurs is not fully understood. B4GALT1 is involved in the assembly of oligosaccharides with specific sequences and structures, specifically oligosaccharides that are part of peptidoglycan. These oligosaccharides are essential for the proper function and stability of proteins. Reduced B4GALT1 activity can result in reduced levels of intact oligosaccharides and subsequent reduced biological activity. One important class of oligosaccharides that B4GALT1 is involved in the assembly of is terminated with a sialylated group, and sialylation is dependent on a preceding galactosylation step. Montasser et al. (2021, supra) found that decreased B4GALT1 activity was associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin.

[0068] Target cells can be located in vitro, ex vivo, or in vivo. One advantage of the material of the present disclosure is that it can be used on cells in situ in vivo, but can also be used on cultured cells. In some embodiments, cells are treated ex vivo and then introduced into a living body (e.g., reintroduced into the living body from which they originally originated). The present disclosure can also be used to edit target RNA sequences in cells derived from transplants or cells within so-called organoids (e.g., liver tissue organoids or "spheroids"). Organoids are considered to be three-dimensional in vitro-derived tissues, but are driven to generate individual, isolated tissues using specific conditions. In therapeutic settings, organoids are useful because they can be generated in vitro from patient cells and then reintroduced into patients as autologous material, which is less likely to be rejected than ordinary transplants.

[0069] Without wishing to be bound by theory, it is believed that RNA editing through ADARs occurs on primary transcripts either in the nucleus during transcription or splicing, or in the cytoplasm, where, for example, mature mRNA, miRNA, or ncRNA may be edited.

[0070] It will be apparent that targeted editing according to the present disclosure can be applied to any adenosine in a B4GALT1 transcript, provided that adenosine deamination reduces B4GALT1 function. However, as outlined herein, it is preferable to target the adenosine at position 1055 in the wild-type B4GALT1 transcript, changing the codon AAU (encoding asparagine) to AIU (or AGU, encoding serine). Generally speaking, RNA editing can be used to create RNA sequences with different properties. Such properties can be coding properties (creating proteins with different sequences or lengths, altering the properties or function of the protein) or binding properties (causing inhibition or overexpression of the RNA itself or a target or binding partner; entire expression pathways can be altered by recoding their corresponding sequences on the miRNA or target RNA). Protein function or localization can be freely modified by functional domains or recognition motifs (including, but not limited to, signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co- or post-translational modification, catalytic sites for enzymes, binding sites for binding partners, signals for degradation or activation, etc.). As disclosed herein, the EONs disclosed herein can also cause splice effects such as exon skipping (e.g., skipping of exon 5), which is not necessarily a bad thing because the resulting mRNA may transiently encode an inactive B4GALT1 protein (because the original DNA encoding the protein remains unaltered). These and other forms of RNA and protein "engineering" are encompassed by the present disclosure, whether for the purposes of disease prevention, delay, or treatment, or for other purposes, in medicine or biotechnology, as diagnostics, prophylactics, therapeutics, research tools, or otherwise. Thus, any RNA editing of a target adenosine in a B4GALT1 transcript that results in a decrease in the turnover rate of B4GALT1 is encompassed by the present disclosure.

[0071] The present disclosure relates to the completely new field of treating CVD using gene editing technology.Gene editing technology is not particularly limited.Suitable technology includes known gene therapy technology, including: DNA editing technology such as CRISPR / Cas, ZFN, TALEN and meganuclease; preferably RNA editing technology, such as ADAR-mediated editing technology, which will be described in more detail herein.

[0072] The amount, dosage, and administration regimen of administered EON may vary depending on cell type, disease to be treated, target population, mode of administration (e.g., systemic or local), severity of disease, and tolerance level of side effects, but these may and should be evaluated by trial and error during in vitro studies, preclinical and clinical trials.Testing is particularly easy when the modified sequence causes easily detectable phenotypic changes or changes in specific biomarkers (levels or activity).Higher doses of EON may compete for binding to ADAR in cells, thereby depleting the amount of entities free to participate in RNA editing, but regular administration tests will reveal such effects, if any, for a given EON and a given target.

[0073] One suitable testing technique involves delivering EONs to a cell line or test organism and then taking biopsies at various time points. The sequence of the target RNA in the biopsy samples can be assessed, allowing for easy tracking of the percentage of cells with the modification. Once this testing is performed, knowledge is retained, allowing subsequent deliveries to be performed without the need for biopsies. Thus, the methods disclosed herein can include identifying the presence of a desired change in the target RNA sequence in a cell, thereby verifying that the target RNA sequence has been modified. This step typically involves sequencing the relevant portion of the target RNA, or a cDNA copy thereof (or, if the target RNA is pre-mRNA, a cDNA copy of its splicing product), as described above, allowing for easy verification of the sequence change. Alternatively, the change can be assessed based on protein function, for example, by measuring the rate of decrease in UDP-Gal before and after treatment, or by assessing the glycosylation level of transferrin in serum, or other potential markers. These measurements are preferably performed in vitro on samples obtained from the treated subject.

[0074] After RNA editing occurs in a cell, the modified RNA may be diluted over time, e.g., due to cell division, the finite half-life of the edited RNA, etc. Thus, in terms of practical treatment, the methods disclosed herein may involve repeated delivery of EONs until enough of the target RNA is modified to provide a tangible benefit to the patient and / or to maintain that benefit over time.

[0075] The EONs disclosed herein are particularly suitable for therapeutic use, and therefore the present disclosure also relates to pharmaceutical compositions comprising the EONs disclosed herein, or vectors or plasmids encoding the EONs disclosed herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier can simply be a saline solution. This can be useful, particularly for pulmonary delivery, as being isotonic or hypotonic. The present disclosure also provides a delivery device (e.g., a syringe, an inhaler, a nebulizer) comprising the pharmaceutical composition disclosed herein.

[0076] The present disclosure also provides the EONs disclosed herein for use in the treatment of CVD. This treatment can be achieved by altering the target B4GALT1 RNA sequence in mammalian, e.g., human, hepatocytes, preferably hepatocytes. Because B4GALT1 expression is found to be high in the liver, but ubiquitously expressed throughout the human body. Similarly, the present disclosure provides the use of the EONs disclosed herein in the manufacture of a medicament for altering the target B4GALT1 RNA sequence in mammalian, preferably human, hepatocytes, more preferably hepatocytes, thereby treating, preventing, or ameliorating CVD, as described herein.

[0077] The present disclosure also provides a method for deaminating at least one specific target adenosine present in a target B4GALT1 RNA sequence in a cell, the method comprising the following steps: providing an EON disclosed herein to the cell (naked or via vector delivery); uptake of the EON (or the vector) by the cell; annealing the EON to a target RNA molecule; allowing an endogenous mammalian ADAR enzyme to deaminate the target adenosine in the target RNA molecule (preferably, an adenosine at position 1055 in the B4GALT1 transcript) to inosine; and optionally identifying the presence of the inosine in the RNA sequence.

[0078] The term CVD includes conditions such as CAD (sometimes called coronary heart disease), stroke, and transient ischemic attack (TIA; i.e., ministroke), peripheral artery disease, and / or aortic disease. The EONs disclosed herein can be used to treat, prevent, or ameliorate any or all of these conditions. In a preferred embodiment, the CVD to be treated by the present disclosure is CAD.

[0079] The methods disclosed herein can be applied to subjects in which the target is adenosine. For example, if the target adenosine is present at position 1055 of the B4GALT1 transcript, patients identified as having or likely to have the c.1055A>G variant are generally not treated.

[0080] The present disclosure also provides a method for deaminating at least one specific target adenosine present in a target B4GALT1 RNA sequence in a cell, the method comprising the following steps: providing a vector or plasmid encoding an EON disclosed herein to the cell; uptake of the vector or plasmid by the cell; annealing the EON to a target RNA molecule; allowing an endogenous mammalian ADAR enzyme to deaminate the target adenosine in the target RNA molecule (preferably, an adenosine at position 1055 in the B4GALT1 transcript) to inosine; and optionally identifying the presence of the inosine in the RNA sequence.

[0081] In a preferred embodiment, depending on the net effect of the A to I conversion, the identifying step may include the following steps: sequencing the target RNA; sequencing cDNA derived from the target RNA; assessing the presence or absence of an A to G conversion in the cDNA derived from the target RNA; assessing the presence or absence of a functional protein; assessing whether the deamination alters pre-mRNA splicing; or, if the deamination results in the target RNA encoding an enzyme with a reduced enzyme turnover rate, using a functional readout. Examples include assessing the rate of UDP-Gal reduction and / or evaluating biomarkers in serum and / or plasma. For example, a decrease in B4GALT1 enzyme turnover can be detected through a decrease in plasma fibrinogen, a decrease in serum LDL-C, or a decrease in serum tetrasialylated transferrin levels with a corresponding increase in lower sialylated levels (e.g., an increase in trisialylated transferrin levels).

[0082] Of course, the most suitable method for identifying the presence of inosine after deamination of target adenosine is dPCR or even sequencing, using the method well known to those skilled in the art, as outlined herein.However, those skilled in the art of liver disease can apply the test for monitoring certain biomarkers related to LDL-C and / or fibrinogen level as mentioned above.

[0083] A suitable functional assay for testing reduced B4GALT1 turnover is described in detail in Montasser et al. (2021, supra). Briefly, one in vivo test is the carbohydrate-deficient transferrin (CDT) test. The CDT test is clinically used to diagnose patients with congenital glycosylation disorders. The CDT test evaluates the sialylation level of the protein transferrin. Under normal conditions, transferrin is mostly tetrasialylated. Reduced B4GALT1 activity results in a decrease in the level of tetrasialylated transferrin and a corresponding increase in the level of hyposialylated transferrin (e.g., trisialylated transferrin). This test can be used to evaluate the efficacy of EON in an in vivo environment, analyzing the combined effects of reduced B4GALT1 turnover rate and the ratio of variant to wild-type B4GALT1. Further testing involves assessing the rate of UDP-Gal reduction in a biochemical assay. While any acceptor can be used in this assay, a convenient acceptor is the monosaccharide N-acetylglucosamine (NGlcNAc). Standard assay conditions are provided in Montasser et al. (2021, supra). This assay may be useful for examining kinetic parameters of purified B4GALT1 mutants or cell isolates after EON-mediated ADAR editing. Further testing could involve knocking in the B4GALT1 enzyme or variants into mouse models to assess serum and / or plasma concentrations of biomarkers for B4GALT1 activity, such as LDL-C and / or fibrinogen.

[0084] The EONs disclosed herein are suitably administered in aqueous solutions (e.g., saline) or suspensions, which may contain additives, excipients, and other ingredients compatible with pharmaceutical use, at concentrations ranging from 1 ng / ml to 1 g / ml, preferably from 10 ng / ml to 500 mg / ml, and more preferably from 100 ng / ml to 100 mg / ml. Dosages ranging from about 1 μg / kg to about 100 mg / kg, preferably from about 10 μg / kg to about 10 mg / kg, and more preferably from about 100 μg / kg to about 1 mg / kg, may be appropriate. Administration may be by inhalation (e.g., via a nebulizer), nasal, oral, injection or infusion, intravenous, subcutaneous, intradermal, intramuscular, intratracheal, intraperitoneal, rectal, intrathecal, intracisternal, parenteral, or the like. Administration may be in the form of solids, powders, tablets, gels, solutions, sustained-release formulations, or any other form compatible with human pharmaceutical use.

[0085] In one aspect, the method disclosed herein comprises the following steps: administering to a subject an EON or pharmaceutical composition disclosed herein; allowing a double-stranded nucleic acid complex between the EON and its specific, complementary target nucleic acid molecule to form in cells within the subject; allowing existing endogenous adenosine deaminase enzymes, such as ADAR1 and / or ADAR2, to participate; and allowing the enzyme to deaminate a target adenosine in the target nucleic acid molecule to inosine, thereby reducing, preventing, or ameliorating CVD.

[0086] The RNA editing molecule present in cells is usually proteinaceous in nature, for example, the ADAR enzyme found in metazoans, including mammals.Preferably, the editing entity in cells is an enzyme, more preferably adenosine deaminase or cytidine deaminase, even more preferably adenosine deaminase.These are enzymes with ADAR activity.One of the most interesting is human ADAR, namely, hADAR1 and hADAR2, including any isoforms thereof.The RNA editing enzymes known in the art, for which the oligonucleotide constructs disclosed herein can be conveniently designed, include adenosine deaminase (ADAR) acting on RNA, such as hADAR1 and hADAR2 in humans or human cells, and cytidine deaminase.It is known that there are two isoforms of hADAR1: a long interferon-induced form of 150 kDa, and a shorter form of 110 kDa, which is produced from a common pre-mRNA through alternative splicing. As a result, the level of the 150 kDa isoform available in cells can be affected by interferon, particularly interferon-γ (IFN-γ). hADAR1 is also induced by TNF-α. This provides an opportunity to develop combination therapy, in which IFN-γ or TNF-α and the EON disclosed herein are administered to patients as a combination product or as separate products, simultaneously or sequentially (in any order). In some disease states, elevated levels of IFN-γ or TNF-α may already exist in certain tissues of patients, which creates an additional opportunity for more specific editing of diseased tissues. Those skilled in the art will understand that the degree to which the editing body inside the cell is redirected to other target sites can be adjusted by changing the affinity of the first nucleic acid strand for the recognition domain of the editing molecule.

[0087] [Chemical modification] All of the chemical modifications listed below that can be used with the EONs disclosed herein can also be used with the sense strand complementary to the EON when the EON and the complementary strand form a so-called heteroduplex RNA-editing oligonucleotide (HEON) complex, as described in PCT / EP2023 / 079290 (unpublished). The exception is that the opposing sense strand does not have an isolated nucleotide. Therefore, modifications involving an isolated nucleotide relate only to the EONs disclosed herein, while all other modifications relate to the EONs disclosed herein and any (protected) sense oligonucleotides that can be used with the EONs in pharmaceuticals. This includes the use of hydrophobic moieties (e.g., tocopherol and cholesterol) and cell-specific ligands (e.g., GalNAc moieties), which are also described in detail herein and in PCT / EP2023 / 079290 (unpublished), and can be attached to the EON, its opposing strand, or both.

[0088] The internucleoside bond in the oligonucleotide disclosed herein may comprise one or more natural internucleoside bonds and / or modified internucleoside bonds.Without limitation, at least one, at least two, or at least three internucleoside bonds from the 5'-end and / or 3'-end of the EON are preferably modified internucleoside bonds.Preferably, the modified internucleoside bond is a PS bond.In one embodiment, all internucleoside bonds of the EON are modified internucleoside bonds.In one embodiment, the EON comprises a PNdmi bond that connects the most terminal nucleoside at the 5'-end and / or 3'-end and the second nucleoside at each of these ends, respectively.The PNdmi bond preferably used in the EON disclosed herein has the following structure: JPEG2025540146000001.jpg47166

[0089] Common limiting factors in oligonucleotide-based therapeutics include the ability of the oligonucleotide to be taken up by cells (when delivered "naked" without the application of a delivery vehicle), biodistribution, and resistance to nuclease-mediated degradation. Those skilled in the art recognize that various chemical modifications can help overcome such limitations, and these have been extensively reported in the art. Examples of such currently commonly used chemical modifications include 2'-O-methyl (often abbreviated as 2'-OMe or 2'-O-Me), 2'-F, and 2'-O-methoxyethyl (often referred to as 2'-methoxyethoxy or 2'-MOE) sugar modifications, and the use of PS linkages between nucleosides. WO2020 / 201406 discloses the use of MP linkage modifications at specific positions around isolated nucleotides within a first nucleic acid strand. The ribose 2' group in all nucleotides of an EON, except for the ribose sugar moiety of an isolated nucleotide, which has certain limitations with respect to compatibility with RNA editing, can be independently selected from the following: 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4' linkage (e.g., locked nucleic acid (LNA)), or other 1'-, 2'-, 3'-, 4'-, or 5'-ribose substitutions. Isolated nucleotides in an EON that do not contain other chemical modifications to the ribose sugar, base, or linkage preferably do not have 2'-OMe or 2'-MOE substitutions, but may have 2'-F, 2',2'-difluoro (diF), or 2'-ara-F (FANA) substitutions, or may be DNA. PCT / EP2023 / 069609 (unpublished) describes modifying the 2' position of the ribose sugar moiety of isolated nucleotides with a 2',2'-disubstituted modification such as diF, which is also applicable. The 2'-4' bond can be selected from many linkers known in the art, such as a methylene linker, an amide linker, or a constrained ethyl linker (cEt).

[0090] The present disclosure provides an EON for use in deaminating a target nucleotide (preferably adenosine) in a target RNA, wherein the EON is complementary to a continuous region of nucleotides in the target RNA that includes the target adenosine, and the nucleotide directly opposite the target nucleotide in a first nucleic acid strand is an isolated nucleotide. When the target nucleotide is adenosine, the isolated nucleotide preferably includes a base with an NH moiety at a position similar to the cyclic nitrogen, or a modified base, or a base analog (e.g., Benner's base Z). Nucleotides in the EON are numbered such that the isolated nucleotide is numbered 0 and the nucleotide 5' to the isolated nucleotide is numbered +1. Numbers increase positively (+) toward the 5' end and negatively (-) toward the 3' end, with the first nucleotide 3' to the isolated nucleotide being numbered -1. The numbering of internucleoside linkages in EON is such that linkage 0 is the 5' linkage of the lone nucleotide, and the linkage positions within the oligonucleotide increase in positive (+) order toward the 5' end and decrease in negative (-) order toward the 3' end.

[0091] Preferably, the EON comprises one or more (chiral pure or chiral mixed) PS bonds. In one embodiment, the PS bonds link the terminal 3, 4, 5, 6, 7, or 8 nucleotides at each end of the first nucleic acid strand. In one embodiment, the EON comprises one or more phosphoramidate (PN) bonds. In one embodiment, the PN bonds link the terminal 2 nucleotides at each end of the EON disclosed herein.

[0092] Nucleosides in EONs may be natural nucleosides (deoxyribonucleosides or ribonucleosides) or unnatural nucleosides. In RNA editing, double-stranded RNA generally serves as a substrate for enzymes with deaminase activity (e.g., ADAR), so ribonucleosides are considered "natural," while deoxyribonucleosides may be considered unnatural or modified for the sake of discussion, since DNA does not exist in an RNA-RNA double-stranded substrate structure. Those skilled in the art will understand that even if a nucleotide has a natural ribose moiety, the base and / or linkage may be unnaturally modified.

[0093] In addition to the specific preferred chemical modifications at certain positions in the compounds disclosed herein, the compounds disclosed herein may also contain one or more (additional) modifications to the nucleobase, scaffold, and / or backbone linkage. These may or may not be present in the same monomer, for example, at the 3' and / or 5' positions. Scaffold modification refers to the presence of modified forms of the ribose moiety (i.e., pentose moiety) naturally occurring in RNA, such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2'-modified sugars, 4'-modified sugars, 5'-modified sugars, and 4'-substituted sugars.Examples of suitable modifications include, but are not limited to, 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)). pyr), 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- 2'-O-[2-(N,N-methylcarbamoyl)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 bridged nucleic acid (BNA) scaffold modifications, e.g., conformationally restricted nucleotide (CRN) monomers, locked nucleic acid (LNA) monomers, xylo-LNA monomers, α-LNA mono mer, 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) constrained ethyl (cEt) BNA monomer, (2′-O,4′-C) constrained methoxyethyl (cMOE) BNA monomer, 2′,4′-BNA. NC (NH) monomer, 2′,4′-BNA NC (NMe) monomer, 2′,4′-BNA NC(NBn) monomers, ethylene-bridged nucleic acid (ENA) monomers, carba-LNA (cLNA) monomers, 3,4-dihydro-2H-pyran nucleic acid (DpNA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, oxo-CBBN monomers, heterocyclic bridged BNA monomers (e.g., triazolyl- or tetrazolyl-linked), amide-bridged BNA monomers (e.g., AmNA), urea-bridged BNA monomers, sulfonamide-bridged BNA monomers, bicyclic carbocyclic nucleotide monomers, TriNA monomers, α-l-TriNA monomers, bicyclic DNA (bcDNA) monomers, F-bcDNA monomers, tricyclic DNA (tcDNA) monomers, F-tc DNA monomers, α-anomeric bicyclic DNA (abcDNA) monomers, oxetane nucleotide monomers, locked PMO monomers derived from 2'-amino-LNA, guanidine-bridged nucleic acid (GuNA) monomers, spirocyclopropylene-bridged nucleic acid (scpBNA) monomers, and their derivatives; cyclohexenyl nucleic acid (CeNA) monomers, altritol nucleic acid (ANA) monomers, hexitol nucleic acid (HNA) monomers, fluorinated HNA (F-HNA) monomers, pyranosyl-RNA (p-RNA) monomers, 3'-deoxypyranosyl DNA (p-DNA), unlocked nucleic acid (UNA); and inverted versions of any of the above monomers. All of these modifications are known to those skilled in the art.

[0094] The base sequence of the EON disclosed herein is complementary to a portion of the base sequence of the target B4GALT1 transcript, including at least the target adenosine to be deaminated to inosine (preferably adenosine at position 1055), and therefore can anneal (or hybridize) with the target transcript. The complementarity of the base sequence can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the double strands can hybridize, taking into account the complementarity between the strands.

[0095] In contrast to what has been described regarding gapmers and their relationship to ribonuclease degradation, and the use of such gapmers in double-stranded complexes (see, for example, EP 3954395 A1), the EONs disclosed herein do not contain consecutive stretches of DNA nucleotides that would cause the target sequence (or sense nucleic acid strand) to be targeted for ribonuclease-mediated degradation. In one embodiment, the EONs disclosed herein do not contain four or more consecutive DNA nucleotides at any position within their sequence. In one embodiment, the EONs disclosed herein are composed of as many (chemically) modified nucleotides as possible to increase resistance to ribonuclease-mediated degradation, while at the same time being as efficient as possible in producing an RNA editing effect. This means that isolated nucleotides and other multiple nucleotides within the EON may be DNA, but also means that there are no consecutive stretches of four or more consecutive DNA nucleotides within the EON. Thus, the EONs disclosed herein are not gapmers. A gapmer is essentially a single-stranded nucleic acid, consisting of a central region (a DNA gap region having at least four consecutive deoxyribonucleotides) and wing regions located directly at its 5'-end (5' wing region) and 3'-end (3' wing region). In contrast, the EON disclosed herein may be any oligonucleotide that produces, induces, induces, or enables the RNA editing effect of deaminating adenosine to inosine in a target RNA molecule, and therefore is as resistant as possible to RNase-mediated degradation to achieve this effect.

[0096] In one embodiment, the EON disclosed herein or the sense strand that can be annealed thereto before entering a target cell is conjugated to a hydrophobic moiety, such as palmityl or an analog thereof, cholesterol or an analog thereof, or tocopherol or an analog thereof. This is preferably conjugated to the 5'-end. When the hydrophobic moieties are conjugated to the 5'-end and the 3'-end, the hydrophobic moieties may be the same or different. The hydrophobic moiety conjugated to the oligonucleotide may be directly conjugated or indirectly conjugated via another substance. When the hydrophobic moiety is directly conjugated, it is sufficient that the moiety is conjugated via a covalent bond, ionic bond, hydrogen bond, or the like. When the hydrophobic moiety is indirectly conjugated, it may be conjugated via a linker. The linker may be cleavable or non-cleavable. A cleavable linker refers to a linker that can be cleaved under physiological conditions, for example, within a cell or an 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 portion of the body 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, phosphodiesters (either one or both), phosphoesters, carbamates, and disulfide bonds, as well as natural DNA linkers. Cleavable linkers also include self-immolative linkers. A non-cleavable linker refers to a linker that is not cleaved under physiological conditions or is cleaved much more slowly than a cleavable linker, such as a linker consisting of a PS bond, a modified or unmodified deoxyribonucleoside linked by a PS bond, a spacer linked through a PS bond, and a modified or unmodified ribonucleoside. When the linker is a nucleic acid such as DNA or an oligonucleotide, there is no limit to its chain length. Typically, however, it can be 2 to 20 bases in length, 3 to 10 bases in length, or 4 to 6 bases in length.The length or composition of the spacer linking the ligand and the oligonucleotide is not limited, and may include, for example, ethylene glycol, TEG, HEG, an alkyl chain, propyl, 6-aminohexyl, or dodecyl.

[0097] The present disclosure also provides a pharmaceutical composition comprising the EON disclosed herein, further comprising a pharmaceutically acceptable carrier and / or other additives, and optionally dissolved in a pharmaceutically acceptable organic solvent or the like. The dosage form in which the EON or the 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 in a single dose or multiple doses. It may be administered daily or at appropriate time intervals determined using general knowledge in the art, and may be adjusted based on the disorder and the effectiveness of the active ingredient.

[0098] In one embodiment, an EON disclosed herein comprises at least one nucleotide having a sugar moiety comprising a 2'-OMe modification. In one embodiment, an EON disclosed herein comprises at least one nucleotide having a sugar moiety comprising a 2'-MOE modification. In one embodiment, an EON disclosed herein comprises at least one nucleotide having a sugar moiety comprising a 2'-F modification. In one embodiment, an isolated nucleotide has a 2'-H in the sugar moiety and is therefore referred to as a DNA nucleotide, although additional modifications may be present in its base and / or its linkage to the adjacent nucleoside. In one embodiment, an isolated nucleotide has a 2'-F in the sugar moiety. In one embodiment, an isolated nucleotide has a diF substitution in the sugar moiety. In one embodiment, an isolated nucleotide has 2'-F and 2'-C-methyl in the sugar moiety. In one embodiment, an isolated nucleotide comprises a 2'-F in the arabinose configuration (FANA) in the sugar moiety. In one embodiment, an EON is an antisense oligonucleotide capable of forming a double-stranded nucleic acid complex with a target RNA molecule, wherein the double-stranded nucleic acid complex is capable of recruiting adenosine deaminase to deaminate a target adenosine in a target B4GALT1 RNA molecule, wherein the nucleotide opposite the target adenosine in the EON is an isolated nucleotide, and wherein the isolated nucleotide has the following formula (II): JPEG2025540146000002.jpg40166 where: X is O, NH, OCH2, CH2, Se, or S; B is a nitrogenous 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 each independently selected from H, OH, F, or CH3; R3 is a portion of the EON located 5' to the isolated nucleotide and consisting of 7 to 30 nucleotides; and R4 is a portion of the EON located 3' to the isolated nucleotide and consisting of 4 to 25 nucleotides. The nucleotides on the 3' and / or 5' sides of the isolated nucleotide may be DNA, more preferably the nucleotide on the 3' side (-1 position).

[0099] In one embodiment, the EON disclosed herein comprises at least one MP internucleoside linkage according to formula (III): JPEG2025540146000003.jpg40166

[0100] A preferred position for the MP bond in the EONs disclosed herein is the -2 bond position, thereby linking the nucleoside at the -1 position with the nucleoside at the -2 position, although MP bond positions at other positions are not expressly excluded.

[0101] The EONs disclosed herein may also include one or more linkage modifications according to the structure of formula (IV): JPEG2025540146000004.jpg53166 where: 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 20Alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugated group. In a preferred embodiment, X=O and R=methyl, and the bond modification is referred to as mesyl phosphoramidate (MsPA or PNms). In a preferred embodiment, PN mesyl (i.e., PNms) bond is present at the -2 bond position in the EON disclosed herein (for example, in EON B4GALT1-65 (SEQ ID NO: 1079)) (in this case, it is present instead of PS, PO, or MP bond).

[0102] In preferred embodiments, the EONs disclosed herein comprise an internucleoside linkage of the structure of formula (IV), where X=O and R=CH, which linkage is generally referred to herein as a PNms linkage (mesyl phosphoramidate). In other preferred embodiments, R is equal to one of the following structures (a), (b), (c), (d), (e), (f), (g), (h), or (i): JPEG2025540146000005.jpg158166

[0103] Also disclosed herein is an EON capable of mediating the deamination of adenosine by recruiting a deaminase within a cell after forming a double-stranded complex with a region of a target RNA nucleic acid molecule within a cell, said region comprising a target adenosine, said deaminase being capable of deaminating said target adenosine to inosine, and said EON comprising a moiety having a structure according to formula (V): JPEG2025540146000006.jpg41166Where: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-C 20 Alkyl, substituted C1-C 20alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or a conjugated group. In a preferred embodiment, X=O and R=methyl.

[0104] The EONs disclosed herein may contain a substitution of one of the non-bridging oxygens in the phosphodiester bond. This modification slightly destabilizes base pairing but confers significant resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include PS, phosphonoacetate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonates (including 3'-alkylene phosphonate, 5'-alkylene phosphonate, and chiral phosphonate), phosphinate, phosphoramidate (including 3'-amino phosphoramidate and aminoalkyl phosphoramidate), thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, or boranophosphate. Particularly preferred are internucleoside linkages modified to contain PS. Particularly preferred are internucleoside linkages modified to contain MP. Particularly preferred are internucleoside linkages modified to include PNms. Particularly preferred are internucleoside linkages modified to include PNdmi. The normal internucleoside linkage between nucleotides can be modified by mono- or dithiolation of the phosphodiester bond to produce PS esters or phosphorodithioate esters, respectively. Other modifications of the internucleoside linkage are possible, including amidation and peptide linkers. To generate the most effective and most stable oligonucleotide compound, those skilled in the art can determine which linkage modifications the EON should include at each binding position of the EON disclosed herein for which target RNA nucleic acid molecule.

[0105] In one embodiment, the EON disclosed herein comprises at least one nucleotide having a sugar moiety containing a 2'-fluoro (2'-F) modification. A preferred position for the nucleotide having the 2'-F modification is the -3 position in the EON, which may also occur in conjunction with the same 2' modification in an isolated nucleotide, as described above.

[0106] In one embodiment, an EON disclosed herein comprises at least one phosphonoacetate or phosphonoacetamide internucleoside linkage.

[0107] In one embodiment, the EON disclosed herein comprises at least one nucleotide that includes an LNA ribose modification or a UNA ribose modification. In one embodiment, the EON disclosed herein comprises at least one nucleotide that includes a TNA ribose modification.

[0108] Those skilled in the art know that oligonucleotides, such as the EONs outlined herein, are generally composed of repeating monomers. These monomers are most often 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). They consist of a pentose sugar (ribose), a 5'-linked phosphate group (which is linked via a phosphate ester), and a 1'-linked base. The sugar is often referred to as the "scaffold" of the nucleotide because it connects the base and the phosphate group.

[0109] Thus, modifications to the pentose sugar are often referred to as "scaffold modifications." The original pentose sugar may be completely replaced by another moiety that similarly links the base and phosphate group. Thus, while a pentose sugar is often the scaffold, it is understood that a scaffold does not necessarily have to be a pentose sugar. Examples of scaffold modifications that may be applied to the monomers of the EONs disclosed herein are disclosed in WO2020 / 154342, WO2020 / 154343, and WO2020 / 154344.

[0110] In one embodiment, the EON disclosed herein may comprise one or more nucleotides with a 2'-MOE ribose modification. In another embodiment, the EON disclosed herein comprises one or more nucleotides without a 2'-MOE ribose modification, and the 2'-MOE ribose modification is located at a position that does not prevent an enzyme with adenosine deaminase activity from deaminating the target adenosine. In another embodiment, the EON disclosed herein comprises a 2'-OMe ribose modification at a position that does not comprise a 2'-MOE ribose modification, and / or the EON comprises a deoxynucleotide at a position that does not comprise a 2'-MOE ribose modification. In one embodiment, the EONs disclosed herein include one or more nucleotides containing a 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, TNA, 2'-fluoro (2'-F), 2',2'-disubstituted modifications (e.g., 2',2'-difluoro (diF) modifications, 2'-fluoro-2'-C-methyl modifications, or other modifications, including the 2'-spirocyclic modifications described, for example, in Grosse et al. (2022. ACS Med Chem Lett DOI:10.1021 / acsmedchemlett.2c00372)), or a 2'-4' linkage (i.e., LNA, i.e., bridged nucleic acids, such as those described in WO2018 / 007475). In another embodiment, other applicable nucleic acid monomers include arabinonucleic acids and 2'-deoxy-2'-fluoroarabinonucleic acids (FANA), for example, to improve affinity. The 2'-4' linkage can be selected from linkers known in the art, such as methylene linkers or constrained ethyl linkers. A wide variety of 2' modifications are known in the art. Further examples are disclosed in further detail in, for example, WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2018 / 134301, WO2019 / 219581, WO2019 / 158475, and WO2022 / 099159.In all cases, the modification must be compatible with editing, so that the EON acts as an editing-inducing oligonucleotide that forms a double-stranded complex with the target RNA and recruits a deaminating enzyme, which then deaminates the target adenosine. If the monomer contains a UNA ribose modification, the monomer can have a 2' position that contains the same modifications described above, such as 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, 2'-F, 2',2'-diF, 2'-fluoro-2'-C-methyl, arabinonucleic acid, FANA, or a 2'-4' linkage (i.e., a bridged nucleic acid such as LNA).

[0111] A base (sometimes called a nucleobase) is generally adenine, cytosine, guanine, thymine, or uracil, or a derivative thereof. A base (sometimes called a nucleobase) is defined as a moiety that can bind to another nucleobase through hydrogen bonds, polar bonds (e.g., through a CF moiety), or aromatic electron interactions. Cytosine, thymine, and uracil are pyrimidine bases, generally linked to the scaffold through their 1-nitrogen. Adenine and guanine are purine bases, generally linked to the scaffold through their 9-nitrogen. As used herein, the terms "adenine," "guanine," "cytosine," "thymine," "uracil," and "hypoxanthine" refer to the nucleobase itself. The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to a nucleobase linked to (deoxy)ribose.

[0112] The nucleobases in the EONs disclosed herein can be adenine, cytosine, guanine, thymine, inosine, or uracil, or any other moiety capable of interacting with another nucleobase through hydrogen bonding, polar bonding (e.g., C—F), or aromatic electronic interactions. The nucleobases at any position within the nucleic acid strand can be modified forms of adenine, cytosine, guanine, or uracil, such as, for example, hypoxanthine (nucleobase at inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseudouracil, 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-2,6 -diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, 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 derivatives thereof, super A, super T, super G, amino-modified nucleobases or derivatives thereof; and modified or universal bases (e.g., 2,6-difluorotoluene), or deletions such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, and azaribose).

[0113] In one embodiment, the nucleotide analog is an analog of a nucleic acid nucleotide. In one embodiment, the nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine. In one embodiment, the nucleotide analog is not guanosine or deoxyguanosine. In one embodiment, the nucleotide analog is not a nucleic acid nucleotide. In one embodiment, the nucleotide analog is not adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine.

[0114] Nucleotides are typically linked to adjacent nucleotides through condensation of their 5'-phosphate moiety to the 3'-hydroxyl moiety of the adjacent nucleotide monomer. Similarly, their 3'-hydroxyl moiety is typically linked to the 5'-phosphate of the adjacent nucleotide monomer. This forms a phosphodiester bond. The phosphodiester and scaffold form an alternating copolymer. The bases are attached to this copolymer, i.e., the scaffold moiety. Because of this characteristic, the alternating copolymer formed by the linked scaffolds of an oligonucleotide is often referred to as the "backbone" of the oligonucleotide. Because phosphodiester bonds connect adjacent monomers, they are often referred to as "backbone linkages." It is understood that even if the phosphate group is modified and replaced with an analogous moiety, such as PS, such moiety is still referred to as the backbone linkage of the monomer. This is referred to as "backbone linkage modification." In a general sense, the backbone of an oligonucleotide comprises alternating scaffolds and backbone linkages.

[0115] The EONs disclosed herein may include linkage modifications, such as, but not limited to, modified forms of phosphodiesters present in RNA, including, but not limited to, PS, chiral pure PS, (R)-PS, (S)-PS, MP, chiral pure MP, (R)-methylphosphonate, (S)-methylphosphonate, phosphorylguanidine (e.g., PNdmi), chiral pure phosphorylguanidine, (R)-phosphorylguanidine, (S)-phosphorylguanidine, phosphorodithioate (PS2), phosphonacetate (PACE), Phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methyl phosphorothioate, methylthiophosphonate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, borano PS, methylboranophosphate, methylborano PS, methylboranophosphonate, methylboranophosphothioate, phosphate, phosphotriester, aminoalkylphosphotriester, and derivatives thereof. Other modifications include phosphoramidites, phosphoramidates, N3'→P5' phosphoramidates, phosphorodiamidates, phosphorothiodiamidates, sulfamates, diethylene sulfoxides, amides, sulfonates, siloxanes, sulfides, sulfones, formacetyls, alkenyls, methylenehydrazinos, sulfonamides, triazoles, oxalyls, carbamates, methyleneiminos (MMIs), and thioacetamido nucleic acids (TANAs); and derivatives thereof. Various salts, mixed salts, and free acid forms are also included, including 3'→3' and 2'→5' linkages.

[0116] In one embodiment, the EON disclosed herein contains a substitution of one of the non-bridging oxygens in the phosphodiester bond. This modification slightly destabilizes base pairing but confers significant resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include PS, phosphonoacetate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkylphosphonates (including 3'-alkylenephosphonate, 5'-alkylenephosphonate, and chiral phosphonate), phosphinate, phosphoramidate (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, or boranophosphate. Particularly preferred are internucleoside linkages modified to contain PS. Many of these non-natural bond modifications (e.g., PS) are chiral, meaning that they have both Rp and Sp configurations, and are known to those skilled in the art. In one embodiment, the chirality of the PS bond is controlled, meaning that each bond has either the preferred Rp or Sp configuration. The selection of the Rp or Sp configuration at a particular binding position may depend on the target sequence, the EON sequence, and the binding efficiency and induction efficiency resulting in RNA editing. However, if this is not particularly desired, the composition may contain an EON having both the Rp and Sp configurations at a particular binding position as an active compound. A mixture of EONs is also possible, where one configuration is preferred at some positions but not at other positions.

[0117] Again, in all cases, the modification must be compatible with editing, so that the EON serves as an editing oligonucleotide, and when bound to its target sequence, the nature of dsRNA generates, which can recruit adenosine deaminase.In all embodiments, the enzyme with adenosine deaminase activity is preferably ADAR1 or ADAR2.In a highly preferred embodiment, the EON is an RNA editing oligonucleotide that targets pre-mRNA or mRNA, wherein the target nucleotide is adenosine in the target RNA, and the adenosine is deaminated to inosine, which is read as guanosine by the translational machinery.The present disclosure also provides a pharmaceutical composition comprising the EON characterized herein and a pharmaceutically acceptable carrier.

[0118] Other chemical modifications of the EONs disclosed herein include replacing any one or more hydrogen atoms with deuterium or tritium, examples of which can be found, for example, in WO2014 / 022566 or WO2015 / 011694.

[0119] The present disclosure also provides an EON disclosed herein or a pharmaceutical composition comprising an EON disclosed herein, for use in treating, preventing, or ameliorating CVD, such as CAD. In one embodiment, the present disclosure provides an EON disclosed herein or a pharmaceutical composition comprising an EON disclosed herein, for use in treating, preventing, or ameliorating a disease in which B4GALT1 functions in a wild-type state. In one embodiment, the present disclosure provides an EON disclosed herein or a pharmaceutical composition comprising an EON disclosed herein, for use in treating, preventing, or ameliorating a disease associated with high LDL-C and / or fibrinogen levels. In one embodiment, the present disclosure provides an EON disclosed herein or a pharmaceutical composition comprising an EON disclosed herein, for use in treating or preventing CVD, such as CAD.

[0120] The EONs disclosed herein preferably do not contain a 5'-terminal O6-benzylguanosine or a 5'-terminal amino modification, and preferably are not covalently linked to a SNAP tag domain (modified O6-alkylguanosine-DNA-alkyltransferase). The EONs disclosed herein preferably do not contain a boxB RNA hairpin sequence. In one embodiment, the EONs disclosed herein contain 0, 1, 2, or 3 wobble base pairs with the target sequence and / or 0, 1, 2, 3, 4, 5, 6, 7, or 8 mismatch base pairs with the target RNA sequence. When the isolated nucleotide is a uridine, no mismatch occurs. One alternative to a uridine is to place an isouridine opposite the target adenosine, which is considered a mismatch because it does not pair like U and A do. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the EON directly opposite the target adenosine.

[0121] It should be noted that when EON is delivered through vector (for example, AAV vector), there is no chemical modification in the EON that acts on target RNA molecule.Although it is preferable to use " naked " EON with chemical modification as outlined herein, EON delivered through other means (for example, through AAV vector expression) or editing molecules with circular or hairpin structure (recruitment part, for example, disclosed in WO2016 / 097212, WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560 and WO2022 / 078995) are also encompassed in the present disclosure.Because they can also be applied to edit adenosine in target B4GALT1 RNA molecule to generate reduced function B4GALT1 protein.

[0122] It is noteworthy that even if an EON contains chemical modifications as detailed herein, it can still be delivered via a delivery vehicle. Suitable delivery vehicles include nanoparticle delivery vehicles such as polymer nanoparticles, dendrimers, inorganic nanoparticles and nanocrystals, organic nanocrystals, and liposomes. Preferred nanoparticles are lipid nanoparticles (LNPs), which are nano-sized lipid vesicles that carry the EONs of the present disclosure and aid in their delivery to target cells. Even when LNPs or other similar types of carriers are used, the EON is still considered naked because it is not transcribed from an encoding polynucleotide (e.g., in the case of a plasmid or vector, the EON is not considered "naked"). Therefore, even if a chemically modified EON is encapsulated by a carrier, preferably an LNP, it is still considered naked because it is produced in such a form in a laboratory environment and later encapsulated in a carrier using methods known to those skilled in the art. The present disclosure also relates to delivery vehicles, preferably LNPs, which include "naked" and chemically modified EONs, including those disclosed herein, more preferably those disclosed in any of SEQ ID NOs: 3-42, 59-1069, and 1078-1190, preferably EONs selected from the group consisting of SEQ ID NOs: 23, 19, 31, 27, 35, 39, 69, 70, 71, 72, 73, 93, 94, 95, 1079, 1084, 1093, 1095, 1100, 1102, 1115, 1121, 1123, 1124, and 1139-1190. Those skilled in the art will understand that even if a delivery moiety or appendage to the EON (e.g., a GalNAc moiety to target hepatocytes in the liver) is used, or if the GalNAc-(linker)-EON is encapsulated in a delivery vehicle such as an LNP, the EON is still considered naked.

[0123] The EON disclosed herein can utilize endogenous cellular pathways and naturally occurring ADAR enzymes (=endogenous) to specifically edit target adenosines in target RNA sequences.When the EON disclosed herein is present in a double-stranded complex with target RNA molecules, it can recruit endogenous ADAR to form a complex with it, and then promote the deamination of (single) specific target adenosine nucleotides in target RNA sequences.Ideally, only one adenosine is deaminated.Preferably, when the EON disclosed herein forms a complex with ADAR, it causes the deamination of single target adenosine.

[0124] Analysis of natural targets of ADAR enzymes has shown that they generally contain mismatches between the two strands that form the RNA helices edited by ADAR1 or ADAR2. 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 Acid Res 39(13):5669-5681). Characterization of the optimal pattern of paired / mismatched nucleotides between EONs and target RNAs is also likely to be important for the development of effective ADAR-based EON therapies.

[0125] As outlined above, the EONs disclosed herein utilize specific nucleotide modifications at predetermined locations to ensure stability and proper ADAR binding and activity. As described in detail herein, these modifications may vary and include modifications in the EON backbone, in the sugar moiety of the nucleotide, and in the nucleobase or phosphodiester bond. They may also be variably distributed throughout the EON sequence. Specific modifications may be required to support interactions with various amino acid residues in the RNA-binding domain of ADAR enzymes and those in the deaminase domain. For example, internucleotide PS bonds or 2'-OMe or 2'-MOE modifications may be tolerated in some EONs, but should be avoided in others to avoid interfering with the critical interactions between the enzyme and the phosphate and 2'-OH groups. Specific nucleotide modifications may also be required to enhance editing activity for substrate RNAs whose target sequences are not optimal for ADAR editing. Previous studies have established that certain sequence contexts are more susceptible to editing. For example, the target sequence 5'-UAG-3' (with a central target A) contains the most favorable nearest neighbor nucleotide for ADAR2, while the 5'-CAA-3' target sequence is unfavorable (Schneider et al. 2014. Nucleic Acids Res 42(10):e87). Structural analysis of the ADAR2 deaminase domain suggests the possibility of enhancing editing by carefully selecting the nucleotide opposite the target trinucleotide. For example, the 5'-CAA-3' target sequence pairs with the 3'-GCU-5' sequence on the opposite strand (creating a central AC mismatch), but this is unfavorable because the guanosine base clashes sterically with the amino acid side chain of ADAR2. While other adenosines in the B4GALT1 transcript may be targeted and reduce protein function, in a preferred embodiment, the adenosine at position 1055 is deaminated.The present disclosure also provides RNA-editing oligonucleotides (generally referred to herein as EONs) that can result in the deamination of adenosines in B4GALT1 transcripts, resulting in a B4GALT1 enzyme with a reduced turnover rate. This means that the deamination is not strictly limited to the adenosine at position 1055; other adenosines (single or multiple) can be targeted, which can also result in reduced B4GALT1 enzyme function. Other adenosines may be identified, for example, by population genetic screening or in silico analysis, that are also important for B4GALT1 function and can also be targeted through RNA editing according to the teachings of the present disclosure. All RNA events and oligonucleotides that can be used for such targeting are encompassed by the present disclosure, regardless of the exact nucleic acid molecule or EON.

[0126] Mutagenesis studies of human ADAR2 revealed that a single mutation at residue 488 (E488Q) from glutamic acid to glutamine increased the deamination rate constant by 60-fold compared to the wild-type enzyme (Kuttan & Bass. 2012. Proc Natl Acad Sci USA. 109(48):3295-3304). During the deamination reaction, ADAR flips the base to be edited from its RNA duplex and places it in the enzyme active site (Matthews et al. 2016. Nat Struct Mol Biol. 23(5):426-433). When ADAR2 edits an adenosine in a favorable context (A:C mismatch), the nucleotide opposite the target adenosine is called a lone cytidine. The crystal structure of ADAR2 E488Q bound to dsRNA revealed that the glutamine (Gln) side chain at position 488 can donate a hydrogen bond to the N3 position of the lone cytidine, leading to an improved catalytic rate of ADAR2 E488Q. In the wild-type enzyme, a glutamic acid (Glu) exists at position 488 instead of glutamine (Gln), and the amide group of the glutamine is absent, becoming a carboxylic acid instead. Therefore, to achieve the same contact with the lone cytidine using the E488Q mutant in the wild-type context, protonation is required for this contact to occur. When using endogenously expressed ADAR2 to correct disease-associated mutations, it is important to maximize the editing efficiency of the wild-type ADAR2 enzyme present in cells. WO 2020 / 252376 discloses the use of EONs, specifically modified RNA bases at the lone cytidine position, to mimic the hydrogen bonding pattern observed with the E488Q ADAR2 mutant. It was hypothesized that by replacing the nucleotide opposite the target adenosine in the EON with a cytidine analogue that functions as a hydrogen bond donor at N3, it would be possible to stabilize the same contacts that would improve catalytic rate in the mutant enzyme.Two cytidine analogs were of particular interest: pseudoisocytidine (also referred to as "piC"; Lu et al. 2009. J Org Chem. 74(21):8021-8030; Burchenal et al. (1976) Cancer Res 36:1520-1523) and Benner's base Z (also referred to as "Zd"; Yang et al. 2006. Nucl Acid Res. 34(21):6095-6101), which were initially selected because they donate a hydrogen bond at N3 with minimal perturbation to the geometry of the nucleobase. Benner's base is also referred to as 6-amino-5-nitro-3-yl-2(1H)-pyridone. The presence of cytidine analogs in EONs can be in addition to modifications to the ribose 2' group. The ribose 2' groups in the EON can be independently selected from the following: 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or a 2'-4' linkage (i.e., bridged nucleic acids such as LNA), or other 2' substituents. The 2'-4' linkage can be selected from linkers known in the art, such as a methylene linker or a constrained ethyl linker.

[0127] In one embodiment, the EON comprises one or more sugar moieties that are mono- or di-substituted at the 2′, 3′, and / or 5′ positions, as exemplified below: —OH; —H; —F; substituted or unsubstituted, straight or branched lower (C1-C 10 ) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-aryl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy.

[0128] In one embodiment, the nucleotide analog or equivalent in EON comprises one or more base modifications or substitutions.Modified bases include synthetic and natural bases, such as inosine, xanthine, hypoxanthine, and other aza, deaza, hydroxy, halo, thio, thiol, alkyl, alkenyl, alkynyl, thioalkyl derivatives of pyrimidine and purine bases, which are known or will be in the future in the art.Purine nucleobases and / or pyrimidine nucleobases can be modified, for example, by amination or deamination of heterocycles, to change their properties.The exact chemical structure and format can vary from oligonucleotide construction to oligonucleotide construction and application to application, and can be determined according to the wishes and preferences of those skilled in the art.

[0129] The EONs disclosed herein are typically longer than 10 nucleotides, preferably greater than 11, 12, 13, 14, 15, 16, and even more preferably greater than 17 nucleotides. In one embodiment, the EONs disclosed herein are longer than 20 nucleotides. The EONs 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, the EONs disclosed herein contain 18-70 nucleotides, more preferably 18-60 nucleotides, and even more preferably 18-50 nucleotides. Thus, in particularly preferred embodiments, the EON disclosed herein comprises 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, or 50 nucleotides. In one embodiment, the EON is 27, 28, 29, or 30 nucleotides in length.

[0130] In one embodiment, an inverted deoxyT or dideoxyT nucleotide is incorporated at each or both ends of the EON disclosed herein. [Example]

[0131] Example 1. Editing of Target Adenosines in Human B4GALT1 Target RNA Molecules Using an In Vitro Biochemical Editing Assay First, the first set of B4GALT1-targeting EONs (shown in Figure 2) was tested for editing of human B4GALT1 target (pre-)mRNA in an in vitro biochemical editing assay. To obtain B4GALT1 target RNA, PCR was performed using the B4GALT1 G-block (IDT), which contains the T7 promoter sequence and (part of) the HFE sequence, as a template, with the forward primer 5'-CTC GAC GCA AGC CAT AAC AC-3' (SEQ ID NO: 43) and the reverse primer 5'-TGG ACC GAC TGG AAA CGT AG-3' (SEQ ID NO: 44). The 5'-3' G-block sequence (SEQ ID NO: 45) is as follows, in which the target adenosine is underlined and bold, and the primer sequence is underlined:

[0132] JPEG2025540146000007.jpg27166

[0133] The PCR product is then used as a template for in vitro transcription using the MEGAscript T7 Transcription Kit. RNA is purified on a urea gel and then extracted in 50 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.1% SDS, 0.3 M NaCl buffer, followed by phenol-chloroform purification. The purified RNA is used as a target in biochemical editing assays.

[0134] First, EON RM4439-RM4454 and RM4826-RM4849 were annealed with the B4GALT1 target RNA, respectively, in a buffer solution (5 mM Tris-Cl pH 7.4, 0.5 mM EDTA, and 10 mM NaCl) at a target RNA to oligonucleotide ratio of 1:3 (600 nM oligonucleotide and 200 nM target). The sample was heated to 95°C for 3 minutes and then slowly cooled to room temperature. The editing reaction was then carried out. The annealed oligonucleotide / target RNA was mixed with protease inhibitors (cOmplete™ Mini, EDTA-free Protease I, Sigma-Aldrich), ribonuclease inhibitor (RNasin, Promega), polyA (Qiagen), tRNA (Invitrogen), and editing reaction buffer (15 mM Tris-Cl pH 7.4, 1.5 mM EDTA, 3% glycerol, 60 mM KCl, 0.003% NP-40, 3 mM MgCl2, and 0.5 mM DTT) to a final concentration of 6 nM oligonucleotide and 2 nM target RNA. The reaction was initiated by adding purified ADAR2 (GenScript) to the mixture to a final concentration of 6 nM and incubated at 37°C for a predetermined time. Each reaction was stopped by adding 95 μl of 95°C 3 mM EDTA solution. A 6 μl aliquot of the stopped reaction mixture was then used as a template for cDNA synthesis using the Maxima Reverse Transcriptase Kit (Thermo Fisher Scientific) with random hexamer primers (Thermo Fisher Scientific). Initial denaturation of the RNA was performed in the presence of primers and dNTPs at 95°C for 5 minutes, followed by slow cooling to 10°C. First-strand synthesis was then performed according to the manufacturer's instructions, in a total volume of 20 μl, using an extension temperature of 62°C. For pyrosequencing analysis, the product was amplified by PCR using the Amplitaq Gold 360 DNA Polymerase Kit (Applied Biosystems) according to the manufacturer's instructions, using 1 μl of cDNA as template.PCR is then performed using the following thermal cycling protocol: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 7 min.

[0135] During cDNA synthesis in the reverse transcription reaction, inosine base pairs with cytidine, so the nucleotide incorporated into the edited portion during PCR is guanosine. The percentage of guanosine (edited) and adenosine (unedited) is determined by pyrosequencing. Pyrosequencing of PCR products and data analysis are performed using a PyroMark Q48 Autoprep instrument (QIAGEN) according to the manufacturer's instructions, using 10 μl of PCR product input and 4 μM sequencing primers. Analysis performed by the instrument provides results for selected nucleotides as the percentage of adenosine and guanosine detected at that position. Therefore, the degree of A to I editing at a selected position is measured by the percentage of guanosine at that position.

[0136] Example 2: Editing of human B4GALT1 transcripts in HepG2 cells Editing of endogenous human B4GALT1 transcripts was investigated in human cells. To this end, human HepG2 hepatocellular carcinoma cells were cultured in EMEM + 10% FBS + 1% P / S. Cells were maintained at 37°C in a 5% CO atmosphere. In the first experiment, EONs designated RM4826-RM4849 (see Figure 2) were tested for editing efficiency. A total of 0.75 x 10 5HepG2 cells were seeded and treated with 1 or 5 μM EON, and 1 μM AG1856 (also known as saponin, a triterpene glycoside; see WO2021 / 122998) was added per well. This mixture was left on the cells for 72 hours. The cells were then harvested, and total RNA was isolated from the transfected cells using the ReliaPrep™ RNA Miniprep Kit. After removing the culture medium, the cells were washed once with PBS. After completely aspirating the PBS, 100 μL of lysis buffer was added to the well to lyse the cells and recover the intracellular material. After adding 35 μL of isopropanol, the mixture was loaded onto a column and subjected to multiple washing steps and DNase I treatment. After elution in a total volume of 15 μL of DNase / RNase-free water, RNA yield was determined using spectrophotometric analysis (NanoDrop) and stored at −80°C.

[0137] Maxima reverse transcriptase (RT, Thermo Fisher Scientific) was used to generate complementary DNA (cDNA). Typically, 500 ng of total RNA was used in a reaction mixture containing 4 μL of 5x RT buffer, 2 μL of dNTP mix (10 mM each), 0.5 μL of Oligo(dT), 0.5 μL of random hexamers, and 0.5 μL of Maxima reverse transcriptase (all Thermo Fisher Scientific). DNase- and RNase-free water was added to a total volume of 20 μL. The sample was loaded into a T100 thermal cycler (Bio-Rad) and initially incubated at 25°C for 10 minutes, followed by a 30-minute cDNA reaction at 50°C and a 5-minute stop step at 85°C. The sample was cooled to 4°C and then stored at -20°C.

[0138] To determine editing efficiency, cDNA samples were subjected to two multiplex digital PCR (dPCR) assays. HepG2 cDNA samples were diluted 5-fold before dPCR analysis. The first dPCR assay was designed to distinguish between cDNA species containing the original adenosine and those containing edited inosine (which is converted to guanidine during cDNA synthesis). The first dPCR assay also quantified the amount of B4GALT1-specific cDNA molecules in the mixture using a primer / probe set targeting exons 1 and 2. The second dPCR assay was designed to measure exon 5 skipping of B4GALT1 and the housekeeping gene HPRT1. Primer and probe sequences are listed in Table 1.

[0139] JPEG2025540146000008.jpg146166

[0140] Digital PCR was performed using a QIAcuity 4 (5-plex) system, a QIAcuity PCR kit, and a 96-well 8.5K nanoplate (Qiagen). A total of 1.2 μL of diluted cDNA mixture was used in a dPCR mixture containing: 3 μL of 4x QIAcuity Mastermix, 0.6 μL of each primer (10 μM stock concentration), and 0.3 μL of each probe (10 μM stock concentration), plus DNase- and RNase-free water for a total volume of 12 μL. The dPCR mixture was prepared in a preplate, then transferred to a 96-well 8.5K nanoplate and sealed with a nanoplate seal. The plate was then transferred to the QIAcuity 4 instrument. First, a priming and rolling step was performed to form and separate the chamber compartments, followed by an amplification step using the following cycling protocol: 95°C for 2 minutes for enzyme activation, 95°C for 15 seconds for denaturation, and 60°C for 30 seconds for annealing / extension, for 40 cycles. After the amplification step, an image acquisition step was performed for all wells. Data were analyzed using QIAcuity Suite Software (Qiagen).

[0141] The total copy amount per ng of RNA was determined by summing the A-containing, G-containing, and exon 5 skip-containing segments per ng of RNA. The percentage A-to-I editing was determined by dividing the number of G-containing segments per ng of RNA by the sum (G+A-containing segments) and multiplying by 100. The percentage of exon 5 skipping was determined by dividing the number of exon 5 skip-containing segments per ng of RNA by the total copy number.

[0142] The results of RNA editing of the endogenous B4GALT1 transcript are shown in Figure 3 and show that, although efficiency varied significantly between EONs, increasing EON concentration from 1 μM to 5 μM resulted in higher editing levels. No editing was observed in the negative controls (AG1856 alone and untreated samples (NT)). RM4826, RM4830, RM4834, RM4838, RM4842, and RM4846 appeared to exhibit the highest levels of RNA editing.

[0143] Because EON binding to target transcripts may or may not interfere with splicing and induce exon skipping, we investigated whether exon 5 of the B4GALT1 pre-mRNA was skipped during splicing. The c.1055A target adenosine resides within exon 5 of the B4GALT1 gene. The results, shown in Figure 4, indicate that several EONs induced very high levels of exon skipping (in some cases exceeding 70%), although this level did not appear to correlate with the editing levels observed in the six most successful editing EONs. The highest exon skipping percentages were observed in RM4832, RM4834, RM4836, RM4838, RM4839, RM4840, and RM4841. Lower exon skipping levels were observed in RM4826, RM4827, RM4828, RM4829 (5 μM samples were lost), RM4842, RM4843, RM4844, RM4845, RM4846, RM4847, RM4848, and RM4849. Interestingly, for EONs with lower skipping percentages, these lower exon skipping levels were observed in samples with higher concentrations (5 μM) of EON than in samples with lower concentrations (1 μM). It remains unclear whether exon skipping induced by treatment with RNA-editing oligonucleotides, as outlined herein, is an unwanted effect or a bonus effect. The primary reason for targeting c.1055A in the B4GALT1 transcript is to reduce B4GALT1 protein function. Skipping exon 5 of the transcript, which is a frame-maintaining event, may also result in a protein with reduced activity (shorter), which may be an additional beneficial effect of EON treatment.

[0144] Example 3. Editing of human B4GALT1 transcripts in liver spheroids. Next, we investigated whether RNA editing of endogenous B4GALT1 transcripts could be achieved in hepatic spheroids cultured from primary human hepatocytes. Female primary human hepatocytes (PHH) (BioIVT) were used to generate spheroids. Following the supplier's protocol, PHH cell suspensions (15,000 cells / mL) were seeded into Nuclon Sphera low-attachment U-bottom 96-well plates at 1,500 cells / well. The plates were then incubated at 37°C in a 5% CO2 atmosphere for 5 days.

[0145] After incubation, the spheroids were transferred to a flat-bottom 96-well plate and pooled. Pooling resulted in eight spheroids per well, with a total medium volume of 100 μL per well. To maintain the spheroid cultures, 100 μL of maintenance medium was added to each well and incubated for an additional 48 hours at 37°C in a 5% CO atmosphere. The maintenance medium consisted of INVITROGRO Spheroid Maintenance Medium (BioIVT) combined with Spheroid Medium Supplement A, which was also used for spheroid treatment. 100 μL of medium was then removed from each well (containing day 7 spheroids) and 100 μL of treatment medium was added. Each well was treated with 1 or 5 μM EON and 1 μM AG1856 saponin (see above) for 72 hours. For this initial spheroid experiment, four of the best performing EONs (see above) were selected: RM4834, RM4838, RM4842, and RM4846. Negative controls were saponin only and NT samples.

[0146] After exposure to EON / saponin, spheroids were harvested and washed once with PBS, followed by the addition of 300 μL of lysis buffer. RNA isolation, RNA yield determination, cDNA synthesis, editing efficiency using dPCR, and evaluation of exon 5 skipping were performed as described in Example 2.

[0147] The results are shown in Figure 5. Consistent with the results observed in HepG2 cells, all four EONs were able to mediate relatively high levels of RNA editing of the endogenous B4GALT1 transcript in spheroids cultured in PHH, reaching levels exceeding 30% (Figure 5A). All four EONs functioned in a similar range. As shown in Example 2, exon 5 skipping was also evaluated in these spheroids, and the results are shown in Figure 5B. Interestingly, treatment with RM4834 and RM4838 resulted in relatively high levels of exon 5 skipping (over 40%), while RM4842 and RM4846 only induced a skipping percentage of approximately 8%. Neither editing nor exon skipping was observed in samples treated with saponin alone (NT+AG in Figure 5) or in untreated samples. This demonstrates that significant levels of RNA editing can be achieved using a variety of antisense oligonucleotides, which unexpectedly vary significantly in their ability to affect splicing of the human B4GALT1 pre-mRNA.

[0148] Example 4. Effect on fibrinogen levels after in vitro editing of human B4GALT1 transcripts. To investigate the effect of RNA editing of the c.1055A target adenosine in the human B4GALT1 transcript on fibrinogen secretion, we tested whether such RNA editing reduces fibrinogen levels in the supernatants of human hepatocellular carcinoma cell lines HepG2 and Huh-7 cells after treatment with EON. To this end, HepG2 cells are cultured in EMEM + 10% FBS + 1% P / S, and Huh-7 cells are cultured in RPMI + 10% FBS + 1% P / S. Cells are maintained at 37°C in a 5% CO2 atmosphere. A total of 0.75 x 10 5HepG2 cells and, separately, 0.5 x 10 5 Huh-7 cells were seeded into 24-well plates and treated with 5 μM EON and 1 μM AG1856 (see above) per well for 72 hours. The cell culture supernatant was then collected. The samples were centrifuged at 2000 g for 10 minutes. The supernatant was then transferred to a new tube. The samples were diluted 1:500, and fibrinogen levels were measured using a high-sensitivity fibrinogen ELISA (ab241383, Abcam) as follows: 50 μL of sample or standard was added to each well. 50 μL of antibody cocktail was then added to the well. After 1 hour of incubation at room temperature, the wells were washed three times with wash buffer. 100 μL of TMB Development Solution was then added to each well, and the plate was incubated for 10 minutes in the dark. Finally, 100 μL of Stop Solution was added to each well. Plates are read at 450 nm using a plate reader (SpectraMax M5). A standard curve is generated using the 4PL method in GraphPad (version 9.0.1). Raw values ​​are blank corrected, and average values ​​are calculated from duplicate measurements. Fibrinogen levels are determined by interpolation using the standard curve. Fibrinogen levels are normalized to the RNA yield in NT+ media conditions.

[0149] Example 5. Editing of human B4GALT1 transcripts in primary human hepatocytes. Next, a new set of EONs was designed based on the best-performing editors from the initial editing screen (EON01 (RM4826), EON05 (RM4830), EON09 (RM4834), EON13 (RM4838), EON17 (RM4842), and EON21 (RM4846)). This new set of EONs and their respective chemical modifications are shown in Figure 6. Some of these EONs differ in their 5'-end portions. This is because some EONs are complementary to exon 6 (i.e., after intron 5 is spliced ​​from the pre-mRNA), while others are complementary to the 5'-end portion bearing intron 5 (i.e., before splicing). See, for example, the difference in the 5'-end portions between B4GALT1-32 (RM106386) and B4GALT1-218 (RM106292). Each of the EONs shown in Figure 6 (except B4GALT1-134(-)) contains a triantennary GalNAc modification (L001=OP-042; Hongene Biotech) at the 5' end to stimulate hepatocyte entry and is linked to the most 5' nucleotide via a TEG linker (L103). EONs and their adducts were prepared according to standard protocols well known to those skilled in the art.

[0150] The new set of EONs was used in conjunction with EON01 (RM4826) and EON05 (RM4830) to examine the editing of endogenous human B4GALT1 transcripts in primary human hepatocytes (PHH). A non-treated (NT) sample was also used as a negative control. For this purpose, a total of 0.5 x 10 5PHH cells (BioIVT) were seeded using Invitrogen CP medium supplemented with TORPEDO Antibiotic Mix. The cells were maintained at 37°C in a 5% CO2 atmosphere. Four hours after seeding, the medium was replaced with Invitrogen HI medium supplemented with TORPEDO Antibiotic Mix and cultured. The next day, the cells were treated with 5 μM EON + 1 μM AG1856 per well. This mixture was left on the cells for 72 hours. Afterwards, the cells were washed once with PBS, and 100 μL of lysis buffer was added. Total RNA was isolated using the Direct-zol™ RNA Microprep Kit (Zymo Research). After adding 100 μL of ethanol (95–100%), the mixture was loaded onto a column and subjected to multiple washing steps and DNase I treatment. After elution in a total volume of 15 μL of DNase / RNase-free water, RNA yields were determined using spectrophotometry (NanoDrop) and stored at −80° C. RT reactions and dPCR were subsequently performed using the indicated primers and probes as described in Example 2.

[0151] The results are shown in Figure 7. (A) The editing percentages obtained in these PHHs are illustrated, clearly showing that EON05 (without a GalNAc moiety) remains one of the most successful EONs. This may be explained by the possibility that the interaction of GalNAc with the respective receptor is not critical for promoting entry in vitro but may have a significant additive effect in an in vivo environment. In any case, some of the new EONs clearly showed beneficial effects over others, with B4GALT1-33, for example, being one of the most successful. Figure 7(B) shows the percentage of exon 5 skipping in the same samples, clearly demonstrating the significant differences between some EON treatments. For example, after incubation with B4GALT1-212 and -213, exon skipping was close to zero, while B4GALT1-175 and -176 EONs induced relatively high skipping percentages. Notably, while EON01 and EON05 acted similarly in editing, there was a notable difference between these two (non-GalNAc-containing) EONs in inducing exon 5 skipping.

[0152] In subsequent experiments, it was then investigated whether RNA editing in PHHs could also be achieved without the addition of saponin, i.e., by simply co-incubating oligonucleotides in the cell culture medium (=gymnotic uptake, or "gymnosis"). The overall experimental setup was identical to that described above, with the only exception that no saponin was added along with EON. All subsequent RNA purification and dPCR steps were the same as described above.

[0153] The results of this experiment are shown in Figure 8, where (A) shows the percentage of RNA editing after gymnotic incorporation of the indicated EONs, which is clearly (and as expected) lower than that shown in Figure 7 (in which saponin was used as the "transfection agent"), but still significantly above background. It should be noted, as noted above, that low levels of editing do not necessarily imply low downstream effects on the protein and its functionality. The percentage of editing induced by EONs ranged around 1%, with only B4GALT1-132 exhibiting levels as high as 4%. The percentage of exon skipping (exon 5) was also determined in these samples. Consistent with the reduced editing levels, the level of exon skipping was also reduced (see Figure 8(B)), a pattern similar to that observed in experiments in which saponin was used to improve oligonucleotide entry into cells.

[0154] Example 6. Editing of B4GALT1 transcripts in primary mouse hepatocytes. A similar experiment to that described in Example 5 was performed using primary mouse hepatocytes (PMH) without the application of saponin. Primary mouse hepatocytes were isolated from mouse livers using a Liver perfusion kit (Miltenyi) and a GentleMACS Octo Dissociator with Heaters (Miltenyi) according to the manufacturer's instructions. More specifically, the liver was removed from the mouse, washed with PBS, and transferred to a GentleMACS Octo Dissociator with Heaters. The automated program 37C_m_LIPK_1 was run, which consists of various steps including priming, initial perfusion, washing, equilibration, and enzyme perfusion. After the perfusion process was completed, the perfused liver lobe and spent enzyme solution were transferred to a gentleMACS C tube (Miltenyi). The gentleMACS C tube was then placed back into the Octo Dissociator, and the program LIPK_HR_1 was run to further release hepatocytes from the perfused liver. Finally, the cell solution was passed through a MACS 100 μM SmartStrainer (Miltenyi) to enrich for hepatocytes. A debris removal kit (Miltenyi) was used to remove debris from the cell solution. The cell pellet was resuspended in 5 mL of DMEM + 5% FBS + 1% P / S. Cells were counted and a total of 0.75 × 10 5 Cells were seeded in 1000 x g of GALT1 cells. The cells were treated with 5 μM EON in Williams E medium + 2 mM glutamine + 1% P / S, and the mixture was left on the cells for 72 hours. RNA isolation, RNA yield determination, cDNA synthesis, editing efficiency using dPCR, and evaluation of exon 5 skipping effects were performed as described in Example 2. However, only a single dPCR mixture was used, combining assays for editing, exon 5 skipping, and B4GALT1 expression in exons 1-2, using mouse-specific primers and probes, as shown in Table 2.

[0155] JPEG2025540146000009.jpg100166

[0156] The results of these experiments are shown in Figure 9, where (A) shows the editing percentage using the same EON. These EONs are 100% complementary to the target sequence in the human B4GALT1 (pre-) mRNA transcript, except for the lone nucleotide opposite the target adenosine. The mouse (Mus musculus) target sequence contains a G at the 5' position from the target A, meaning that the nucleotide at position -5 (usually a uridine) in the EON in Figure 6 forms a G:U base pair with the mouse target transcript sequence. As shown in WO 2017 / 220751, this is not necessarily a disadvantage. In addition to the mismatch at the lone position, additional mismatches, bulges, and / or wobble may contribute to improved recognition by the ADAR enzyme in cells of the formed double-stranded RNA complex, which is considered a more suitable target configuration. If this is the case, the data shown in Figure 9(A) support this. This is because higher editing levels were observed in these PMHs under these conditions (without saponin) than in PHHs (see Figure 8(A)). EON05 (RM4830; SEQ ID NO: 23) performed best, reaching editing levels of over 10%. Consistent with the results in PHHs, the extent to which the GalNAc moiety contributes to cell entry in this in vitro configuration remains unclear. Nevertheless, RNA editing levels by the most successful EONs were in the 5% range. The percentage of exon 5 skipping shown in Figure 9(B) followed a similar pattern to that observed in PHHs described above.

[0157] Example 7. Editing of human B4GALT1 transcripts in primary human hepatocytes using EON entrapped in lipid nanoparticles as a delivery vehicle. In the next experiment, we investigated whether different types of delivery methods, in which EONs are incorporated into so-called lipid nanoparticles (LNPs), contribute to the delivery of oligonucleotides to target cells. For this purpose, we selected EON05 (RM4830; SEQ ID NO: 23; see Figure 2), EON13 (RM4838; SEQ ID NO: 31; see Figure 2), and B4GALT1-134(-) (RM107261; SEQ ID NO: 1069) (which is identical to B4GALT1-134 but does not contain the GalNAc moiety and TEG linker, and is also referred to herein as EON134; see Figure 6).

[0158] EON-containing LNP formulations were produced as disclosed in WO2015 / 048020. Lipid stocks were first prepared in ethanol. These lipids were then mixed in a molar ratio of DLin-MC3-DMA (50%), cholesterol (38.5%), DSPCV (10%), and PEG-200-DMG (1.5%). The formulation process can be summarized as follows: Each EON was mixed with lipids in a microfluidic device in a 1:3.6 ratio. The formulated LNPs were further concentrated using a tangential flow filtration (TFF) system, buffer exchanged by dialysis for 12 hours, and then sterilized by sterile filtration. The final solution concentration was 1 mg / mL. EON-containing LNP formulations were stored in glass vials at 2–8 °C until further use.

[0159] PHHs were obtained and seeded as described in Example 5. A total of 0.5 × 10 cells per well were added 24 hours before incubation with the LNP formulations. 5 Cells were seeded in wells containing EON05-LNP, EON13-LNP, and EON134-LNP at concentrations ranging from 0.01 to 10 μM in INVITROGRO HI medium + TORPEDO Antibiotic mix + 10% FBS for 72 hours. Recovery, RNA isolation, cDNA preparation, and dPCR procedures were performed as described in Example 2.

[0160] Figure 10(A) shows the percentage editing achieved in PHHs using the three LNP formulations shown at different concentrations, with an untreated sample used as a negative control. This indicates that a concentration of 1 μM works best under these conditions at the time the cells were harvested, and that editing levels of up to 10% can be achieved using the EON05-LNP formulation (without the aid of saponin). Similar to the experiments described above, the percentage of exon 5 skipping was also determined in these samples, which yielded the expected results based on the determined percentage editing (see Figure 10(B)).

[0161] Example 8. Editing of B4GALT1 transcripts in mouse hepatocytes The three LNP preparations described in Example 7 (EON05-LNP, EON13-LNP, and B4GALT1-134(-)(~EON134-LNP)) were also used in in vivo experiments in which mice were intravenously (IV) injected with the LNP formulations or a control. The in vivo experiments were configured as follows, with the oligonucleotide dose level for all mice being 3 mg / kg body weight, except for the repeat dose in Group 6 and the initial dose for mice on days 2 and 4 in Group 5, which was 1.5 mg / kg body weight:

[0162] JPEG2025540146000010.jpg55166

[0163] The negative control vehicle was an LNP formulation that did not contain any oligonucleotides, while Actb-LNP was an LNP formulation containing an oligonucleotide (RM3891) against the human actin B target sequence, and this formulation was also used as a negative control in this experiment.

[0164] On the designated necropsy date, mice were sacrificed according to standard procedures, followed by blood collection and removal of organs, including the liver. RNA was isolated from liver tissue. 1 mL of Trizol (Thermo Fisher Scientific) was added to liver tissue in a 2 mL tube containing 1.4 mm ceramic beads (Thermo Fisher Scientific), and the sample was homogenized for 25 seconds at 6 m / s using a Beadmill 24. The tissue homogenate was transferred to a 1.5 mL Eppendorf tube, and 200 μL of chloroform (VWR) was added per mL of homogenate. After centrifugation at 12,000 g for 15 minutes at 4°C, 300 μL of the aqueous layer was transferred to a new 1.5 mL Eppendorf tube. 300 μL of isopropanol was then added, and RNA isolation proceeded using the ReliaPrep™ RNA Miniprep Kit. The mixture was loaded onto a column and subjected to multiple washing steps and DNase I treatment. Elution was performed using DNase / RNase-free water in a total volume of 50 μL. RNA yield determination, cDNA synthesis, editing efficiency using dPCR, and evaluation of exon 5 skipping were performed as described in Example 2.

[0165] Figure 11(A) shows the percentage of editing in the livers of mice from each treatment group compared to the Actb-LNP control (RM3891-LNP) and PBS on days 2, 4, 7, and 30, as indicated. These results demonstrate that editing levels as high as 2% were attainable two days after administration, which was significantly higher than that observed in the livers of mice treated with the negative control. The percentage of editing decreases over time, as seen in samples from days 4, 7, and 30. Figure 11(B) shows the percentage of exon 5 skipping in the same samples, again confirming the results observed in the cells and liver spheroids described above.

[0166] Example 9. Editing of human B4GALT1 transcripts in primary human hepatocytes. To further explore the potential of the present disclosure, an additional set of 960 EONs was designed, each individually or independently, to vary the chemical modification and length of the oligonucleotides to ultimately improve editing of B4GALT1 transcripts. These 960 EONs, along with their respective RM numbers and chemical modifications, are shown in Figure 12. From this set and from the other designed EONs described herein, the best performing candidates are used with or without a GalNAc moiety (and a TEG linker) and formulated into LNP formulations as needed. These best performing candidates are then advanced into (pre)clinical development to ultimately treat CVD in patients in need thereof.

[0167] Example 10. Editing of human B4GALT1 transcripts in primary human hepatocytes. Additionally, another set of 22 EONs was designed and tested alongside some of the more successful editing-inducible EONs from previous screens: B4GALT1-05=EON05=RM4830=SEQ ID NO:23; B4GALT1-13=EON13=RM4838=SEQ ID NO:31; B4GALT1-69=RM107689=SEQ ID NO: 1064; and B4GALT1-84=RM107704=sequence number 1068.

[0168] This new set of 22 EONs is shown in Figure 13, along with their respective RM numbers and chemical modifications.

[0169] Using these EONs, editing of endogenous human B4GALT1 transcripts was examined in PHH. Non-treated (NT) and saponin-only samples were also used as negative controls. Cells were cultured, seeded, and treated as described in Example 5 above, except that cells were co-treated with 0.5 μM AG1856 per well. RNA purification, cDNA synthesis, and dPCR were performed as described in Example 2 using the indicated primers and probes.

[0170] The results are illustrated in Figure 14 and show that all EONs tested resulted in relatively high editing levels, with B4GALT1-13, B4GALT1-65 (SEQ ID NO: 1079), B4GALT1-71 (SEQ ID NO: 1084), B4GALT1-80 (SEQ ID NO: 1093), and B4GALT1-82 (SEQ ID NO: 1095) performing best.

[0171] Example 11. Exon 5 skipping in human B4GALT1 transcripts after EON treatment As discussed herein, the purpose of EONs is to induce editing of human B4GALT1 transcripts by introducing A>I deamination, thereby generating the N352S mutation (or "variant") in the protein sequence. The target adenosine at position 1055 in the transcript is relatively close to the 3' end of exon 5 in the pre-mRNA. It was observed that some of the EONs disclosed herein (in addition to inducing the desired RNA editing) also induce skipping of exon 5 from the pre-mRNA, resulting in an out-of-frame transcript and consequently downregulating active B4GALT1. Thus, while splice modulation is often undesirable, in this case it may be considered a side effect (or unexpected bonus effect) and may be beneficial in the treatment of CVD. Therefore, we investigated the extent to which the position of the complementary sequence of the EON with the target RNA influences exon 5 skipping. To this end, a large set of EONs was designed, with 12 EONs of the same length but with different chemical modifications (including GalNAc and TEG linkers) included in nine separate sets. Thus, 9 × 12 EONs were tested for their ability to induce exon skipping, potentially revealing how their location correlates with exon skipping efficiency. Figure 15(A) shows the exon 5 / exon 6 boundary and the relative location of target A in exon 5. The bottom of the diagram shows the locations of the nine sets of EONs. These are shown in the 3'→5' direction, with the 5' end of the EON extending into intron 5, primarily targeting the pre-mRNA. EONs were distributed at 5 × 10 per well. 4The cells were tested in a 96-well format in PHH cells, incubated with 5 μM EON concentrations, and co-treated with 1 μM AG1856. RM105550 (=B4GALT1-13 with a GalNAc moiety but no TEG linker; identical to RM106564; SEQ ID NO: 1100) was used as a control. Cells were harvested after 72 hours, and exon 5 skipping was then determined as described above. Figure 15(B) shows the results obtained for all individual EONs. A subset is shown at the bottom of the graph. The results clearly show that more exon skipping is observed when the EON is complementary to sequences relatively far from the 3' end of exon 5. However, skipping efficiency significantly decreased when the complementarity was "shifted" toward the exon 5 / intron 5 boundary. This indicates that the location of the EON and where it hybridizes to the target sequence not only influences RNA editing but also exon 5 skipping, which in combination can contribute to downregulating the function of the B4GALT1 protein, as desired and as discussed herein.

[0172] Example 12. Editing of human B4GALT1 transcripts in primary human hepatocytes using EONs with different 2'-fluoro modifications. We tested whether the location of 2′-F modifications within EONs influences their effect on RNA editing and / or exon 5 skipping. To this end, a set of 20 EONs was designed based on the sequence and modifications described for B4GALT1-13 (RM106564; SEQ ID NO: 1100, which is identical to RM105550 in Figure 15). The different B4GALT1-13-based EONs and their 2′-F modifications are shown in Figure 16 (SEQ ID NOs: 1101-1120). This shows that RM106949 (SEQ ID NO: 1101) completely lacks 2′-F modifications, except for one. Using these EONs, experiments similar to those described in Example 11 were performed, except that purified RNA was used for determining the percentage of exon 5 skipping, cDNA synthesis, and dPCR to measure RNA editing of the target adenosine. Figure 17(A) shows the percentage editing (black bars) and percentage exon 5 skipping (white bars) for each of the 20 EONs compared to B4GALT1-13 (RM106564; SEQ ID NO: 1100 (RM4838; see also SEQ ID NO: 31)). Notably, RM106564 exhibited approximately 22% editing and 43% exon skipping, whereas RM106949 exhibited significant exon skipping (>50%) but little RNA editing. This suggests that the 2'-OMe modification, present in place of the 2'-F modification, prevents proper deamination of this target by ADAR enzymes without interfering with splice regulation. Importantly, some EONs, such as RM106950 (SEQ ID NO: 1102) and RM106963 (SEQ ID NO: 1115), showed editing percentages greater than 30%, while exon 5 skipping was less than 30%, indicating that the location of the 2'-F modification is important for achieving different effects. Similar experiments were performed with 17 EONs (SEQ ID NOs: 1122-1138) based on the B4GALT1-21 EON (RM106566; SEQ ID NO: 1121 (RM4846; see also SEQ ID NO: 39)).The results of the same experiment are shown in Figure 17(B) and show that some EONs, such as RM106970 (sequence number 1123) and RM106971 (sequence number 1124), performed comparable to RM106566 in editing (up to 35%) and exon 5 skipping (approximately 2%).

Claims

1. 1. An RNA editing oligonucleotide (EON) capable of forming a double-stranded complex with a region of a target RNA nucleic acid molecule in a human cell, comprising: the double-stranded complex is capable of recruiting an endogenous ADAR enzyme naturally present in the cell; the region includes a target adenosine; the nucleotide in the EON opposite the target adenosine is a lone nucleotide; The ADAR enzyme deaminates the target adenosine to inosine; and The target RNA nucleic acid molecule is a transcription product molecule of the human β-1,4-galactosyltransferase 1 (B4GALT1) gene. RNA-editing oligonucleotides.

2. 2. The EON according to claim 1, wherein the B4GALT1 transcript molecule is a pre-mRNA or mRNA molecule; EON.

3. 3. The EON according to claim 1 or 2, The cells are human liver cells, preferably hepatocytes. EON.

4. An EON according to any one of claims 1 to 3, The target adenosine is present in the B4GALT1 transcript at a position that, if replaced by a guanosine, would encode a B4GALT1 protein variant with a reduced enzyme turnover rate. EON.

5. An EON according to any one of claims 1 to 4, the target adenosine is present at position c.1055A in the B4GALT1 transcript; and The deamination results in a change from asparagine (N; Asn) to serine (S; Ser) at position 352 in the human wild-type B4GALT1 amino acid sequence. EON.

6. An EON according to any one of claims 1 to 5, wherein at least one nucleotide comprises one or more non-natural chemical modifications in a ribose moiety, a linker moiety, or a base moiety; provided that the isolated nucleotide is not a cytidine containing a 2'-OMe ribose substitution. EON.

7. 7. The EON according to claim 6, the one or more modifications in the linkage are each independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonyl phosphoramidate, or PNdmi internucleoside linkage; EON.

8. 8. An EON according to claim 6 or 7, The one or more modifications in the ribose moiety are mono- or di-substitutions at the 2', 3', and / or 5' positions of the ribose and are selected from the group consisting of: -OH; -F; substituted or unsubstituted, straight or branched lower (C １ -C １０ ) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-aryl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy; each independently selected from EON.

9. An EON according to any one of claims 1 to 8, The EON comprises or consists of an EON selected from the group set forth in SEQ ID NOs: 3 to 42, 59 to 1069, and 1078 to 1190; Preferably, selected from the group consisting of SEQ ID NOs: 23, 19, 31, 27, 35, 39, 69, 70, 71, 72, 73, 93, 94, 95, 1079, 1084, 1093, 1095, 1100, 1102, 1115, 1121, 1123, 1124, and 1139-1190; EON.

10. An EON according to any one of claims 1 to 9, the isolated nucleotide is a deoxynucleotide having a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleobase or an isouracil nucleobase; EON.

11. A nucleic acid molecule encoding an EON according to any one of claims 1 to 5, A vector, preferably a viral vector, more preferably an adeno-associated viral (AAV) vector.

12. A nanoparticle delivery vehicle formulation comprising an EON according to any one of claims 1 to 10.

13. 13. The nanoparticle delivery vehicle formulation of claim 12, wherein the nanoparticle delivery vehicle is a lipid nanoparticle (LNP), Nanoparticle delivery vehicle formulations.

14. A pharmaceutical composition comprising an EON according to any one of claims 1 to 10, a vector according to claim 11, or a nanoparticle delivery vehicle formulation according to claim 12 or 13, and a pharmaceutically acceptable carrier.

15. An EON according to claims 1 to 10, a vector according to claim 11, a nanoparticle delivery vehicle formulation according to claim 12 or 13, or a pharmaceutical composition according to claim 14, for use in the treatment of cardiovascular disease (CVD), EON, vector, nanoparticle delivery vehicle formulation, or pharmaceutical composition.

16. Use of an EON according to claims 1 to 10, a vector according to claim 11, a nanoparticle delivery vehicle formulation according to claim 12 or 13, or a pharmaceutical composition according to claim 14, In the manufacture of a medicament for the treatment of CVD, use.

17. 1. An in vitro, ex vivo, or in vivo method of editing a B4GALT1 transcript molecule, comprising: The method comprises the step of contacting the B4GALT1 transcript molecule or a part thereof with an AON according to any one of claims 1 to 10, thereby editing the B4GALT1 transcript molecule. method.

18. 1. A method of treating, delaying, or ameliorating CVD in a patient in need thereof, comprising: The method comprises contacting B4GALT1 transcript molecules in cells of a subject with an EON according to any one of claims 1 to 10, thereby treating the patient. method.

19. 1. A method for deaminating a target adenosine in a B4GALT1 transcript molecule in a cell, comprising: The method includes the following steps: (i) providing the cell with an EON according to any one of claims 1 to 10; (ii) allowing the EON to be taken up by the cells; (iii) annealing the EON to the B4GALT1 transcript molecule; (iv) allowing an endogenous ADAR enzyme naturally present in the cell to deaminate the target adenosine in the target RNA molecule to inosine; and (v) identifying the presence of said inosine within said target RNA molecule. may include method.

20. 20. The method of claim 19, the cells are human cells, preferably hepatocytes, more preferably hepatocytes; the target adenosine is present at position c.1055A in the B4GALT1 transcript; and The deamination results in a change from asparagine (N; Asn) to serine (S; Ser) at position 352 in the human wild-type B4GALT1 amino acid sequence. method.

21. 21. The method of claim 19 or 20, wherein step (v) is: a) sequencing a B4GALT1 pre-mRNA or mRNA molecule, or a cDNA derivative thereof; b) assessing the presence of the 352Ser B4GALT1 protein variant; or c) using a functional readout, preferably assessing the rate of depletion of UDP-Gal or assessing the glycosylation level of transferrin in serum; Including, method.

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