Antisense oligonucleotides for the treatment of hereditary HFE hemochromatosis

Abstract

The present disclosure relates to the field of diseases caused by iron overload, such as homeostatic iron regulatory protein (HFE) hemochromatosis. The present disclosure provides oligonucleotides for RNA editing technology that target and deaminate the c.845G>A nucleotide in the transcript of the p.Cys282Tyr (C282Y) mutant human HFE gene, reducing iron overload, particularly in the liver.

Description

[Technical Field]

[0001] This invention relates to the field of medicine, which relates to diseases caused by iron overload, such as homeostatic iron regulatory protein (HFE)-associated hemochromatosis. The invention relates to the use of nucleotide editing technology in targeting homeostatic iron regulatory (HFE) genes and transcripts to result in amino acid changes that restore the normal function of the HFE protein in regulating iron homeostasis in the body. [Background technology]

[0002] Iron overload disorders represent an important class of human diseases. Among the major iron overload conditions, notably the most commonly and well-studied is HFE-associated hemochromatosis (HH). The most prevalent HH-causing mutation in humans is the C282Y substitution in HFE, resulting in disruption of iron homeostasis (Milman NT et al. 2019. Gastroenterology Res. 12(5):221-232; Anderson GJ & Bardou-Jacquet E. 2021. Ann Transl Med. 9(80):731; Barton JC & Edwards CQ. 2018. GeneReviews. Seattle (WA) University of Washington. 1993, updated December 6, 2018; Brissot P et al. 2018. Nat Rev Dis Primers. 4:18016; Cancado RD et al. 2022. Hematol Transfus Cell Ther. 44(1):95-99; Ye Q et al. 2016. PloS One. 11(9):e0163423). The disease is characterized by decreased expression of the iron-regulating hormone hepcidin, leading to increased dietary iron absorption and iron deposition in multiple tissues, including the liver, pancreas, joints, heart, and pituitary gland. The phenotype of HH is highly variable, with some individuals showing little or no evidence of elevated body iron, while others exhibit severe iron accumulation, tissue damage, and clinical sequelae. Genetically most susceptible individuals exhibit at least some evidence of iron accumulation (elevated transferrin saturation and serum ferritin). Because it is highly prevalent in the Caucasian population (1:200 to 1:500), only a minority of affected individuals exhibit clinical symptoms, yet it remains an important clinical entity. Early symptoms include abdominal pain, weakness, lethargy, weight loss, joint pain, erectile dysfunction in men, decreased libido due to hypogonadism in women, muscle loss, osteoporosis, diabetes, and an increased risk of cirrhosis when serum ferritin levels are above 1000 ng / mL.Other findings may include progressively increasing skin pigmentation, congestive heart failure and / or arrhythmias, arthritis, and hypogonadism. Individuals affected with HH have inadequately high absorption of iron from normal diets by the mucosa of the small intestine, leading to damage to target organs and excessive parenchymal storage of iron that can cause organ failure.

[0003] Men are much more likely to develop serious disease than women, who lose iron primarily through menstrual blood loss. Other forms of blood loss, immune system effects, the amount of bioavailable iron in the diet, and Lifestyle factors, such as heavy alcohol consumption, may also contribute to iron accumulation and disease development. Cirrhosis is more common among C282Y homozygotes who consume more than 60 g of alcohol per day. Iron overload-related symptoms are common in men between the ages of 40 and 60 and in women after menopause. Although HH occasionally manifests at younger ages, liver fibrosis or cirrhosis is rare before the age of 40. The onset of cirrhosis is generally thought to determine whether an individual will have a normal life expectancy or a reduced life expectancy despite iron deficiency therapy, primarily due to the development of hepatocellular carcinoma. Treating patients with cirrhosis to achieve iron deficiency does not eliminate the 10%–30% risk of primary liver cancer. In general, death in individuals with clinical HH is often caused by liver failure, primary liver cancer, extrahepatic cancer, congestive heart failure, or arrhythmia. Early screening studies showed that 38% to 50% of C282Y homozygotes developed iron overload, and 10% to 33% eventually developed hemochromatosis-related symptoms or organ damage. Some C282Y heterozygotes had elevated serum transferrin saturation and serum ferritin concentrations. However, they typically do not develop iron overload complications, which may arise due to environmental influences, lifestyle, or other mutations (such as H63D in HFE or in other iron homeostasis genes). Individuals homozygous for the C282Y mutation may be asymptomatic for 10 years, after which men may develop symptoms at approximately age 40 and women at approximately age 50. Patients with the C282Y mutation had a worse quality of life compared with patients with other genotypes, as measured by the Short Health Questionnaire (SF-36) (Fonseca et al. 2018. BMC Med Genet. 19(1):3).

[0004] Phlebotomy (phlebotomy) is the standard treatment for patients with hemochromatosis. It is highly effective, safe, and inexpensive in preventing hemochromatosis-related damage. Early diagnosis and initiation of phlebotomy are critical to preventing tissue and cellular damage caused by reactive oxygen species derived from iron overload. Nevertheless, phlebotomy is not always sufficient, and elderly patients often cannot tolerate the regimen. In rare cases of severe iron overload and / or poor venous availability where phlebotomy is ineffective, iron chelators are an adjuvant treatment or alternative, but iron chelation therapy is not indicated for classic hemochromatosis. Erythrocytapheresis has been used to treat patients with hemochromatosis but is more expensive and less applicable than phlebotomy. Two studies have demonstrated the importance of early intervention and adequate hemochromatosis treatment to prevent morbidity caused by hemochromatosis associated with C282Y homozygosity for HFE. Ong et al. (Lancet Haematol. 2017; 4(12):e607-614) conducted a randomized controlled trial in patients with the C282Y homozygous genotype and moderate serum ferritin levels (300-1000 μg / L). The cohort was divided into two groups: iron reduction by erythrocyte apheresis (treatment) or sham treatment by plasma apheresis (control). They found improvements in the Modified Fatigue Impact Scale (MFIS) scores in the treatment group compared with the control group. In a large cohort study in the UK Biobank including 2,890 patients with the C282Y homozygous genotype, Pilling et al. (BMJ. 2019;364:k5222) concluded that hemochromatosis is significantly more prevalent in both men and women and that the clinical diagnosis is associated with concomitant morbidity (liver disease, rheumatoid arthritis, osteoarthritis, and diabetes) (Cancado et al. 2022, supra).

[0005] The present disclosure aims to provide one or more alternative and / or improved compounds or compositions for use in the treatment of hereditary HH. Summary of the Invention

[0006] Disclosed herein is an RNA editing oligonucleotide (EON) that can form a double-stranded (ds) complex with a region of endogenous human HFE transcription molecule in cells, where the region of the HFE transcription molecule comprises a target adenosine, and the ds complex can recruit endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the HFE transcription molecule.Preferably, the HFE transcription molecule is a pre-mRNA or mRNA molecule.Preferably, the cell is a human liver cell, more preferably a hepatocyte.In a preferred embodiment, the target adenosine is the c.845G>A mutation in HFE gene. When the EON is in its naked form, it is preferred that at least one nucleotide contains one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety, or one or more additional non-naturally occurring chemical modifications, provided that the orphan nucleotide, the nucleotide in the EON directly opposite the target adenosine, is not a cytidine containing a 2'-OMe ribose substitution.

[0007] Further disclosed herein is a vector, preferably a viral vector, more preferably an adeno-associated viral (AAV) vector, comprising a nucleic acid molecule encoding an EON capable of forming a ds complex with a region of an endogenous human HFE transcription molecule in a cell, wherein the region of the HFE transcription molecule includes a target adenosine, and the ds complex is capable of recruiting an endogenous ADAR enzyme to deaminate the target adenosine to inosine.

[0008] Further disclosed herein is a pharmaceutical composition comprising an EON as disclosed, or a vector as disclosed, and a pharmaceutically acceptable carrier.

[0009] Further disclosed herein is an EON capable of forming a ds complex with a region of an endogenous human HFE transcription molecule within a cell, wherein the region of the HFE transcription molecule includes a target adenosine, and wherein the ds complex is capable of recruiting an endogenous ADAR enzyme to deaminate the target adenosine to inosine, for use in treating HFE chromatosis.

[0010] Disclosed are methods for editing an HFE polynucleotide, the method comprising contacting an HFE polynucleotide with an EON capable of effecting adenosine deaminase (ADAR)-mediated adenosine-to-inosine conversion related to iron homeostasis, thereby editing the HFE polynucleotide. Disclosed are methods for treating HFE hemochromatosis in a patient in need thereof, the method comprising contacting an HFE polynucleotide in a cell of the subject with an EON capable of effecting ADAR-mediated adenosine-to-inosine conversion related to iron homeostasis, thereby treating the patient. [Brief explanation of the drawings]

[0011] One or more embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which: [Figure 1-1]Figure 1 shows the human HFE target RNA sequence (5' to 3', SEQ ID NO: 52) at the top, with the target adenosine in bold and the tyrosine codon at position 282 of the human HFE protein underlined. Below the target sequence are given the sequences (also 5' to 3') of the first 51 EONs (SEQ ID NOs: 1 to 51, respectively, from top to bottom) designed to edit the target adenosines. The chemical modifications in the EONs are as follows: m5Ue is a 2'-MOE-modified 5-methyl-uridine; Ge is a 2'-MOE-modified guanosine; m5Ce is a 2'-MOE-modified 5-methyl-cytidine; Gm, Am, Um, and Cm are 2'-OMe-modified guanosine, adenosine, uridine, and cytidine, respectively; Af, Uf, Gf, and Cf are 2'-F-modified adenosine, uridine, guanosine, and cytosine, respectively; Zd is a 2'-F-modified adenosine, uridine, guanosine, and cytosine, respectively. Cytidine analogs, also called nucleosides with Benner's base (further outlined herein), have a deoxy moiety (=DNA) at the ribose position; C2f is a 2',2'-difluoro-modified cytidine; Ad and Cd are deoxyadenosine and deoxycytidine, respectively; an asterisk "*" indicates a phosphorothioate (PS) bond; "!" indicates a PNdmi bond; and "^" indicates a methylphosphonate (MP) bond. All other bonds are phosphodiester bonds. [Figure 1-2] Same as above. [Figure 2] Figure 2 shows the editing efficiency over time in an in vitro biochemical editing assay using EON RM4700 to RM4726 (as indicated), divided into three panels (A), (B), and (C) for visualization. [Figure 3] Figure 3 shows the percentage editing determined after 72 hours of exposure to 5 μM EON RM4700, RM4723, and RM4725 (as indicated) in EBV-immortalized B lymphocytes from a donor (GM14715) homozygous for the C282Y (c.845G>A) mutation in the HFE gene, separated by 1 μM saponin AG1856. Negative controls included the use of scrambled oligonucleotides, untreated (NT) samples, samples without reverse transcriptase (-RT), and a water control. [Figure 4] Figure 4 shows the percentage editing determined after 72 hours of exposure to 5 μM EON RM4700 through RM4723, and RM4725 (as indicated) in the presence of 1 μM saponin AG1856, split in EBV-immortalized B lymphocytes derived from a donor (GM14631) homozygous for the C282Y (c.845G>A) mutation in the HFE gene. Negative controls included the use of scrambled oligonucleotides, untreated (NT) samples, samples treated with AG1856 saponin only, samples without reverse transcriptase (-RT), and a water control. [Figure 5-1] Figure 5 shows the human HFE target RNA sequence (5' to 3', SEQ ID NO: 52) at the top, with the target adenosine in bold and the tyrosine codon underlined. Below the target sequence, the sequences (also 5' to 3') of 49 additional EONs (SEQ ID NOs: 66 to 164) designed to edit the target adenosines, shown at the top of Figure 1, are given. The chemical modifications in the EONs are as given in Figure 1, with Gd being deoxyguanosine, Ae being 2'-MOE-modified adenosine, and L004 being a 3'-linked triantennary GalNAc moiety as described in WO2022 / 271806. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 6] Figure 6 shows the percentage of editing (left y-coordinate, black bars) determined in EBV-immortalized B lymphocytes derived from a donor (GM14715) homozygous for the C282Y (c.845G>A) mutation in the HFE gene after 72 hours of exposure to the EONs indicated below the graph at 5 μM in the presence of 2 μM saponin AG1856. Negative controls were the untreated (NT) sample and saponin alone. RM4717 is the positive control EON for Figures 1 and 3 (HFE-34). Hepcidin expression levels in these EON-treated cells were determined in the same samples and are shown as the y-coordinate, right, open bars. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure describes an alternative approach that targets the p.Cys282Tyr (C282Y) mutation in HFE to generate wild-type HFE protein, potentially restoring proper iron processing and thereby preventing, reversing, or treating hereditary HH. 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) HFE transcripts (pre-mRNA and / or mRNA) in vivo, preferably using endogenous deaminating enzymes, to generate HFE proteins whose function in hepcidin regulation is restored. While the C282Y mutation described above is by far the most common mutation found in the HFE gene that can cause iron overload in homozygous genotypes, RNA editing technology as disclosed herein can also be applied to other target adenosines in the HFE gene that can be targeted to restore HFE gene function or even to produce a gain-of-function effect.

[0013] 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 repertoire of RNA-encoding genomes by several orders of magnitude. RNA-editing enzymes have been described in eukaryotic species throughout the animal and plant kingdoms, and these processes play a critical role in managing cellular homeostasis in metazoans ranging from the simplest life forms (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 are carried out by enzymes called adenosine deamidase acting on RNA (ADAR) and APOBEC / AID (cytidine deamidase acting on RNA), respectively.

[0014] ADARs are multidomain proteins that contain 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. The catalytic domain's key function is to convert A to I at a nearby, predefined location in the target RNA by deamination of the nucleobase, but it also recognizes and binds to portions of the dsRNA helix. The cellular translation machinery reads inosine as guanosine, meaning that if the edited adenosine is present in the coding region of an mRNA or pre-mRNA, it can be recorded in the protein sequence. The A to I conversion can occur in the 5' non-coding sequence of the target mRNA, creating a new translation initiation site upstream of the original initiation site and thereby generating an N-terminally extended protein, or it can occur in the 3' UTR or other non-coding portion of the transcript, affecting RNA processing and / or stability. Furthermore, the A to I conversion may occur at splice elements in introns or exons in pre-mRNA, resulting in altered splicing patterns. As a result, exons may be included or skipped. Enzymes that catalyze adenosine deamination belong to the ADAR enzyme family, including human deamination enzymes hADAR1, hADAR2, and hADAR3. However, deamination activity has not been demonstrated for hADAR3.

[0015] The use of oligonucleotides to edit target RNAs, applied to adenosine deaminase, has been described (e.g., Woolf et al. 1995. PNAS 92:8298-8302; Montiel-Gonzalez et al. 2013. PNAS 110(45):18285-18290; Vogel et al. 2014. Angewandte Chemie Int Ed 53:267-271). A disadvantage of the method described in Montiel-Gonzalez et al. (2013) is that it requires a fusion protein consisting of the box B recognition domain of the bacteriophage lambda N-protein genetically fused to the adenosine deamination domain of a truncated native ADAR protein. This requires target cells to be transduced with the fusion protein or transfected with a nucleic acid construct encoding the engineered adenosine deaminase fusion protein for expression, which is a major hurdle. The system described in Vogel et al. (2014) suffers from a similar drawback in that it is unclear how to apply the system without first genetically modifying ADAR, then transfecting or transforming cells with target RNA and providing the cells with this genetically modified protein. US9,650,627 describes a similar system. The oligonucleotides described in Woolf et al. (1995), which were 100% complementary to the target RNA sequence, suffer from a significant lack of specificity: almost all adenosines in the target RNA strand complementary to the antisense oligonucleotide are edited.

[0016] ADARs are known to act on any dsRNA. Through a process sometimes referred to as "promiscuous editing," the enzymes edit multiple A's in dsRNA. Therefore, there is a need for methods and means to circumvent this promiscuous editing and target only specific adenosines in target RNA molecules for therapeutic applications. Vogel et al. (2014) demonstrated that such off-target editing can be suppressed by using 2'-O-methyl (2'-OMe)-modified nucleosides in oligonucleotides opposite the adenosines that should not be edited, while using unmodified nucleosides opposite the specifically targeted adenosines on the target RNA. However, because this study was performed without using recombinant ADAR enzymes covalently linked to AONs, the effect of specific editing at the target nucleotide was not demonstrated. Several publications have now demonstrated that it is feasible to recruit endogenous ADARs (thus, without the need for exogenous and / or recombinant materials) while maintaining the specificity of targeting a single adenosine within a target RNA molecule and deaminating it to inosine. WO2016 / 097212 discloses antisense oligonucleotides (AONs) for targeted editing of RNA, characterized by the presence of a sequence complementary to the target RNA sequence (referred to herein as the "targeting portion") and a stem-loop / hairpin structure (referred to herein as the "recruiting portion"), preferably non-complementary to the target RNA. Such oligonucleotides are called "self-looping AONs." The recruiting portion acts on the dsRNA formed by hybridization of the targeting portion with the target sequence to recruit natural ADAR enzymes present in cells. Due to the recruiting portion, the presence of a conjugated entity or a modified recombinant ADAR enzyme is not required.WO2016 / 097212 describes the recruitment moiety as a stem-loop structure that mimics either a Z-DNA structure known to be recognized by the dsRNA-binding domain of a natural substrate (e.g., a GluB receptor) or an ADAR enzyme, or a Z-DNA-binding domain. 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 described is an intramolecular stem-loop structure formed within the AON itself, and is thought to attract (endogenous) ADAR. Similar systems containing stem-loop structures for RNA editing have been described in WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995.

[0017] WO2017 / 220751 and WO2018 / 041973 describe next-generation AONs that do not contain such stem-loop structures but are (almost perfectly) complementary to the target region. In one embodiment, one or more mismatched nucleotides, wobble, or bulge exist between the oligonucleotide and the target sequence. While the only mismatch may be at the nucleoside site opposite the target adenosine, in other embodiments, AONs (or RNA-editing oligonucleotides, abbreviated as "EONs") have been described with multiple bulges and / or wobble when binding to the target sequence region. When the sequence of the EON is carefully selected to attract / recruit ADARs, in vitro, ex vivo, and in vivo RNA editing appears feasible using EONs lacking stem-loop structures and endogenous ADAR enzymes. An "orphan nucleoside," defined as a nucleoside in an EON located directly opposite the target adenosine in a target RNA molecule, does not have a 2'-OMe modification. The orphan nucleoside is a deoxyribonucleoside (DNA), and the remainder of the EON may still have nucleotides immediately surrounding the orphan nucleoside that contain 2'-O-alkyl modifications (such as 2'-OMe) at the sugar moiety, or chemical modifications that further improve RNA editing efficiency and / or increase resistance to nucleases (such as DNA compared to RNA). Even this effect can be further improved using sense oligonucleotides (SONs) that "protect" the EON from degradation (as described in WO2018 / 134301).The use of chemical modifications and specific structures in oligonucleotides that can be used in ADAR-mediated editing of specific adenosines in target RNA is described in WO2019 / 111957, WO2019 / 158475, WO2020 / 165077, WO2020 / 201406, WO2020 / 211780, WO2021 / 008447, WO2021 / 020550, WO2021 / 060527, WO2 It has been the subject of numerous publications in this field, including WO2021 / 117729, WO2021 / 136408, WO2021 / 182474, WO2021 / 216853, WO2021 / 242778, WO2021 / 242870, WO2021 / 242889, WO2022 / 007803, WO2022 / 018207, WO2022 / 026928, and WO2022 / 124345.While the use of specific sugar moieties has been disclosed, for example, in WO2020 / 154342, WO2020 / 154343, WO2020 / 154344, WO2022 / 103839, and WO2022 / 103852, there has been little interest in stereo-defined oligonucleotides (generally for oligonucleotides that can be used, for example, for gapmers relating to diverse target sequences, for exon skipping in siRNA, or specifically for RNA editing oligonucleotides). The use of linker moieties is described in WO2011 / 005761, WO2014 / 010250, WO2014 / 012081, WO2015 / 107425, WO2017 / 015575 (HTT), WO2017 / 062862, WO2017 / 160741, WO2017 / 192664, WO2017 / 192679 (DMD), WO2017 / 198775, WO2017 / 210647, WO2018 / 067973, WO2018 / 098264, WO2018 / 223056(PNPLA3), WO2018 / 223073(APOC3), WO2018 / 223081(PNPLA3), WO2018 / 237194, WO2019 / 032607(C9orf72), WO2019 / 0559 51, WO2019 / 075357(SMA / ALS), WO2019 / 200185(DM1), WO2019 / 217784(DM1), WO2019 / 219581, WO2020 / 118246(DM1), WO2020 / 160 336 (HTT), WO2020 / 191252, 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. Following these disclosures, a vast number of publications have been concerned with targeting specific RNA target molecules, or specific adenosines within such RNA target molecules, to repair mutations caused by premature stop codons or other disease-causing mutations.Examples of such disclosures targeting adenosines within specific target RNA 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 / 231679 ( GJB2); WO2019 / 071274 and WO2021 / 231680 (MECP2); WO2021 / 231685 and WO2021 / 231692 (OTOF, autosomal recessive non-syndromic hearing loss); WO2021 / 231691 (XLRS); WO2021 / 231698 (argininosuccinate lyase deficiency); WO2021 / 130313 and WO2021 / 231830 (ABCA4); and WO2021 / 243023 (SERPINA1).

[0018] Disclosed herein are EONs that can cause (or "trigger") RNA editing of a target adenosine in a human HFE transcript (pre-mRNA and / or mRNA), thereby restoring the resulting HFE protein to its wild-type function, for example, in hepcidin regulation. In a preferred embodiment, the EON causes deamination of the adenosine present at position 845 of the mutant mRNA, thereby producing inosine. In other words, the UAC codon encoding tyrosine (mutant form) at amino acid position 282 is converted to a UIC codon, which is read as UGC by the translational machinery and encodes cysteine ​​(wild-type form). In another embodiment, the EONs herein cause deamination of another adenosine present in the HFE transcript, which may be any adenosine that, when deaminated to inosine, results in a gain-of-function HFE protein. Other mutations may be present in the HFE gene (and transcript) that can be targeted via RNA editing, thereby restoring normal HFE function. As disclosed herein, a preferred mutation to target is the c.845G>A mutation in the human HFE gene, which results in the p.Cys282Tyr HFE protein variant.

[0019] In a preferred embodiment, the EON herein is a single-stranded (ss) oligonucleotide comprising an "orphan nucleotide" located opposite the target adenosine, the orphan nucleotide being chemically modified as disclosed herein, and the remainder of the oligonucleotide being chemically modified to prevent nuclease degradation, also as disclosed herein. In another embodiment, the EON herein relates to any kind of oligonucleotide or heteroduplex oligonucleotide complex, which may or may not be bound to a hairpin structure (internal or terminal), may bind to ADAR or its catalytic domain, or the oligonucleotide is expressed via a vector such as AAV, or the oligonucleotide is in a circular form. It should be understood that any kind of oligonucleotide-based RNA editing is encompassed by the present invention, provided that it is related to the deamination of nucleotides in HFE transcripts, preferably the mutation causing C282Y, and causes the restoration of HFE function.

[0020] In preferred embodiments, the EONs herein are "naked" oligonucleotides that contain various chemical modifications in the ribose sugar, the base, and / or the internucleoside linkages of one or more nucleotides in the sequence, and that are capable of hybridizing to an HFE transcript or portion thereof containing a target adenosine and recruiting endogenous ADARs for deamination of the target adenosine.

[0021] In particular, when an EON includes a chemical modification, it may still be delivered via a delivery vehicle, as detailed herein. Suitable delivery vehicles include, for example, lipid nanoparticles (LNPs), which are nanosized lipid vehicles carrying the EON described herein and aid in delivery to target cells. When an LNP is applied, or any other similar type of carrier, the EON is still considered "naked" because it is not transcribed from an encoding polynucleotide (such as in the case of a plasmid or vector, in which the EON is not considered "naked" but is considered transcribed). Thus, even though a chemically modified AON is encapsulated in a carrier, preferably an LNP, it is still considered naked because it is prepared, such as in laboratory conditions, and then encapsulated in a carrier using methods known to those skilled in the art. The present disclosure also relates to delivery vehicles, preferably LNPs, comprising a chemically modified AON as disclosed herein, even more preferably those disclosed in any of SEQ ID NOS: 1-51 and 66-164. Those skilled in the art will understand that when a delivery moiety or attachment to the EON is used (such as a GalNAc moiety that targets hepatocytes in the liver, e.g., the L004 GalNAc moiety shown in Figure 5), and even when the GalNAc-EON is encapsulated in a delivery vehicle such as an LNP, the EON is still considered "naked."

[0022] Embodiment

[0023] The present disclosure provides an EON capable of forming a ds complex with a region of an endogenous human HFE transcription molecule in a cell, where the region of the HFE transcription molecule contains a target adenosine, and the ds complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the HFE transcription molecule. In a preferred embodiment, the HFE transcription molecule is a pre-mRNA or mRNA molecule. In one embodiment, the cell is a human liver cell, preferably a hepatocyte. In one embodiment, the target adenosine is the c.845G>A mutation in the HFE gene. In one embodiment, deamination of the target adenosine results in restoration of wild-type HFE protein, although this is not necessarily the case when the transcript contains a mutation other than c.845G>A. In one embodiment, the EON comprises or consists of the nucleotide sequence of any one of the EON sequences set forth in Figures 1 or 5, with alternative chemical modifications as detailed herein. In one embodiment, each EON comprises a chemical modification as set forth in Figures 1 or 5. In one embodiment, at least one nucleotide contains one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety, or one or more additional non-naturally occurring chemical modifications, except that the orphan nucleotide, which is the nucleotide opposite the target adenosine in the EON, is not a cytidine containing a 2'-OMe ribose substitution. In one embodiment, the orphan nucleotide is a cytidine analog, such as a deoxynucleotide containing a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleobase (also known as Benner's base). In one embodiment, the orphan nucleotide is a uridine analog, such as a deoxynucleotide containing an isouracil nucleobase. In one embodiment, the EON contains one or more mismatches, wobbles, or bulges, and there can be one mismatch when the target adenosine has an opposite cytidine in the EON. If the orphan nucleotide is a cytidine, it does not contain a 2'-OMe ribose substitution.In one embodiment, the one or more additional modifications in the linking moiety are each independently selected from PS, phosphonoacetate, phosphorodithioate, MP, sulfonyl phosphoramidate, or PNdmi internucleotide linkages. In one embodiment, the one or more additional modifications in the ribose moiety are -OH; -F; substituted or unsubstituted, linear or branched lower ribose (C1-C2) optionally interrupted by one or more heteroatoms. 10 ) mono- or di-substitutions at the 2', 3', and / or 5' positions of the ribose, each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl; -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.

[0024] In one embodiment, the present disclosure provides a vector, preferably a viral vector, more preferably an AAV vector, comprising a nucleic acid molecule encoding the EON herein. In an embodiment, the present disclosure provides a pharmaceutical composition comprising the EON herein or the vector herein and a pharmaceutically acceptable carrier. In one embodiment, the present disclosure provides the EON herein, the vector herein, the LNP formulation herein, or the pharmaceutical composition herein for use in treating an iron overload disorder, preferably HFE hemochromatosis. In one embodiment, the present disclosure provides the use of the EON herein, the vector herein, or the LNP formulation herein in the manufacture of a medicament for treating an iron overload-related disorder, preferably HFE hemochromatosis.

[0025] In one embodiment, the present disclosure provides a method for editing an HFE polynucleotide, the method comprising contacting an HFE polynucleotide with an EON capable of causing ADAR-mediated adenosine-to-inosine conversion, which is involved in iron homeostasis, thereby editing the HFE polynucleotide, preferably wherein the EON is as disclosed herein. In one embodiment, the present disclosure provides a method for treating HFE hemochromatosis in a patient in need thereof, the method comprising contacting an HFE polynucleotide in a cell of the subject with an EON capable of causing ADAR-mediated adenosine-to-inosine conversion, which is involved in iron homeostasis, thereby treating the patient, preferably wherein the EON is as disclosed herein, a vector encoding the EON herein, or an LNP formulation herein. In one embodiment, the present disclosure provides a method for treating HFE hemochromatosis, the method comprising administering to a patient in need thereof a therapeutically effective amount of the EON described herein, the vector described herein, or the pharmaceutical composition described herein.

[0026] In one embodiment, the present disclosure provides a method for deaminating a target adenosine in an HFE pre-mRNA or mRNA molecule in a cell, the method comprising the steps of: (i) providing a cell with an EON, LNP formulation, or vector as disclosed herein; (ii) allowing the cell to take up the EON, LNP, or vector, respectively; (iii) allowing the EON to anneal to an HFE pre-mRNA or mRNA molecule; (iv) allowing an endogenous ADAR enzyme to deaminate the target adenosine in the target RNA molecule to inosine; and, optionally, (v) confirming the presence of inosine in the target RNA molecule. In a preferred embodiment, the target adenosine is the c.845G>A mutation in a human HFE pre-mRNA or mRNA molecule. The step of confirming the presence of inosine at the target adenosine position preferably includes (a) determining the sequence of the HFE pre-mRNA or mRNA molecule; (b) assessing the presence of wild-type HFE protein; or (c) using a functional readout device to preferably assess serum or plasma ferritin concentration or serum transferrin saturation percentage. Such assessment can be performed in vitro on a sample removed from the subject being treated. For example, ferritin concentration can be measured before and after EON treatment to determine the level of EON activity (and, of course, RNA editing of the target transcript).

[0027] definition

[0028] The term "nucleoside" refers to a nucleobase linked to a (deoxy)ribosyl sugar, but without a phosphate group. A "nucleotide" is composed of a nucleoside and one or more phosphate groups. The term "nucleotide" thus refers to the respective nucleobase-(deoxy)ribosyl-phospholinker, as well as any chemical modification of the ribose moiety or phosphate group. Thus, the term includes nucleotides containing locked ribosyl moieties (including a 2'-4' bridge containing a methylene group or any other functional group), unlocked nucleic acids (UNA), threose nucleic acids (TNA), nucleotides containing linkers containing phosphodiesters, phosphonoacetates, phosphotriesters, PS, phosphoro(di)thioates, MP, methylthiophosphonates, phosphoramidate linkages, and the like. Sometimes the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, and inosine and hypoxanthine are used interchangeably to refer to the corresponding nucleobase on the one hand and to refer to the nucleoside or nucleotide on the other hand. Thymine (T) is 5-methyluracil (m 5 Also known as 5-methyluridine, thymine is a uridine derivative; thymine, 5-methyluridine, and uridine are interchangeable throughout this document. Unless the context clearly requires otherwise, the terms nucleobase, nucleoside, and nucleotide are sometimes used interchangeably, for example, 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 in the art.

[0029] Whenever oligonucleotides, oligos, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA) editing oligonucleotides, EONs, and RNA (antisense) oligonucleotides are used, both oligoribonucleotides and deoxyoligoribonucleotides are intended, unless the context dictates otherwise. Oligonucleotides may be completely devoid of RNA or DNA nucleotides (as found in nature) or may be composed entirely of modified nucleotides. Whenever "oligoribonucleotides" are used, they may contain bases A, G, C, U, or I. Whenever "deoxyoligoribonucleotides" are used, they may contain bases A, G, C, T, or I. However, oligonucleotides of the present invention may contain a mixture of ribonucleosides and deoxyribonucleosides. When deoxyribonucleotides are used, they have no modification at the 2' position of the sugar, and therefore the nucleotides are often abbreviated as dA, dC, dG, or T, where the "d" denotes the deoxy nature of the nucleoside. On the other hand, ribonucleosides that are normal RNA or modified at the 2' position are often abbreviated without the "d" and are often abbreviated with the respective modification as described herein.

[0030] Any reference to nucleotides in an oligonucleotide includes cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine. Any reference to adenine includes N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine, and 7-methyladenine. Any reference to uracil includes dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudouracil, 4-thiouracil, and 5-hydroxymethyluracil. Any reference to guanine includes 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. Whenever a nucleoside or nucleotide is referred to, other modifications are included, including ribofuranose derivatives such as 2'-deoxy, 2'-hydroxy, and 2'-O-substituted variants such as 2'-OMe, as well as 2'-4' bridged variants. Whenever an oligonucleotide is referred to, the linkage between two mononucleotides may be a phosphodiester bond, as well as modifications thereof, including phosphonoacetate, phosphotriester, PS, phosphoro(di)thioate, MP, phosphoramidate linker, phosphorylguanidine, thiophosphorylguanidine, sulfonophosphoamidate, and the like.

[0031] The term "comprising" encompasses "including" as well as "consisting of." For example, a composition "comprising X" may consist solely of X, or may include something additional, e.g., X+Y. The term "about" in relation to a numerical value x is arbitrary and means, for example, x±10%.

[0032] The word "substantially" does not exclude "completely." For example, a composition that is "substantially free of Y" may be completely free of Y. Where applicable, the word "substantially" may be omitted from the definition of the invention.

[0033] The term "complementary" as used herein refers to the fact that an EON hybridizes to a second nucleic acid strand under physiological conditions (e.g., when an oligonucleotide (=guide oligonucleotide) as a first nucleic acid strand forms a heteroduplex RNA-editing oligonucleotide complex, or 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 has perfect pairing with its opposite nucleotide in the opposite sequence. In other words, even if an EON is complementary to a target sequence, there may be mismatches, wobble, and / or bulges between the oligonucleotide and the target sequence, and the EON will still hybridize to the target sequence under physiological conditions, allowing cellular RNA-editing enzymes to edit the target adenosine. The term "substantially complementary," therefore, also means that there is sufficient nucleotide matching between the EON and the target sequence that the EON hybridizes to the target RNA under physiological conditions, despite the presence of mismatches, wobble, and / or bulges. As provided herein, if an EON is capable of hybridizing to its target under physiological conditions, the EON may be complementary but may contain one or more mismatches, wobble, and / or bulges with the target sequence.

[0034] The term "downstream," in reference to a nucleic acid sequence, means further along the sequence in the 3' direction, and 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.

[0035] References to "hybridization" typically refer to specific hybridization and exclude non-specific hybridization, which can occur under experimental conditions selected using techniques well known in the art to ensure that the most stable interaction between probe and target is where the probe and target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0036] The term "mismatch" is used herein to refer to opposite 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 historical sense, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the EON contains 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, it does not necessarily mean that the interaction is unstable, which means that the term "mismatch" may be somewhat outdated based on the present invention, where Hoogsteen base pairing may be considered a mismatch based on the origin of the nucleotides, but is still relatively stable. For example, an isolated G:G pairing in a double-stranded RNA may be quite stable, but is still defined as a mismatch.

[0037] The term "splice variant" relates 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 prevented, causing aberrant splicing and subsequent translation out of frame, resulting in premature termination of the encoded protein. Often, such short-end proteins are rapidly degraded and do not have any functional activity.

[0038] The EON herein (and the complementary nucleic acid strand when two oligonucleotides form an HEON) may 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 orphan nucleotides in the EON are preferably cytidine or analogs thereof (e.g., nucleotides with Benner's bases), or uridine or analogs thereof (e.g., isouridine), and / or contain, in one embodiment, a diF modification at the 2'-sugar position, or in another embodiment, a deoxyribose (2'-H, DNA), and in yet a further embodiment, at least one, and in another embodiment, both, adjacent nucleotides adjacent to the orphan nucleotide do not contain a 2'-OMe modification. Complete modification of all nucleotides of an oligonucleotide with natural bases results in an oligonucleotide that is non-functional with respect to RNA editing (as is known in the art), presumably because it interferes with ADAR activity at the target position. In general, adenosines in target RNA can be protected from editing by providing a 2'-OMe group at the opposite nucleotide (at least when no other chemical substitutions or modifications are present within the nucleotide) or by providing guanine or adenine as the opposite base, as these two nucleobases can also reduce editing of the opposite adenosine.

[0039] A variety of chemistries and modifications are known in the art of oligonucleotides that can be readily used in accordance with the present disclosure. Regular internucleoside linkages between nucleotides may be altered by mono- or dithiolation of phosphodiester bonds to produce PS esters or phosphorodithioate esters, respectively. Other internucleoside linkage modifications are also possible, including amidation and peptide linkers.

[0040] In certain embodiments, the EONs herein comprise 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.

[0041] It is known in the art that RNA editing entities (such as human ADAR enzymes) edit dsRNA structures with specificity that varies depending on several factors. One important factor is the degree of complementarity between the two strands that form the dsRNA sequence. Perfect complementarity between the two strands often results in the catalytic domain of human ADAR reacting to any adenosine it encounters and indiscriminately deaminating the adenosine. The specificity of hADAR1 and 2 can be increased by introducing chemical modifications and / or ensuring several mismatches in the dsRNA. This likely aids in the positioning of the dsRNA-binding domain in ways that are yet to be elucidated. Furthermore, the deamination reaction itself can be facilitated by providing an oligonucleotide containing a mismatch opposite the adenosine to be edited. Following the explanations herein, those skilled in the art will be able to design the complementary portion of the oligonucleotide as needed.

[0042] The intracellular RNA editing protein most interested in being used with EON herein is human ADAR2. Those skilled in the art will understand that the degree to which intracellular editing entities are directed to other target sites can be controlled by changing the affinity of the first nucleic acid strand for the recognition domain of the editing molecule. The precise modification can be determined through some trial and error and / or through computational methods based on the structural interaction between the recognition domain of the EON and the editing molecule. Additionally, or alternatively, the degree of recruitment and redirection of intracellular editing entities can be controlled by the dose and dosage regimen of the EON. This is often determined by experimenters (in vitro) or by clinicians in phase I and / or phase II clinical trials.

[0043] The present disclosure also relates to modifying target RNA sequences in eukaryotic, preferably metazoan, more preferably mammalian, and most preferably human cells. The present disclosure is particularly suited to modifying RNA sequences in cells and tissues where HFE is expressed and where the protein functions. The pathogenic mechanisms by which mutant HFE gene products affect iron homeostasis are not fully understood. Hepcidin, produced in the liver, is the "master regulator" of iron homeostasis in the body, and its primary role is to inactivate ferroportin. Ferroportin plays a key role in regulating iron transport (efflux) across the plasma membrane in enterocytes, hepatocytes, and macrophages. Normal HFE and transferrin receptor 2 complexes on the hepatocyte plasma membrane stimulate hepcidin production / activation, subsequently inhibiting intestinal iron uptake. In hemochromatosis, defective HFE complexes cause reduced hepcidin production / activation, resulting in increased intestinal iron uptake, largely independent of the body's iron status. Thus, HH is characterized by low plasma levels of hepcidin, termed "hepcidin deficiency." Intracellular iron accumulation triggers oxidative stress, DNA damage, cell necrosis, and, over time, fibrosis. This progression is typically seen in the liver, where early fibrosis may eventually lead to cirrhosis. Because HFE is primarily produced and plays an important role in liver cells, preferred target cells for the EONs herein are liver cells, more preferably hepatocytes. Target cells can be located in vitro, ex vivo, or in vivo. One advantage of the EONs disclosed herein is that they can be used with cells in situ in vivo, as well as with cells in culture. In some embodiments, cells are treated ex vivo and then introduced into the body (e.g., reintroduced into the organism from which they originally originated). The EONs herein can also be used to edit target RNA sequences in cells derived from transplants or in so-called organoids, e.g., liver tissue organoids. Organoids can also be envisioned as 3-dimensional in vitro derived tissues, but are derived using specific conditions to generate individual, isolated tissues.They are useful in therapeutic settings because they can be derived from the patient's cells in vitro, allowing the organoids to be reintroduced into the patient as autologous material that is less likely to be rejected than regular transplants.

[0044] Without wishing to be bound by theory, it is believed that hADAR2-mediated RNA editing occurs on primary transcripts in the nucleus during transcription or splicing, or in the cytoplasm, where, for example, mature mRNA, miRNA, or ncRNA may be edited.

[0045] It should be clear from the present disclosure that targeted editing can be applied to any adenosine within an HFE transcript, provided that adenosine deamination results in an increase or restoration of HFE protein function. However, as outlined herein, it is preferred to target the adenosine at position 845 in the mutant HFE transcript (=c.845G>A mutation) and result in a change from a UAC codon (encoding tyrosine) to a UIC (or UGC) codon (encoding cysteine). Generally speaking, RNA editing may be used to create RNA sequences with different properties. Such properties may be coding (producing proteins with different sequences or lengths, resulting in altered protein properties or function) or binding (causing inhibition or overexpression of the RNA itself or a target or binding partner; recoding miRNAs or their cognate sequences on target RNAs may alter entire expression systems). Protein function or localization may be optionally altered by functional domains or recognition motifs, including, but not limited to, signal sequences, targeting or localization signals, recognition sites for protein cleavage or co- or post-translational modification, catalytic sites for enzymes, binding sites for binding partners, degradation or activation signals, etc. These and other forms of RNA and protein "engineering" are encompassed by the present invention, whether to prevent, delay, or treat disease, or as a diagnostic, prophylactic, therapeutic, research tool, or any other purpose in medicine or biotechnology. Thus, any RNA editing of a target adenosine in an HFE transcript that results in the improvement or restoration of HFE protein function is encompassed by the present invention.

[0046] The present disclosure discloses a completely new field of using gene editing technology to treat iron overload or HH.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, and preferably RNA editing technology, such as ADAR-mediated editing technology, as further detailedly summarized herein.

[0047] The amount, dose, and dosage regimen of the EON to be administered may vary depending on the cell type, the disease to be treated, the target population, the administration method (e.g., systemic vs. local), the severity of the disease, and the acceptable level of side effects, but can and should be evaluated through trial and error in in vitro studies, preclinical, and clinical trials. Testing is particularly simple when the modified sequence results in an easily detectable phenotypic change or a change in the level or activity of a specific biomarker. Higher doses of EON may compete for binding to ADAR in cells, thereby depleting the amount of entities free to perform RNA editing, but routine administration testing will reveal any such effects for a particular EON and a particular target.

[0048] One suitable testing technique involves delivering EONs to a cell line or test organism, followed by taking biopsy samples at various subsequent time points. The sequence of the target RNA can be assessed in the biopsy samples, and the proportion of cells with the modification can be easily tracked. Once this testing is performed, the findings can be retained, and future deliveries can be performed without the need to take biopsies. The methods of the invention may therefore include a step of confirming the presence of the desired change in the cellular target RNA sequence, thereby confirming that the target RNA sequence has been modified. As noted above, this step typically involves sequencing the relevant portion of the target RNA or its cDNA copy (or, if the target RNA is pre-mRNA, a cDNA copy of its spliced ​​product), so that the sequence change can be easily confirmed. Alternatively, changes may be assessed by measuring or assessing, for example, serum or plasma ferritin concentration or serum transferrin saturation percentage before and / or after treatment, or any other potential markers of protein function, preferably in vitro, with these measurements performed on samples from the treated subject.

[0049] After RNA editing occurs in a cell, the modified RNA may fade over time due to, for example, cell division, the limited half-life of the edited RNA, etc. Therefore, in an actual treatment period, the methods of the present invention may involve repeatedly delivering EONs until the target RNA is sufficiently modified that a definite benefit is imparted to the patient and / or the benefit is maintained over time.

[0050] Because the EONs herein are particularly suitable for therapeutic use, the present disclosure also relates to pharmaceutical compositions comprising the EONs herein, or vectors or plasmids encoding the EONs herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier can simply be saline, which can be particularly effective in isotonic or hypotonic solutions for pulmonary delivery. The present disclosure also provides delivery devices (e.g., syringes, inhalers, nebulizers) containing the pharmaceutical compositions of the present invention.

[0051] The present disclosure also provides the EONs herein for use in methods for repairing mutations in the RNA sequence of a target HFE in a mammal, preferably a human liver cell, as described herein. Similarly, the present disclosure provides use of the EONs herein in the manufacture of a medicament for causing an alteration in the RNA sequence of a target HFE in a mammal, preferably a human liver cell, as described herein, thereby treating, preventing, or ameliorating a disease associated with iron overload, such as HFE hemochromatosis.

[0052] The present disclosure also provides a method for deaminating at least one specific target adenosine present in an RNA sequence of a target HFE in a cell, the method comprising the steps of: providing an EON herein to a cell; allowing the cell to take up the EON; allowing the EON to anneal to the target RNA molecule; allowing a mammalian ADAR enzyme containing a native dsRNA binding domain as found in a wild-type enzyme to deaminate the target adenosine in the target RNA molecule (preferably, an adenosine at position 845 in a mutant HFE transcript) to inosine; and, optionally, confirming the presence of inosine in the RNA sequence.

[0053] The present disclosure also provides a method for deaminating at least one specific target adenosine present in an RNA sequence of a target HFE in a cell, the method comprising the steps of: providing a cell with a vector or plasmid encoding the EON herein; allowing the cell to take up the vector or plasmid; allowing the EON to anneal to the target RNA molecule; allowing a mammalian ADAR enzyme containing a native dsRNA-binding domain as found in the wild-type enzyme to deaminate the target adenosine in the target RNA molecule (preferably, an adenosine at position 845 in the mutant HFE transcript) to inosine; and, optionally, confirming the presence of inosine in the RNA sequence.

[0054] Because the target RNA after deamination should encode a functional protein, in a preferred embodiment, depending on the final deamination effect of the A to I conversion, the confirmation step may include the following steps: sequencing the target RNA; assessing the presence or absence of a functional protein; assessing whether deamination alters pre-mRNA splicing; or using a functional readout device. An example is the assessment of ferritin or hepcidin levels after RNA editing. Ferritin levels are generally considered the best biomarker for body iron content. Serum transferrin saturation percentage is an indicator of blood iron content and organ iron supply. High serum transferrin saturation may be a first indicator of HFE hemochromatosis and may be present, even if serum ferritin levels are still within the normal range. Confirmation of deamination to inosine may therefore be a functional readout using a suitable biomarker. The functional assessment of HFE hemochromatosis described herein will generally follow methods known to those skilled in the art. A very suitable method to confirm the presence of inosine after deamination of the target adenosine is, of course, dPCR, or even sequencing, using methods well known to those skilled in the art. However, those skilled in the art of liver disease may also apply tests to monitor specific biomarkers related to iron overload, as described above.

[0055] The EONs herein are suitably administered in aqueous solutions, e.g., saline, or suspensions, optionally containing 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. Doses may suitably range 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. Administration may be by intranasal or oral inhalation (e.g., via nebulization), intravenous, subcutaneous, intradermal, intramuscular, intratracheal, intraperitoneal, rectal, intrathecal, intracisternal, parenteral injection or infusion, and the like. Administration may be in the form of a solid, powder, pill, gel, solution, delayed release formulation, or any other form compatible with pharmaceutical use in humans.

[0056] In one embodiment, the method herein comprises administering to a subject an EON or a pharmaceutical composition herein, allowing the formation of a ds nucleic acid complex between the EON and its specific complementary target nucleic acid molecule in a cell in the subject, allowing the engagement of an endogenous adenosine deaminase enzyme, such as ADAR2, and allowing the enzyme to deaminate the target adenosine in the target nuclear target molecule to inosine, thereby alleviating, preventing, or reversing the iron overload-related disease. Diseases that may be treated by this method preferably include, but are not limited to, the genetic diseases listed herein and any other disease in which deamination of adenosine in HFE transcripts restores protein function in a patient in need thereof.

[0057] RNA editing molecules present in cells, such as ADAR enzymes found in metazoans, including mammals, are often naturally proteinaceous. Preferably, the cell editing entity is an enzyme, more preferably adenosine deaminase or cytidine deaminase, even more preferably adenosine deaminase. These are enzymes with ADAR activity. Of particular interest are human ADARs, including any of their isoforms, hADAR1 and hADAR2. RNA editing enzymes known in the art, for which the oligonucleotide constructs described in the present invention may be conveniently designed, include adenosine deaminase (ADAR) and cytidine deaminase, which act on RNA in humans or human cells, such as hADAR1 and hADAR2. hADAR1 is known to exist in two isoforms: an interferon-inducible 150 kDa long form and a 100 kDa short form, which are produced by alternative splicing from a common pre-mRNA. As a result, the level of the 150 kDa isoform available in cells may be affected by interferons, particularly interferon-gamma (INF-γ). hADAR1 is also induced by TNF-α. This provides an opportunity to develop combination therapies in which INF-γ or TNF-α and the EON described in the present invention are administered to patients as a combined product or as separate products, simultaneously or sequentially in any order. In certain disease states, elevated INF-γ or TNF-α levels may already be occurring in certain tissues of the patient, providing additional opportunities for more specific editing of diseased tissues. Those skilled in the art will understand that the degree to which intracellular editing entities are directed to other target sites may be controlled by varying the affinity of the first nucleic acid strand for the recognition domain of the editing molecule.

[0058] chemical modification

[0059] When the EON and the complementary strand form a so-called heteroduplex RNA-editing oligonucleotide (HEON) complex, as described in GB2215614.5 (unpublished), except when the opposite sense strand does not have an orphan nucleotide, all of the chemical modifications listed below that may be used in the EON herein may also be used on the sense strand complementary to the EON. Thus, modifications related to orphan nucleotides relate only to the EON herein, while all other modifications relate to the EON herein and any (protected) sense oligonucleotides that may be used with the EON in pharmaceutical products. This includes the use of hydrophobic moieties (such as tocopherol and cholesterol) and cell-specific ligands (such as GalNAc moieties) that may be attached to either the EON or its opposite strand, or both, as also described herein and detailed in GB2215614.5 (unpublished).

[0060] The internucleoside linkages in the oligonucleotides herein may contain one or more naturally occurring internucleoside linkages and / or modified internucleoside linkages. Without limitation, at least one, at least two, or at least three internucleoside linkages from the 5' and / or 3' ends of the EON are preferably modified internucleoside linkages. A preferred modified internucleoside linkage is a PS linkage. In one embodiment, all internucleoside linkages of the EON are modified internucleoside linkages. In one embodiment, the EON includes a PNdmi linkage connecting the last nucleoside at the 5' and / or 3' ends and the nucleoside immediately preceding the last nucleoside at each of these ends. Preferably, the PNdmi linkage used in the EON herein has the following formula: [ka]

[0061] Common limiting factors in oligonucleotide-based therapeutics are the ability of the oligonucleotide to be taken up by cells (either by itself or when delivered "naked" without being applied to a delivery vehicle), its biodistribution, and its 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 described in the art. Examples of such chemical modifications currently in common use include 2'-O-methyl (often abbreviated as 2'-OMe or 2'-O-Me), 2'-F, and 2'-O-methoxyethyl (often also referred to as 2'-methoxyethoxy or 2'-MOE) modifications of sugars, and the use of PS linkages between nucleosides. WO2020 / 201406 discloses the use of MP linkage modifications at specific positions surrounding orphan nucleotides in a first nucleic acid strand. Except for the ribose sugar moiety of orphan nucleotides, which have certain limitations in terms of compatibility with RNA editing, the ribose 2' functional group in all nucleotides of an EON can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4'-linkages (e.g., locked nucleic acids (LNA)), or other ribosyl 1'-, 2'-, 3'-, 4'-, or 5'-substitutions. Orphan 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. GB2214347.3 (unpublished) describes modification of the 2' position of the ribose sugar moiety of orphan nucleotides with a 2'2'-disubstitution such as diF, which is also applicable to the invention described herein. The 2'-4' linkage can be selected from many linkers known in the art, such as a methylene linker, an amide linker, or a restricted ethyl linker (cEt).

[0062] 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 stretch of nucleotides in the target RNA containing the target adenosine, and the nucleotide in a first nucleic acid strand opposite the target nucleotide is an orphan nucleotide. When the target nucleotide is adenosine, the orphan nucleotide preferably contains a base, a modified base, or a base analog having an NH moiety in a position similar to the ring nitrogen (e.g., Benner's base Z). Nucleotide numbering in the EON is 0 for the orphan nucleotide, and +1 for the nucleotide 5' to the orphan nucleotide. Counting increases further toward the 5' end and increases toward the 3' end, with the first nucleotide 3' to the orphan nucleotide being -1. Numbering of internucleoside linkages in the EON is such that linkage number 0 is the linkage 5' to the orphan nucleotide, and linkage positions in the oligonucleotide increase toward the 5' end and decrease toward the 3' end.

[0063] 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 two terminal nucleotides on each end of the EON.

[0064] Nucleosides in EONs may be natural nucleosides (deoxyribonucleosides or ribonucleosides) or unnatural nucleosides. It should be noted that in RNA editing, where double-stranded RNA is generally a substrate for enzymes with deaminating activity (such as ADARs), ribonucleosides are considered "natural," while deoxyribonucleosides may be considered unnatural or modified for the purposes of discussion, simply because DNA does not exist in the RNA-RNA duplex substrate form. Those skilled in the art will recognize that when a nucleotide has a natural ribose moiety, it may still have unnatural modifications in the base and / or linkage.

[0065] In addition to the specific preferred chemical modifications at specific positions in the compounds herein, the compounds may also comprise or consist of one or more (additional) modifications to the nucleobase, scaffold, and / or backbone linkages, e.g., at the 3' and / or 5' positions, which may or may not be present in the same monomer. Scaffold modifications refer to the presence of modified versions of the ribosyl moiety (i.e., pentose moiety) that naturally occurs 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'-OMe, 2'-O-(2-cyanoethyl), 2'-MOE, 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(2-aminopropyl), 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl), and the like. 2'-O-alkyl or 2'-O-(substituted) alkyl; 2'-deoxy (DNA); 2'-O-(haloalkyl)methyl such as 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2 2'-O-alkoxycarbonyl, such as [N,N-dimethylcarbamoyl]ethyl (DCME); 2'-halo, e.g., 2'-F, FANA; 2'-O-modified RNA monomers, such as 2'-O-[2-(methylamino)-2-oxoethyl] (NMA); conformationally constrained nucleotide (CRN) monomers, locked nucleic acid (LNA) monomers, xylo-LNA monomers, α-LNA monomers, α-l-LNA monomers, β-d-LNA monomers, 2'-amino-LNA monomers, 2'-(alkylamino)-LNA monomers, 2'-(acylamino)-LNA monomers, 2'-N-substituted 2'-amino-LNA monomers, 2'-thio-LNA monomers, (2'-O,4'-C)-restricted ethyl (cEt) BNA monomers, (2'-O,4'-C)-restricted methoxyethyl (cMOE) BNA monomers, 2',4'-BNANC (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-tcDNA monomers, alpha Bicyclic or bridged nucleic acid (BNA) scaffold modifications include anomeric bicyclic DNA (abcDNA) monomers, oxetane nucleotide monomers, 2'-amino LNA-derived locked PMO monomers, guanidine-bridged nucleic acid (GuNA) monomers, spirocyclopropylene-bridged nucleic acid (scpBNA) monomers, and derivatives thereof; cyclohexenyl nucleic acid (CeNA) monomers, altriol 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.

[0066] The base sequence of the EON herein is complementary to a portion of the base sequence of the target HFE transcript, including at least the target adenosine to be deaminated to inosine (preferably adenosine at position 845), 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 (such as temperature, salt concentration, and the like) under which the two strands can hybridize, taking into account the complementarity between the strands.

[0067] In contrast to what has been described about gapmers and their association with RNase degradation and the use of such gapmers in double-stranded complexes (see, e.g., EP 3954395 A1), the EONs herein do not contain stretches of DNA nucleotides that would target the target sequence (or sense nucleic acid strand) for RNase-mediated degradation. In one embodiment, the EON does not contain four or more consecutive DNA nucleotides anywhere in its sequence. In certain embodiments, the EON is composed of as many (chemically) modified nucleotides as possible to increase resistance to RNase-mediated degradation, while at the same time being as efficient as possible in producing an RNA editing effect. This means not only that orphan nucleotides and some other nucleotides in the EON may be DNA, but also that there are no stretches of four or more consecutive DNA nucleotides in the EON. Thus, the EONs herein are not gapmers. Gapmers reduce expression of target transcripts but do not result in RNA editing of specific adenosines in the target transcript. A gapmer is essentially a ss nucleic acid consisting of a central region (a DNA gap region having at least four consecutive deoxyribonucleotides) and wing regions located immediately adjacent to its 5'-end (5' wing region) and 3'-end (3' wing region). In contrast, the EON herein may be any oligonucleotide that produces an RNA editing effect by deaminating a target adenosine in a target RNA molecule to inosine, thereby being as resistant to RNase-mediated degradation as possible to produce the RNA editing effect.

[0068] In one embodiment, the EON, or the sense strand that may be annealed to it before entering a target cell, is linked to a hydrophobic moiety such as palmityl or its analog, cholesterol or its analog, or tocopherol or its analog. It is preferably linked to the 5' end. When the hydrophobic moieties are linked to the 5' end and the 3' end, the hydrophobic moieties may be the same or different. The hydrophobic moiety linked to the oligonucleotide may be directly linked or indirectly linked via another substrate. When the hydrophobic moiety is directly linked, it is sufficient if the moiety is linked via a covalent bond, ionic bond, hydrogen bond, or the like. When the hydrophobic moiety is indirectly linked, it may be linked via a linking functional group (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 within an animal body (e.g., a human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases or by physiological conditions specific to a part 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, amide, ester, phosphodiester of one or both esters, phosphoester, carbamate, and disulfide bonds, as well as natural DNA linkers. Cleavable linkers also include self-immolative linkers. Non-cleavable linkers refer to linkers that are not cleaved under physiological conditions or are much slower than cleavable linkers, such as linkers consisting of PS bonds, modified or unmodified deoxyribonucleotides linked by PS bonds, spacers linked via PS bonds, and modified or unmodified ribonucleotides. When the linker is a nucleic acid such as DNA or an oligonucleotide, there is no limit to the chain length. However, it may be 2 to 20 bases long, 3 to 10 bases long, or 4 to 6 bases long.There is no limitation in the length or composition of the spacer linking the ligand and oligonucleotide, and it may include, for example, ethylene glycol, TEG, HEG, an alkyl chain, propyl, 6-aminohexyl, or dodecyl.

[0069] The present disclosure 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 for administering the EON or pharmaceutical composition may depend on the disorder to be treated and the tissue that needs to be targeted, and can be selected by common 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, which may be determined using common knowledge in the art and adjusted based on the disorder and the efficacy of the active ingredient.

[0070] In one embodiment, an EON comprises at least one nucleotide having a sugar moiety comprising a 2'-OMe modification. In one embodiment, an EON comprises at least one nucleotide having a sugar moiety comprising a 2'-MOE modification. In one embodiment, an EON comprises at least one nucleotide having a sugar moiety comprising a 2'-F modification. In one embodiment, an orphan 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 the linkage to the base and / or adjacent nucleoside of the orphan nucleotide. In one embodiment, an orphan nucleotide has a 2'-F in the sugar moiety. In one embodiment, an orphan nucleotide has a diF substitution in the sugar moiety. In one embodiment, an orphan nucleotide has 2'-F and 2'-C-methyl in the sugar moiety. In one embodiment, an orphan nucleotide comprises a 2'-F in the arabinose configuration (FANA) in the sugar moiety. In one embodiment, the 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 for deamination of a target adenosine in the RNA molecule of the target HFE, and wherein the nucleotide in the EON opposite the target adenosine is an orphan nucleotide, and the orphan nucleotide has the structure: [ka] wherein 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 both independently selected from H, OH, F, or CH3; R3 is a portion of an EON on the 5' side of an orphan nucleotide consisting of 7 to 30 nucleotides; and R4 is a portion of an EON on the 3' side of an orphan nucleotide consisting of 4 to 25 nucleotides. The nucleotide on the 3' and / or 5' side of the orphan nucleotide may be DNA, and is more preferably the 3' nucleotide (-1 position).

[0071] In one embodiment, the first nucleic acid strand comprises at least one methylphosphonate (MP) internucleoside linkage according to the following structure: [ka]

[0072] Although MP linkages at other positions are not expressly excluded, the preferred position of the MP linkage in the EON herein is the bond at position -2, thereby linking the nucleotide at position -1 to the nucleoside at position -2.

[0073] In one embodiment, the EON comprises at least one nucleotide having a sugar moiety that includes a 2'-fluoro (2'-F) modification. A preferred position for the nucleotide having the 2'-F modification is position -3 in the EON, which may occur with the same 2' modification in an orphan nucleotide as described above.

[0074] In one embodiment, the EON comprises at least one phosphonoacetate or phosphonoacetamide internucleoside linkage.

[0075] In one embodiment, the EON comprises at least one nucleotide that includes a locked nucleic acid (LNA) ribose modification or an unlocked nucleic acid (UNA) ribose modification. In certain embodiments, the EON comprises at least one nucleotide that includes a TNA ribose modification.

[0076] Those skilled in the art will understand that oligonucleotides, such as EONs, as outlined herein, generally consist 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). These consist of a pentose sugar, a ribose, a 5'-linked phosphate group attached via a phosphate ester, and a 1'-linked base. The sugar connects the base and phosphate and is therefore often referred to as the "scaffold" of the nucleotide.

[0077] Modifications to the pentose sugar are therefore often referred to as "scaffold modifications." The original pentose sugar may be replaced in its entirety with another moiety that also links the base and phosphate. Therefore, while a pentose sugar is often the scaffold, it is understood that a scaffold is not necessarily a pentose sugar. Examples of scaffold modifications that may be applied to the monomers of the EONs of the present invention are disclosed in WO2020 / 154342, WO2020 / 154343, and WO2020 / 154344.

[0078] In one embodiment, an EON herein may contain one or more nucleotides with a 2'-MOE ribose modification. Also, in one embodiment, an EON contains one or more nucleotides without a 2'-MOE ribose modification, where the 2'-MOE ribose modification is located at a position that does not prevent an enzyme with adenosine deamination activity from deaminating the target adenosine. In another embodiment, an EON contains a 2'-OMe ribose modification at a position that does not contain a 2'-MOE ribose modification, and / or the oligonucleotide contains a deoxynucleotide at a position that does not contain a 2'-MOE ribose modification. In one embodiment, the EON comprises one or more nucleotides containing a 2'-position that includes 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, TNA, 2'-fluoro (2'-F), 2',2'-difluoro (diF) modification, 2'-fluoro-2'-C-methyl modification, or a 2'-4'-linkage (i.e., a bridged nucleic acid such as a locked nucleic acid (LNA) or, for example, examples listed in WO2018 / 007475). In another embodiment, other nucleic acid monomers that may be applied, for example, for the purpose of improving affinity, are arabinonucleic acid and 2'-deoxy-2'-fluoroarabinonucleic acid (FANA). The 2'-4' linkage can be selected from linkers known in the art, such as a methylene linker or a restricted ethyl linker. A wide variety of 2' modifications are known in the art. Further examples are disclosed in more detail in, for example, WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2018 / 134301, WO2019 / 219581, WO2019 / 158475, and WO2022 / 099159. In all cases, the modification should be compatible with editing, and the EON performs its role as an editor, generating an oligonucleotide that can form a double-stranded complex with the target RNA and recruit a deaminating enzyme that can then deaminate the target adenosine.When the monomer includes an unlocked nucleic acid (UNA) ribose modification, the monomer can have a 2' position that includes 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 a locked nucleic acid (LNA)).

[0079] A base, sometimes referred to as a nucleobase, is generally adenine, cytosine, guanine, thymine, or uracil, or a derivative thereof. A base, sometimes referred to as a nucleobase, is defined as a moiety that can bind to another nucleobase via an H-bond, a polar bond (such as via a CF moiety), or an aromatic electron interaction. Cytosine, thymine, and uracil are pyrimidine bases that generally bind to the scaffold via their 1-nitrogen. Adenine and guanine are purine bases that generally bind to the scaffold via their 9-nitrogen. As used herein, the terms "adenine," "guanine," "cytosine," "thymine," "uracil," and "hypoxanthine" refer to such nucleobases. The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to a nucleobase bound to a (deoxy)ribosyl sugar.

[0080] The nucleobase in the EON herein can be adenine, cytosine, guanine, thymine, or uracil, or any other moiety capable of interacting with another nucleobase via an H-bond, a polar bond (such as C—F), or an aromatic electronic interaction. The nucleobase at any position in the nucleic acid strand can be hypoxanthine (the nucleobase in 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-aminomethyluracil, 5-hydroxymethyluracil, 5-formylcytosine ... cytosine), 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-clamp and its derivatives, Super A (Super A), Super T, Super G, modified forms of adenine, cytosine, guanine, or uracil, such as amino-modified nucleobases or derivatives thereof; and degenerate bases or universal bases, such as 2,6-difluorotoluene, or absent, such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, azaribose).

[0081] In some embodiments, the nucleotide analog is an analog of a nucleic acid nucleotide. In some embodiments, the nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine. In some embodiments, the nucleotide analog is not guanosine or deoxyguanosine. In some embodiments, the nucleotide analog is not a nucleic acid nucleotide. In some embodiments, the nucleotide analog is not adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine.

[0082] Nucleotides are generally linked to adjacent nucleotides via condensation of their 5'-phosphate moiety to the 3'-hydroxy moiety of the adjacent nucleotide monomer. Similarly, their 3'-hydroxy moiety is generally 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 grafted onto this copolymer, i.e., the scaffold moiety. Because of this property, the alternating copolymer formed by the linked scaffold of an oligonucleotide is often referred to as the "backbone" of the oligonucleotide. Because phosphodiester bonds link adjacent monomers together, they are often referred to as "backbone linkages." When the phosphate group is modified so that it becomes an analog moiety instead, such as PS, it is understood that such a moiety is still referred to as the backbone linkage of the monomer. This is referred to as a "backbone linkage modification." In general terms, the backbone of an oligonucleotide includes alternating scaffolds and backbone linkages.

[0083] The EON herein can include linkage modifications, including, but not limited to, PS, chiral pure PS, (R)-PS, (S)-PS, MP, chiral pure methylphosphonate, (R)-methylphosphonate, (S)-methylphosphonate, phosphorylguanidine (such as PNdmi), chiral pure phosphorylguanidine, (R)-phosphorylguanidine, (S)-phosphorylguanidine, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methyl phosphorothioate, methylthiophosphonate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, borano PS, methylboranophosphate. Modified versions of the phosphodiesters present in RNA may be used, such as methylboranophosphate, methylboranoPS, methylboranophosphonate, methylboranophosphothioate, phosphate, phosphotriester, aminoalkylphosphotriester, and their derivatives. Other modifications include phosphoramidite, phosphoramidate, N3'→P5' phosphoramidate, phosphorodiamidate, phosphorothiodiamidate, sulfamate, diethylene sulfoxide, amide, sulfonate, siloxane, sulfide, sulfone, formacetyl, alkenyl, methylenehydrazino, sulfonamide, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamide nucleic acid (TANA); and their derivatives. Various salts, mixed salts, and free acid forms, as well as 3'→3' and 2'→5' linkages, are also included.

[0084] In one embodiment, the EON 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, or phosphonoacetates, or phosphorodithioates, or phosphotriesters, or aminoalkylphosphotriesters, or H-phosphonates, or methyl and other alkyl phosphonates, including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates, or phosphinates, or phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, or thionophosphoramidates, or thionoalkylphosphonates, or thionoalkylphosphotriesters, or selenophosphates, or boranophosphates. Particularly preferred are internucleoside linkages modified to contain PS. Many of the non-naturally occurring modifications of these linkages, such as PS, are chiral, meaning that Rp and Sp configurations exist, as known to those skilled in the art. In one embodiment, the chirality of the PS bond is controlled, meaning that each bond can be either the Rp or Sp configuration. The selection of the Rp or Sp configuration at a particular binding position may depend on the target sequence and the efficiency of binding and induction of RNA editing. However, if this is not particularly desired, the composition may contain an AON as an active ingredient having both the Rp and Sp configurations at a particular binding position. A mixture of such EONs is also feasible, with certain positions preferably having one of the configurations and other positions not being problematic.

[0085] Again, in all cases, the modification should be compatible with editing, and the EON, when binding to its target sequence, due to the nature of the resulting dsRNA, plays its role as an editor, generating an oligonucleotide that can recruit adenosine deaminase. In all aspects of the present invention, the enzyme with adenosine deaminase activity is preferably ADAR1, ADAR2, or ADAT. In a highly preferred embodiment, the EON is an RNA editing oligonucleotide that targets pre-mRNA or mRNA, wherein the target nucleotide in the target RNA is adenosine, which is deaminated to inosine, which is read as guanosine by the translational machinery. The present disclosure also provides a pharmaceutical composition comprising an EON as characterized herein and a pharmaceutically acceptable carrier.

[0086] Other chemical modifications of the EONs herein include the replacement of any one or more deuterium or tritium hydrogen atoms, examples of which can be found, for example, in WO2014 / 022566 or WO2015 / 011694.

[0087] The present disclosure provides an EON herein, or a pharmaceutical composition comprising an EON herein, for use in the treatment or prevention of a disorder associated with iron overload. In one embodiment, the present disclosure provides an EON herein, or a pharmaceutical composition comprising an EON herein, for use in the treatment or prevention of a disease associated with iron overload, such as HH. In one embodiment, the present disclosure provides an EON herein, or a pharmaceutical composition comprising an EON herein, for use in the treatment or prevention of HH.

[0088] The EON herein preferably does not contain a 5'-terminal O6-benzylguanosine or a 5'-terminal amino modification and is preferably not covalently linked to a SNAP-tag domain (engineered O6-alkylguanosine-DNA-alkyltransferase). The EON herein preferably does not contain a boxB RNA hairpin sequence. In one embodiment, the EON herein contains 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 orphan nucleotide is uridine, no mismatch exists. One alternative to uridine is to place an isouridine opposite the target adenosine, which will not pair like G pairs with U. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the EON directly opposite the target adenosine.

[0089] It should be noted that when an EON is delivered via a vector, such as an AAV vector, there is no chemical modification in the EON that acts on the target RNA molecule. Although it is preferable to use a "naked" EON with the chemical modifications outlined herein, EONs delivered via other means, such as AAV vector expression, or that edit circular molecules or have hairpin structures (e.g., recruitment moieties as disclosed in WO2016 / 097212, WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995) can also be applied to edit adenosines in the RNA molecule of a target HFE to produce an HFE protein with restored function, and are therefore encompassed by the present invention.

[0090] The EON herein can utilize endogenous cellular pathways and naturally available ADAR enzymes to specifically edit target adenosines in target RNA sequences.The EON herein can recruit ADAR and its complex, and then promote the deamination of a (single) specific target adenosine nucleotide in the target RNA sequence.Ideally, only one adenosine is deaminated.When the EON herein forms a complex with ADAR, it preferably causes the deamination of a single target adenosine.

[0091] Analysis of natural targets of ADAR enzymes showed that they generally contain mismatches between the two strands that form the RNA helices edited by ADAR1 or 2. These mismatches have been suggested to enhance the specificity of the editing reaction (Stefl et al. 2006. Structure 14(2):345-355; Tian et al. 2011. Nucleic Acids Res 39(13):5669-5681). Characterizing the optimal pattern of paired / mismatched nucleotides between EONs and target RNAs may also be important for the development of efficient ADAR-based EON therapies.

[0092] As outlined above, the EONs herein utilize specific nucleotide modifications at predetermined positions to ensure stability and proper ADAR binding and activity. These modifications may vary and, as summarized in detail herein, may include modifications to the EON backbone, the sugar moiety of the nucleotide, and the nucleobase or phosphodiester linkage. They may be distributed variably throughout the EON sequence. Specific modifications may be required to support interactions between different amino acid residues in the RNA-binding domain of the ADAR enzyme and their deaminase domain. For example, internucleotide PS linkages, 2'-OMe, or 2'-MOE modifications may be tolerated in some portions of the EON, while they should be avoided in other portions to avoid disrupting the enzyme's critical interactions with the phosphate and 2'-OH groups. Specific nucleotide modifications may be required to enhance editing activity on substrate RNAs whose target sequences are not optimal for ADAR editing. Previous studies have demonstrated 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, whereas the 5'-CAA-3' target sequence is not preferred (Schneider et al. 2014. Nucleic Acids Res 42(10):e87). Structural analysis of the ADAR2 deaminase domain suggests that careful selection of the nucleotide opposite the target trinucleotide may facilitate editing. For example, a 5'-CAA-3' target sequence paired with a 3'-GCU-5' sequence on the opposite strand (forming a central AC mismatch) is not preferred because the guanosine base sterically clashes with the amino acid side chain of ADAR2. While other adenosines in the HFE transcript may be targeted to impair protein function, in a preferred embodiment, the adenosine at position 845 is deaminated. The present disclosure provides RNA editing oligonucleotides, generally referred to herein as EONs, that can result in the deamination of adenosines in HFE transcripts, resulting in HFE proteins that are fully functional in regulating iron levels.This means that the present invention is not strictly limited to deamination of adenosine at position 845, but that other adenosines (single or multiple) may be targeted, thereby resulting in improved function of the HFE protein. Other adenosines may be identified, for example, by genetic screening in populations or in silico, that are important (or may be more important) for HFE function and may be targeted via RNA editing in accordance with the teachings of this disclosure. Regardless of what the exact nuclear molecule or EON looks like, all such RNA events and oligonucleotides that can be used for such targeting are encompassed by this disclosure.

[0093] Mutagenesis studies of human ADAR2 revealed that a single mutation at residue 488, from glutamate to glutamine (E488Q), increased the deamination rate constant by 60-fold compared to the wild-type enzyme (Kuttan and Bass. 2012. Proc Natl Acad Sci USA. 109(48):3295-3304). During the deamination reaction, ADAR flips the edited base from its RNA duplex into the enzymatic 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 often referred to as an "orphan cytidine." The crystal structure of ADAR2 E488Q bound to double-stranded RNA (dsRNA) revealed that the glutamine (Gln) side chain at position 488 can provide an H-bond to the N3 position of the orphan cytidine, resulting in an increased catalytic rate of ADAR2 E488Q. In the wild-type enzyme, glutamate (Glu) is present at position 488 instead of glutamine (Gln), and the amide group of glutamine is absent, replaced by a carboxylic acid. To achieve the same contact of the orphan cytidine with the E488Q mutant, protonation would be required in the wild-type context. To utilize endogenously expressed ADAR2 to correct disease-associated mutations, it is essential to maximize the editing efficiency of the wild-type ADAR2 enzyme present in cells. WO 2020 / 252376 discloses the use of EONs with modified RNA bases, particularly at orphan cytidine positions, that mimic the hydrogen-bonding pattern observed in the E488Q ADAR2 mutant. It was found that replacing the nucleotide opposite the target adenosine in the EON with a cytidine analog that acts as an H-bond donor at N3 could stabilize the same contacts that are thought to provide the increased catalytic rate of the mutant enzyme.Two cytidine analogs are of particular interest: pseudoisocytidine (also called "piC"; Lu et al. 2009. J Org Chem. 74(21):8021-8030; Burchenal et al. 1976. Cancer Res. 36:1520-1523), which was initially selected because it provides hydrogen bond donation at N3 with minimal disruption to the geometry of the nucleobase, and Benner's base Z (also called "dZ"; Yang et al. 2006. Nucl Acid Res. 34(21):6095-6101). Benner's base is also called 6-amino-5-nitro-3-yl-2(1H)-pyridone. The presence of a cytidine analog in an AON may be in addition to modifications of the ribose 2' group. The ribose 2' groups in the AON can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4'-linkages (i.e., bridged nucleic acids such as locked nucleic acids (LNA)), or other 2'-substitutions. The 2'-4' linkages can be selected from linkers known in the art, such as methylene linkers or restricted ethyl linkers.

[0094] In one embodiment, EON herein refers to a substituted or unsubstituted, straight or branched lower (C-C) alkyl group optionally interrupted by one or more heteroatoms; 10 ) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl; -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, and the like, and include one or more sugar moieties mono- or di-substituted at the 2', 3', and / or 5' positions.

[0095] In one embodiment, the nucleotide analogs or equivalents within the EONs herein contain 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, and thioalkyl derivatives of pyrimidine and purine bases, as known or would be known in the art. Purine and / or pyrimidine nucleobases may be modified to alter their properties, for example, by amination or deamination of heterocycles. The exact chemical structure and format may vary from oligonucleotide construct to oligonucleotide construct and application to application, and may be implemented according to the desires and preferences of those skilled in the art.

[0096] The EONs 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 herein are longer than 20 nucleotides. The EONs herein are preferably shorter than 100 nucleotides, even more preferably shorter than 60 nucleotides, and even more preferably shorter than 50 nucleotides. In preferred embodiments, the EONs herein comprise 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, and even more preferably 18 to 50 nucleotides. Thus, in particularly preferred embodiments, the EONs herein comprise 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 herein is 27, 28, 29, or 30 nucleotides in length.

[0097] In one embodiment, the EON herein incorporates an inverted deoxyT or dideoxyT nucleotide at one or both termini.

[0098] As described above, in some embodiments, the present disclosure provides an EON for forming a ds complex with a human HFE RNA molecule in human liver cells. Thus, the therapeutic effect is preferably in vivo on human liver cells. Of course, the method can also be performed in vitro or ex vivo.

[0099] The present disclosure provides an EON or a pharmaceutical composition for use in treating a disease. The present disclosure also provides the use of the EON herein or the pharmaceutical composition herein in the manufacture of a medicament for treating a disease. The present disclosure also provides a method for treating a disease in a patient, comprising administering a therapeutically effective amount of the EON herein or the pharmaceutical composition herein. Preferably, the disease is a disease caused by iron overload caused by the C282Y mutation in HFE. The EON is preferably administered therapeutically rather than prophylactically (after genetic counseling), although it cannot be excluded that doing so would also be beneficial.

[0100] After RNA editing occurs in a cell, the modified RNA may fade over time due to, for example, cell division, the limited half-life of the edited RNA, etc. Thus, in actual treatment, the methods of the invention may involve repeatedly delivering AONs until the target RNA is sufficiently modified to provide a reliable benefit to the patient and / or such that the benefit is maintained over time. [Example]

[0101] Example 1. Editing of Targeted Adenosines in Human HFE Target RNA Molecules Using an In Vitro Biochemical Editing Assay

[0102] First, the first set of HFE-targeted EONs (RM4700 to RM4726 shown in Figure 1) were tested for editing of human HFE target (pre)mRNA in an in vitro biochemical editing assay. To obtain HFE target RNA, PCR was performed using an HFE G-block (IDT) containing sequences for the T7 promoter 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: 53) and the reverse primer 5'-TGG ACC GAC TGG AAA CGT AG-3' (SEQ ID NO: 54). The 5' to 3' G-block sequence (SEQ ID NO: 55) is as follows, with the target adenosine in bold and underlined, and the primer sequence underlined: [ka]

[0103] The PCR product was then used as a template for in vitro transcription. The MEGAscript T7 transcription kit was used for this reaction. RNA was purified on a urea gel and subsequently 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 was used as a target in a biochemical editing assay.

[0104] First, EON RM4700 to RM4726 were annealed with the HFE target RNA. This was done in buffer (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 RT. The editing reaction was then carried out. The annealed oligonucleotide / target RNA was incubated with protease inhibitors (cComplete (商標)The oligonucleotides were mixed with purified ADAR2 (GenScript) to a final concentration of 6 nM and 2 nM target RNA. The mixture was incubated at 37°C for a predetermined time point. Each reaction was stopped by adding 95 μl of 3 mM EDTA solution at 95°C. A 6 μl aliquot of the stopped reaction mixture was then used as a template for cDNA synthesis using a Maxima reverse transcriptase kit (ThermoFisher) containing random hexamer primers (ThermoFisher Scientific). Initial RNA denaturation 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. Using 1 μl of cDNA as template, products were amplified by PCR for pyrosequencing analysis using an Amplitaq Gold 360 DNA Polymerase Kit (Applied Biosystems) according to the manufacturer's instructions. PCR was then performed using the following thermal cycling protocol: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 7 minutes.

[0105] Because inosine base pairs with cytidine during cDNA synthesis in the reverse transcription reaction, the nucleotide incorporated at the edited position during PCR was likely guanosine. The percentage of guanosine (edited) relative to adenosine (unedited) was determined by pyrosequencing. Pyrosequencing of PCR products and data analysis were performed on a PyroMark Q48 Autoprep instrument (QIAGEN) according to the manufacturer's instructions, with an input of 10 μl of PCR product and 4 μM sequencing primers. Analysis performed by this instrument provided results for selected nucleotides as the percentage of adenosine and guanosine detected at that position. The degree of A-to-I editing at a selected position was therefore measured by the percentage of guanosine at that position.

[0106] The results are shown in Figure 2A, B, and C, each containing a subset of data points derived from a particular EON. Although efficiency varied, all tested EONs were clearly able to mediate RNA editing in the biochemical editing assay. The EONs that performed best in this in vitro assay were those with 18 nucleotides 5' to the orphan nucleotide and 11 nucleotides 3' to the orphan nucleotide (e.g., RM4716, RM4717, RM4718, and RM4719). [Example]

[0107] Example 2. Editing of target adenosines within human HFE target RNA molecules in B lymphocytes derived from donors with the C282Y mutation

[0108] Next, we tested EON RM4700 to RM4723 and RM4725 for their ability to mediate RNA editing by recruiting endogenously present ADAR enzymes in B lymphocytes from two different donors homozygous for the C282Y (c.845G>A) mutation in the HFE gene. These donors, designated GM14715 and GM14631, provided the B lymphocytes from Corriel. Human Epstein-Barr virus (EBV)-immortalized B lymphocytes were cultured in RPMI-1640 / 10% FBS / 1% Pen-Strep. Cells were maintained at 37°C in a 5% CO2 atmosphere.

[0109] Total 0.2×10 6Cells were co-treated with 5 μM EON+1 μM AG1856 saponin in a total volume of 200 μl in a 48-well format. Those skilled in the art will recognize that a variety of different saponins have been used in many types of applications, and therapeutic uses of saponins have also been described (Weng A et al. 2009. Planta medica 75(13):1421-1422; Weng A et al. 2010. J Chromatography B 878(7):713-718; Weng A et al. 2012. Molecular Oncology 6(3):323-332; Weng A et al. 2012. J Controlled Disease 164(1):74-86; Thakur et al. 2014. J Chromatography B 955:1-9; Jia et al. 1998. J Natural Products 61(11):1368-1373; Haddad et al. 2004. Helvetica chimica acta 87(1):73-81; Fu et al. 2005. J Natural Products 68(5):754-758; Moniuszko-Szajwaj et al. 2016. Helvetica chimica acta 99(5):347-354; Fuchs H et al. 2017. Biomedicines 5(2):14). It has also been described that a specific saponin (SO1861) from Saponaria officinalis can mediate improved intracellular delivery of peptide and lipid nanoparticles, as well as nucleic acids (Weng A et al. 2015. J Controlled Release 206:75-90; Sama S et al. 2017. Int J Pharmaceutics 534:195-205).WO 2019 / 011914 discloses a saponin (GE1741) isolated from Gypsophila elegans that exhibits improved efficacy in the delivery of small molecules, such as nucleic acid molecules, into cells (see also Sama S et al. 2018. J Biotechnology 284:131-139). WO 2021 / 122998 (and EP 3838910B1 resulting from the priority application) further discloses another class of saponins derived from Agrostemma githago L. that have even improved properties over the previously described SO1861 and GE1741 saponins, particularly with regard to toxicity and endosomal escape (see also Clochard J et al. 2020. Int J Pharm 589:119822). The inventors of the present invention used saponin AG1856 (also known as triterpene glycoside or triterpene saponin) disclosed in WO2021 / 122998 to enhance the effect of RNA editing of HFE transcripts in B lymphocytes as described above.

[0110] Negative controls were a sample treated with EON having a scrambled sequence (sequence not shown), a non-treated sample (NT), a sample without reverse transcriptase (-RT) (see below), and a water sample.

[0111] Seventy-two hours after initial exposure to EON and AG1856, cells were harvested and total RNA was isolated using the SV Total RNA Isolation System kit (Promega). After removing the medium, cells were washed once with PBS. After completely aspirating the PBS, 100 μL of BL+TG (Promega) was added to lyse the cells and collect the cell contents. After adding 35 μL of 2-propanol, the mixture was loaded onto a column and subjected to multiple washing steps and DNase I treatment. After elution in a total volume of 20 μL of DNase / RNase-free water, RNA yield was determined using spectrophotometric analysis (NanoDrop) and stored at -80°C.

[0112] cDNA was generated using Maxima reverse transcriptase (RT, Thermo Fisher). Typically, 500 ng of total RNA was used in a reaction mixture containing 4 μL of 5x RT buffer, 1 μL of dNTP mix (10 mM each), and 1 μL of random hexamers (all Thermo Fisher), with DNase- and RNase-free water added to a total volume of 20 μL. Samples were loaded into a T100 thermocycler (Bio-Rad) and incubated at 25°C for 10 min, followed by a cDNA reaction temperature of 50°C for 30 min and a termination step at 85°C for 5 min. Samples were cooled to 4°C before storage at -20°C.

[0113] To determine editing efficiency, cDNA samples were subjected to digital PCR (dPCR) assays. The first dPCR was designed to distinguish between cDNA species containing the original adenosine or edited inosine, which was converted to guanidine during cDNA synthesis. The second multiplex dPCR used a primer / probe set targeting exons 1 and 2 to quantify total HFE transcript copies (cDNA molecules) in the mixture. The third assay used a primer binding to exons 4 / 7 and a probe overlapping the exon 4 / 6 boundary to quantify exon 5 skipping. The primer and probe sequences are as follows: where "+" indicates the 3' side of the LNA nucleotide. [Table 1]

[0114] A total of 1.3 μL of the cDNA mix was used in a dPCR mixture containing 3 μL of 4x dPCR master mix (Qiagen), 0.6 μL of primers, and 0.3 μL of probe (10 μM stock concentration) in a total volume of 13 μL with DNase- and RNase-free water. 12 μL of this mixture was transferred to an 8.5K partition plate and fluorophore measurements were performed on a Qiaquity instrument. dPCR cycling conditions were as follows: 95°C for 2 minutes of enzyme activation, followed by 40 cycles of 95°C for 15 seconds of denaturation and 63°C for 30 seconds of annealing / extension. The A to I editing percentage was determined by dividing the number of G-containing molecules by the total (G- plus A-containing species) and multiplying by 100.

[0115] The results of an editing experiment using B lymphocytes from donor GM14715 are shown in Figure 3, clearly demonstrating that all tested EONs were capable of mediating RNA editing of mutations in HFE transcripts, albeit with varying efficiencies. In EON RM4717, the EON that performed best in this cell-based assay, a maximum of almost 30% editing was observed. After treatment with EON, exon 4 skipping in HFE transcripts was limited (approximately 2%) (data not shown), and the amount of HFE transcripts remained relatively stable after EON treatment (data not shown). Figure 4 shows the results of an editing experiment using B lymphocytes from donor GM14631, which also showed variable efficiencies, consistent with the results shown in Figure 3. RM4716 showed the highest editing level, almost 50%. Samples from RM4704 and RM4707 were lost during the measurements of this initial experiment (indicated by an X). These two experiments revealed that EONs with 18 nucleotides 5' to the orphan nucleotide and 9, 11, or 13 nucleotides 3' to the orphan nucleotide provided the highest efficiency. RM4725, which contains a long stretch of 2'-F at the 5' end of the EON and a relatively short stretch (only 5 nucleotides) of nucleotides at the 3' end, provided relatively low RNA editing.

[0116] These experiments demonstrate that we were able to achieve ADAR-mediated editing using endogenous ADAR enzymes and RNA-editing mediator oligonucleotides in EBV-immortalized B lymphocytes derived from a donor carrying two alleles of the HFE c.845G>A mutation. [Example]

[0117] Example 3. GM14715 Hepcidin Expression Following Editing and Processing of Target Adenosines in Human HFE Target RNA Molecules in B Lymphocytes

[0118] Building on the initially designed EON (FIG. 1), a further set of EONs was designed with various lengths and chemical modifications. These EONs with their modifications are shown in FIG. 5. Some of these were used in the co-treatment of GM14715 B lymphocytes with 2 μM saponin AG1856 as outlined in Example 2. The editing percentage was determined as described above after 72 hours of treatment. FIG. 6 shows the editing percentage observed with these EONs and compares it with the well-performing RM4717 shown in FIG. 3. The results in FIG. 6 clearly show that the best performer (D282-10; RM106443; SEQ ID NO: 73) conferred over 40% editing of the target adenosine in the HFE transcript, even higher than that initially observed with RM4717.

[0119] To determine the downstream functional effects of the RNA editing effects mediated by these EONs, we investigated whether hepcidin expression levels increase mRNA recovery of the C282Y mutation in B lymphocytes. Using the same samples in which editing percentages were determined, we quantified HAMP expression. To do so, we designed a dPCR assay to detect HAMP mRNA, along with the housekeeping gene GUSB, which was used to normalize HAMP expression data. The data, shown in Figure 6, demonstrate that HAMP mRNA expression increases with higher editing levels. [Table 2] [Example]

[0120] Example 4. Generation of the C282Y mutation with human lymphocytes for in vitro screening of EON

[0121] Because the liver plays a central role in iron homeostasis, it would be desirable to test EONs in a cellular model that more closely reflects the target tissue. To do so, human lymphoid cell lines were generated. Because certain EONs contain a 3'-linked triantennary GalNAc moiety, these cells allow GalNAc-assisted EON uptake via lymphocytes expressing the asialoglycoprotein receptor (ASGR). Using a CRISPR / Cas9 gene editing approach, human induced pluripotent stem cell (iPSC) lines were generated that introduced the C282Y (c.845G>A; rs1800562) mutation into the HFE gene. These iPSC C282Y cells differentiated into mature lymphoid cells, which expressed mature lymphoid markers and ASGR. [Example]

[0122] Example 5. Quantification of intracellular iron levels after editing of target adenosines in HFE target RNA

[0123] Because one of the characteristic clinical symptoms of HH is high iron levels in serum and liver, iron measurement assays are being developed to quantify intracellular and tissue iron levels. The goal is to quantify iron levels after HFE C282Y-edited EON treatment to determine the effect of HFE restoration on iron metabolism. Several iron quantification methods are being explored, including spectrophotometry, in which iron is complexed with chromogens such as ferrozine or ferrene-s, which allow for subsequent colorimetric detection at specific wavelengths. Alternatively, inductively coupled plasma mass spectrometry (ICP-MS) is used to quantify the number of iron atoms in cell lysates. [Example]

[0124] Example 6. Quantification of amino acid recovery in C282Y HFE after editing of targeted adenosines

[0125] To determine the impact of EON-mediated HFE restoration on its protein sequence, a method will be developed that allows for the discrimination and quantification of wild-type and mutant HFE (C282Y) proteins. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) will be explored as a possible peptide quantification technique. This technique allows for the quantification of target HFE peptides in cells and tissues. Ideally, the assay will be able to detect both the restored wild-type HFE peptide sequence, containing the C282 amino acid (or its C294HFE mouse equivalent), and the mutant HFE C282Y peptide.

Claims

1. An RNA editing oligonucleotide (EON) capable of forming a double-stranded complex with a region of an endogenous human HFE transcription molecule in a cell, wherein the region of the HFE transcription molecule contains a target adenosine, and the double-stranded complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the HFE transcription molecule.

2. The EON of claim 1, wherein the HFE transcription molecule is a pre-mRNA or mRNA molecule.

3. 3. The EON according to claim 1 or 2, wherein the cell is a human liver cell, preferably a hepatocyte.

4. 4. The EON of any one of claims 1 to 3, wherein the target adenosine is the c.845G>A mutation in the human HFE gene.

5. 5. An EON according to any one of claims 1 to 4, wherein the EON comprises or consists of the nucleotide sequence of any one of the EON sequences of SEQ ID NOs: 1 to 51 and 66 to 164.

6. 6. The EON of any one of claims 1 to 5, wherein at least one nucleotide comprises one or more non-naturally occurring chemical modifications in the ribose, bond, or base moiety, or one or more additional non-naturally occurring chemical modifications, provided that the orphan nucleotide, which is the nucleotide opposite the target adenosine in the EON, is not a cytidine containing a 2'-OMe ribose substitution.

7. The EON of claim 6, wherein the orphan nucleotide is a deoxynucleotide containing a cytidine analogue, preferably a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleobase.

8. 7. The EON of claim 6, wherein the orphan nucleotide is a deoxynucleotide containing a uridine analog, preferably an isouracil nucleobase.

9. The EON of any one of claims 6 to 8, wherein one or more additional modifications in the linking moiety are independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonyl phosphoramidate, or PNdmi internucleotide linkages.

10. One or more additional modifications in the ribose moiety -OH; · −F; - Substituted or unsubstituted, straight or branched lower alkyl groups (C １ -C １０ ) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -Dimethylaminoethoxyethoxy 10. The EON of claim 6, wherein the EON is a mono- or di-substitution at the 2', 3', and / or 5' position of the ribose, each independently selected from the group consisting of:

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

12. A lipid nanoparticle (LNP) formulation comprising an EON according to any one of claims 1 to 10.

13. A pharmaceutical composition comprising an EON described in any one of claims 1 to 10, a vector described in claim 11, or an LNP formulation described in claim 12; and a pharmaceutically acceptable carrier.

14. 14. An EON according to claims 1 to 10, a vector according to claim 11, an LNP formulation according to claim 12, or a pharmaceutical composition according to claim 13, for use in the treatment of homeostatic iron regulatory protein (HFE) hemochromatosis.

15. Use of an EON according to any one of claims 1 to 10 in the manufacture of a medicament for the treatment of homeostatic iron regulatory protein (HFE) hemochromatosis.

16. A method for editing an HFE polynucleotide, the method comprising contacting an HFE polynucleotide with an EON described in any one of claims 1 to 10, thereby editing the HFE polynucleotide.

17. A method for treating homeostatic iron regulatory protein (HFE) hemochromatosis in a patient in need thereof, the method comprising contacting an HFE polynucleotide with an EON described in any one of claims 1 to 10 in the cells of the subject, thereby treating the patient.

18. A method for treating HFE hemochromatosis, comprising administering to a patient in need thereof a therapeutically effective amount of an EON described in any one of claims 1 to 10, a vector described in claim 11, an LNP formulation described in claim 12, or a pharmaceutical composition described in claim 13.

19. (i) providing a cell with an EON according to any one of claims 1 to 10; (ii) enabling EON uptake by cells; (iii) allowing annealing of the EON to the HFE pre-mRNA or mRNA molecule; (iv) allowing the endogenous ADAR enzyme to deaminate the target adenosine in the target RNA molecule to inosine; and, optionally, (v) confirming the presence of inosine in the target RNA molecule 1. A method for deaminating a target adenosine in an HFE pre-mRNA or mRNA molecule in a cell, the method comprising:

20. 20. The method of any one of claims 16 to 19, wherein the target adenosine is the c.845G>A mutation in an HFE pre-mRNA or mRNA molecule.

21. Step (v) is a) determining the sequence of the HFE pre-mRNA or mRNA molecule; b) assessing the presence of wild-type HFE protein; or c) using a functional readout device to assess preferably serum or plasma ferritin concentration, or serum transferrin saturation percentage; 21. The method of claim 19 or 20, comprising:

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